Molecular physiology of the SERCA and SPCA pumps

Cell Calcium (2002) 32(5–6), 279–3050143-4160/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0143-4160(02)00184-7

Molecular physiology of the SERCAand SPCA pumps

F. Wuytack, L. Raeymaekers, L. Missiaen

Laboratorium voor Fysiologie, K.U.Leuven, Campus Gasthuisberg, Leuven, Belgium

Summary Intracellular Ca2+-transport ATPases exert a pivotal role in the endoplasmic reticulum and in the compart-ments of the cellular secretory pathway by maintaining a sufficiently high lumenal Ca2+ (and Mn2+) concentration inthese compartments required for an impressive number of vastly different cell functions. At the same time this lumenalCa2+ represents a store of releasable activator Ca2+ controlling an equally impressive number of cytosolic functions.This review mainly focuses on the different Ca2+-transport ATPases found in the intracellular compartments of mainlyanimal non-muscle cells: the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. Although it is not our in-tention to treat the ATPases of the specialized sarcoplasmic reticulum in depth, we can hardly ignore the SERCA1pump of fast-twitch skeletal muscle since its structure and function is by far the best understood and it can serve as aguide to understand the other members of the family. In a second part of this review we describe the relatively novelfamily of secretory pathway Ca2+/Mn2+ ATPases (SPCA), which in eukaryotic cells are primarily found in the Golgicompartment.© 2002 Elsevier Science Ltd. All rights reserved.

INTRODUCTION

That Ca2+ ions exert a key role in controlling a plethoraof cell functions and that the endoplasmic reticulum (ER)herein fulfills a central position, needs for readers of thisjournal hardly further comment [1]. In this review wehope to highlight some aspects of Ca2+-transport ATPasesthat replenish the ER and the Golgi Ca2+ stores. Thesepumps not only help to bring the cytosolic free Ca2+

concentration ([Ca2+]c) back to its resting level after cellactivation, but they also help to maintain the high [Ca2+]in the lumen of the ER, the Golgi, and other compart-ments of the secretory pathway that are indispensable fornormal cell function. Some reviews dealing with similaror related topics appeared recently [2–5]. The reader isalso referred to the excellent P-type ATPase database keptby KB Axelsen at http://biobase.dk/∼axe/Patbase.html,and to the Online Mendelian Inheritance in Man (OMIM)database, which forms part of the Entrez search and re-trieval system at http://www.ncbi.nlm.nih.gov/Entrez. Al-

Received 1 September 2002Accepted 1 October 2002

Correspondence to: Dr Frank Wuytack, Laboratorium voor Fysiologie,K.U.Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.Tel.: +32-16-345936; fax: +32-16-345991;e-mail: [email protected]

though it is not our intention to focus on the SERCA1apump, which is after all a very specialized pump func-tioning in a very specialized ER-derived compartment, i.e.the sarcoplasmic reticulum (SR) from fast-twitch skeletalmuscle, we can hardly bypass this pump if we want tounderstand the function of the SERCA and related pumpssince it represents the archetype of the SERCA pumps.

STRUCTURE AND FUNCTION OF THESERCA Ca2+ PUMP

The catalytic cycle

While many of the detailed features of the steps in the cat-alytic cycle are still controversial, most models are basedon the transformation between two major conformationalstates, designated E1 and E2 (Fig. 1). In the E1 conforma-tion, the two Ca2+-binding sites are of high affinity andare facing the cytoplasm. In the E2 state the Ca2+-bindingsites are of low affinity and are facing the lumenal side.Either cytosolic ATP or Ca2+ can bind first to the E1conformation. The 2Ca2+-E1-ATP form undergoes phos-phorylation to form 2Ca2+-E1-P, the high-energy phos-phointermediate in which the bound Ca2+ ions becomeoccluded. This intermediate is also called theADP-sensitiveform, because in the presence of ADP the backward

279

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http://www.ncbi.nlm.nih.gov/Entrez

280 F Wuytack, L Raeymaekers, L Missiaen

Fig. 1 Scheme of the E1/E2 model of the SERCA catalytic cycle.See text for details.

reaction occurs with release of the bound Ca2+ andsynthesis of ATP. Conversion to the low-energy inter-mediate is accompanied by a major conformationalchange to 2Ca2+-E2-P (ADP-insensitive form), wherebythe Ca2+-binding sites are converted to a low-affinitystate and reorient towards the lumenal face. The cycleends with the sequential release of Ca2+ and phosphateand a major conformational change from the E2 to theE1 state.

Overview of the structure

Before the publication of the high-resolution structuresof several conformational forms of the SERCA1 Ca2+

pump by Toyoshima et al. [6] and Toyoshima and No-mura [7], a great deal was known already on the basicstructure–function relationships of P-type ATPases basedon extensive biochemical, mutagenesis and low-resolutionstructural analysis in several laboratories. These studieshave been the subject of several reviews [2,8–10]. Morerecently, the implications of the X-ray crystallographicstructure for the molecular description of transport ofCa2+ and other ions have been reviewed [5,6,11–13]. Thescope of the present review allows the presentation ofonly the major findings.The SERCA Ca2+-pump protein consists of a single

polypeptide chain folded into four major domains (Figs. 2and 3): a transmembrane (M) domain, composed of 10transmembrane helices, and three cytosolic domains. Twoof these domains, the actuator (A) domain and the phos-phorylation (P) domain, are connected to the M domain.The third, the nucleotide-binding (N) domain, is con-nected to the P domain. The links between the A and Mdomain and between the N and P domain are thought tobe flexible, as indicated by the lack of structure in theselinks and by large-scale domain movements suggested bythe difference in the E1-2Ca2+ and Ca2+-free E2 structures.The two Ca2+-binding sites are located in the M domain.Depending on the protein conformation, the binding sitescan exist in a high-affinity state allowing access from the

cytosolic side (E1 state), or in a low-affinity state facing thelumenal side of the membrane (E2 state). The structureshown in Fig. 2 is an E1 state with two bound Ca2+ andwith the cytoplasmic access channel already deformed(PDB database file 1EUL). In the E2 structure stabilized bythapsigargin (PDB database file 1IWO) the mouth of thechannel is already formed.The relationship of the overall structure to the primary

amino acid sequence can be seen in Fig. 3, which showsa linear spread-out of the primary sequence with an indi-cation of the amino acid residues belonging to -helicesand -sheets as determined from the E1 crystallographicstructure. The map also preserves the main relationshipsbetween the major domains and indicates the amino acidswhose mutation has the most pronounced effect on pumpexpression or function. Before summarizing themajor con-formational changes revealed by the two available detailedstructures, the major structural features will be reviewedbased on that of the Ca2+-bound E1 conformation.

The P domain

The centrally located phosphorylation (P) domain is buildup of two parts of the primary sequence, separated bya long stretch that forms the N domain. The short up-stream region is connected to the fourth transmembranehelix (M4) and contains the amino acid Asp351 that be-comes transiently phosphorylated during the catalyticcycle and which is conserved in all P-type ion-transportATPases. The longer C-terminal part is connected to M5.The crystallographic structure confirms the proposalmade only 2 years earlier that the P domain of P-type iontransport ATPases belongs to the superfamily of haloaciddehalogenases (HADs) that in addition includes phospho-glucomutases and serine/threonine phosphatases [14].The common structure of this family is a Rossman fold,a seven-stranded parallel -sheet with eight short he-lices. Site-directed mutagenesis has identified the roleof several P-domain amino acids in the pump functionbesides the indispensable phosphate-accepting Asp351.We mention in particular Thr353 and Lys684, both lo-cated close to Asp351 in the three-dimensional structure.The side-chain hydroxyl of Thr353 interacts with bothnucleotide and phosphate, whereas the main chain car-bonyl may coordinate the catalytic Mg2+ [15]. Lys684 islabeled by adenosine triphosphopyridoxal in the presenceof Ca2+, the binding of Ca2+ being a prerequisite for theformation of close contact between the bound nucleotideand the phosphorylation site. Lys684 is equivalent to alysine in HADs that plays a central role in the catalyticmechanism. Mutagenesis indicates that in SERCA it playsa role in stabilization of the excess of negative charge de-veloped in the transition state complex between Asp351and the -phosphoryl group of ATP [16].

Cell Calcium (2002) 32(5–6), 279–305 © 2002 Elsevier Science Ltd. All rights reserved.

Molecular physiology 281

Fig. 2 Model of the SERCA1 Ca2+ pump in the E1 conformation (PDB file 1EUL). The point of view is from the side but some distance abovethe cytosolic surface of the membrane. This orientation clearly shows the flexible connections between the A (blue) and M domains (red) andbetween the N (green) and P domains (brown). The picture was generated with the Cn3D program (NCBI). The positions of some of thecritical residues are indicated.

The N domain

Like in HADs, the P-domain sequence is characterizedby an unusual interruption by a large insert betweenthe first -sheet and the first -helix. This insert forms

the N domain. Toyoshima et al. determined the posi-tion of the nucleotide in the structure by a differenceanalysis of crystals that had been soaked in a solutionof TNP–AMP [6]. The ATP analogue is located close toPhe487, which had been identified by mutagenesis as a

© 2002 Elsevier Science Ltd. All rights reserved. Cell Calcium (2002) 32(5–6), 279–305


Fig. 3 Major domains, secondary structure elements, and functionally critical residues indicated on the primary amino acid sequence of theSERCA1 Ca2+ pump. The numbering of the residues starting from the N-terminus is indicated in italic. Open symbols: loops; grey symbols:-sheets; reverse contrast: -helices. The major domains are indicated as M (membrane domain), A (actuator domain), N (nucleotide-bindingdomain) and P (phosphorylation domain). Ca2+-coordinating residues are indicated by black triangles (: site I; : site II). Non-circlesymbols connected to full lines indicate residues whose mutation results in profound effects on the pump function. Boxed groups of residuesindicate cluster mutations. The type of effect of the mutation is represented by the shape of the residue symbol as indicated. An additionaleffect is a targeting defect caused by a cluster mutation near the N-terminus. T1, T2: tryptic cleavage sites. Tg, CPA: binding region for theSERCA-specific inhibitors thapsigargin and cyclopiazonic acid. The boxed numbers refer to papers describing the mutation experiments: 1.Daiho T et al. J Biol Chem 1999; 274: 23910; 2. Andersen JP, Vilsen B. Acta Physiol Scand 1992; 146: 151; 3. Vilsen B, Andersen JP. J BiolChem 1992; 267: 25739; 4. Sørensen TL et al. J Biol Chem 2000; 275: 5400; 5. Ma H et al. Biochemistry 1999; 38: 15522; 6. Andersen JPet al. J Biol Chem 2001; 276: 23312; 7. Vilsen B et al. J Biol Chem 1989; 264: 21024; 8. Clarke DM et al. J Biol Chem 1990; 265: 6262; 9.Clarke DM et al. Nature 1989; 339: 476; 10. Vilsen B et al. J Biol Chem 1991; 266: 18839; 11. Andersen JP, Vilsen B. J Biol Chem 1992; 267:19383; 12. Andersen JP et al. J Biol Chem 1992; 267: 2767; 13. Vilsen B, Andersen JP. FEBS Lett 1992; 306: 247; 14. Clarke DM et al. J BiolChem 1993; 268: 18359; 15. Chen L et al. J Biol Chem 1996; 271: 10745; 16. Rice WJ, MacLennan DH. J Biol Chem 1996; 271: 31412; 17.Strock C et al. J Biol Chem 1998; 273: 15104; 18. Vilsen B, Andersen JP. Biochemistry 1998; 37: 10961; 19. Zhang Z et al. Biochemistry2000; 39: 8758; 20. Menguy T et al. J Biol Chem 2002; 277: 13016; 21. Zhang Z et al. FEBS Lett 1993; 335: 261; 22. Zhang Z et al. J BiolChem 1995; 270: 16283; 23. Maruyama K, MacLennan DH. Proc Natl Acad Sci USA 1988; 85: 3314; 24. Maruyama K et al. J Biol Chem1989; 264: 13038; 25. McIntosh DB et al. J Biol Chem 1999; 274: 25227; 26. Clausen JD et al. J Biol Chem 2001; 276: 35741; 27. McIntoshDB et al. J Biol Chem 1996; 271: 25778; 28. Hua S et al. Biochemistry 2002; 41: 2264; 29. Clarke DM et al. J Biol Chem 1990; 265: 22223;30. Vilsen B et al. J Biol Chem 1991; 266: 16157; 31. Vilsen B et al. Acta Physiol Scand 1992; 146: 279; 32. Sørensen TL et al. J Biol Chem2000; 275: 28954; 33. Andersen JP, Sørensen T. Biochim Biophys Acta 1996; 1275: 118; 34. Andersen JP et al. Ann NY Acad Sci 1997; 834:333; 35. Sørensen T et al. J Biol Chem 1997; 272: 30244; 36. Andersen JP. J Biol Chem 1995; 270: 908; 37. Adams P et al. Biochem J 1998;335: 131; 38. Andersen JP. FEBS Lett 1994; 354: 93; 39. Andersen JP, Vilsen B. J Biol Chem 1994; 269: 15931; 40. Falson P et al. J BiolChem 1997; 272: 17258; 41. Menguy T et al. J Biol Chem 1998; 273: 20134; 42. Zhang Z et al. J Biol Chem 2001; 276: 15232.



critical residue for ATP binding. Other nearby and well-studied amino acids are Lys492 and Lys515. Lys492 islabeled with 8-azido-TNP–AMP. It is also one of theresidues that participates in the binding of decavanadate.The close proximity of Lys515 to the adenosine moietyexplains the inhibition of ATP binding by modificationof this residue with fluorescein isothiocyanate [17]. InSERCA1 and SERCA2, but not in SERCA3, an interactionsite with phospholamban (PLB) is located in the sequenceLys397–Asp–Asp–Lys–Pro–Val.The crystallized E1 conformation is not a phosphorylat-

able conformation because the nucleotide-binding site ismore than 25Å from Asp351. Although it cannot be ex-cluded that in the native enzyme the nucleotide-bindingsite and Asp351 are closer together, it is more likely thatlarge domainmovements occur that close the gap betweenboth domains (as described below).

The A domain

The A domain consists of the N-terminal part of the se-quence and a stretch between M2 and M3. It is the small-est of the domains. The Thr81–Gly–Glu–Ser motif in oneof the surface loops is one of the most conserved amongP-type ion-transport ATPases. The A domain is connectedto the M domain by long loops, suggesting that it maymove substantially during the catalytic cycle. Significantmovements are also suggested by the sensitivity of trypticcleavage at Arg198 to Ca2+ and phosphorylation.

The M domain and the M6–M7 loop (L67)

The M domain comprises 10 transmembrane -helices(M1–M10) of varying inclination and length. M2 and M5are very long and extend above the cytosolic face of themembrane. The cytoplasmic half of M6 is not an ideal-helix. It may undergo rotation or winding/unwindingduring Ca2+ transport, possibly linked with displacementof the L67 loop. Also the center of M4 is unwound to ac-commodate the Ca2+-binding site. The lumenal loops areshort, except for the M7–M8 loop. The Ca2+-coordinatingresidues are located in M4, M5, M6 and M8. These helicesare centrally located. They are surrounded on one side(M4) by M1–M3, on the opposite side (M8) by M7–M10.M6 and M7 are far apart and are connected by a longcytosolic loop that runs along the top of the M domainand along the bottom of the P domain. This loop is func-tionally important. It mediates interactions between theM and P domains by a network of hydrogen bonds. It alsoforms an interaction site with PLB.The crystal structure determined by Toyoshima et al. [6]

allowed the localization of the two boundCa2+ ions, whichare transported in each cycle. They are 5.7Å apart at ap-proximately the same depth in the membrane. The bind-

ing sites are termed site I and site II [10]. Site I is locatedbetween M6 and M5, site II between M6 and M4 (Fig. 4).The side-chain oxygen atoms contributing to each of thesesites confirm previous site-directed mutagenesis experi-ments. Site I is formed by the side chains of Asn768, Glu771(M5), Thr799 and Asp800 (M6) and Glu908 (M8). Asp800of M6 also contributes to site II. The other side-chain oxy-gen atoms that contribute to site II are provided by Asn796(M6) and Glu309 (M4). In addition, several coordinatingoxygens that form site II are provided by main-chain car-bonyl oxygen atoms of Val304, Ala305 and Ile307 (M4).This geometry is made possible by unwinding of helixM4 between Ile307 and Gly310. An access pathway to theCa2+-binding site is not apparent in the structure, proba-bly because of an occluded-state conformation.Phe256 in the N-terminal part of M3 is essential for the

extremely high-affinity interactionwith the inhibitor thap-sigargin [18,19]. However, thapsigargin binds only to theE2 conformation, which is stabilized by it, like in the E2crystals obtained by Toyoshima andNomura [7]. The bind-ing site is a cavity surrounded by the M3, M5 and M7 he-lices near the cytoplasmic surface of the membrane. M3also participates in the formation of a gateway to the Ca2+

sites in the E2 structure [7], confirming earlier observa-tions that residues in the N-terminal half of M3 profoundlyaffect the rates of binding and dissociation of Ca2+ [20].Falson et al. [21] have first pointed to the functional

importance of the L67 loop. Particularly Asp813 is indis-pensable for rapid and high-affinity activation of the en-zyme by Ca2+ [22]. This and other residues participate ina hydrogen-bonding network with the cytosolic extensionof M5, with the P domain and with the M8–M9 loop. Theimportance of the L67 loop is also apparent from the lack ofproper protein folding in Pro820mutants. Yet another typeof effect is produced by mutations of Arg822 and Lys819.These mutations interfere only slightly with activation byCa2+, but interfere with the formation of the phosphory-lated enzyme [22].

Domain motions during the catalytic cycle

The comparison of the E1 2.8Å-resolution atomic struc-ture obtained in the presence of Ca2+ (PDB file 1EUL)with the 3.1Å-resolution structure of the Ca2+-free E2form in the presence of thapsigargin (PDB file 1IWO) hasrevealed large conformational changes during the cat-alytic cycle [6,7]. Enormous movements had already beeninferred from the mapping of the detailed E1 structureto 6Å-resolution electron-density distributions of crys-tals of the E2 state [13]. Concomittantly with the move-ment of the whole cytoplasmic head along the plane ofthe membrane in the M10 to M1 direction, the subdo-mains also undergo large displacements with respect toeach other. The E2 state shows a much more compact



Fig. 4 Position of the Ca2+-coordinating residues of SERCA1 (E1 state) as seen from the lumenal face. Coordination to oxygens (red)located at a distance of 2.2–2.6 Å from the center of each site is indicated. Grey: outline of part of the transmembrane -helices; light blue:interrupted helix; dark blue: nitrogen atoms. The z-orientation of interrupted or dotted lines deviates strongly from that of the full lines.Interrupted lines extend above, the dotted line below the plane of the paper. An additional coordinating oxygen is contributed to site I by awater molecule (not shown) located below the plane of the paper. The side chain of Asp800 contributes to both sites. Three coordinatingatoms of site II are main-chain carbonyl oxygens of M4: Val304, Ala305 and Ile307. The side chain of these amino acids is not shown. 3Dmodel drawn with the Swiss-PdbViewer program (Glaxo Wellcome Experimental Research, http://www.expasy.ch/spdbv/mainpage.html).

appearance of the cytoplasmic head piece than the E1state. However, the structure of each of the cytoplasmicsubdomains remains largely unaltered. In contrast, con-siderable rearrangements occur in the M domain. HelicesM1 to M6 are displaced or bent, as expected from the dra-matic change of the affinity and of the orientation of theCa2+-binding sites, while helices M7 to M10 and the lower(lumen-facing) part of M5 can be considered as retaininga fixed position. The lower part of M5 is closely associatedwith M7 due to the presence of one glycine residue inM5 (Gly770) and three glycines in M7. Gly770 can then beseen as a pivoting point around which a bending of theupper part of M5 and the movement of the other regionsoccur. This Ca2+-dependent bending of M5 is a majorfactor in the conformational changes because this partof M5 is closely associated with the centrally located Pdomain.In the E1 crystals the bound nucleotide is far away from

the phosphate-accepting Asp351. Given the flexible con-nection with the N domain, swivelling of the whole N do-main by Brownian motion could periodically close the gapand bring the nucleotide-binding site in close proximity tothe phosphorylation site, allowing transfer of phosphate

to Asp351 [13]. There is indeed a 50 rotation of the Ndomain relative to the P domain in the E2 structure. Thecompact E2 structure is stabilized by hydrogen bonds be-tween the A domain and the N and P domains. A role ofthe stable closed configuration is to stop the reaction cy-cle, which can proceed only in the presence of Ca2+. Theseparation of the N and P domains would be further facil-itated by the secondary binding of ATP to the phosphoen-zyme, thereby favoring its hydrolysis and explaining thestimulation of the pump activity by concentrations of ATPabove the level required for saturation of the catalytic site[23].The A domain of E1 requires a 110 rotation to fit the

E2 structure. Given the detached position and the flexi-ble connections, Xu et al. [13] propose that also the move-ment of this domain is brought about by thermal energy.The presence of the aspartyl phosphate group in the Pdomain would function as a latch that catches the A do-main when it happens to come in close contact. The newstabilized position of the A domain would put strain onthe connections to the M domain, resulting in alterationsof the Ca2+-binding sites and release of Ca2+ to the lu-men. Conversely, the movement of the A domain will be


http://www.expasy.ch/spdbv/mainpage.html


controlled by the positions of M1 to M3 as affected bythe Ca2+-induced structural changes of the Ca2+-bindingsites.The change of the affinity for Ca2+ is accompanied by

complex changes of the binding sites. M5 is bent wherebythe P domain is tilted. The largest movements are in thethree residues of M6 that contribute to site I, which un-dergo a 90 rotation. M6 is indirectly connected to the Pdomain via the L67 loop and hydrogen bonds to M5. M4moves down on dissociation of Ca2+. The rearrangementsof the helices within the M domain on binding or dissoci-ation of Ca2+ are not restricted to the Ca2+-binding sites(M4, M5, M6, M8). The inclination of M4, the top of whichis clamped by the P domain, causes M2 to tilt. M3 is con-nected through a hydrogen bond to the P domain, that it-self reorients 30 with respect to themembrane. M1movesand is bent at the top part as a result of steric collision withM3. Concomitantly, all these rearrangements allow the re-lease of Ca2+ at the lumenal side and the recreation of theentry pathway from the cytoplasmic side.

Interaction sites with phospholamban

PLB is a small 52 amino acids type-II membrane proteinhighly expressed in the SR of cardiac and, in larger ani-mals, of slow skeletal muscle (see below). The cytosolicN-terminal part (domain I) is divided in two subdomains.Domain IA (residues 1–21) forms an -helix in trifluo-roethanol [24] and contains regulatory residues that arephosphorylated by protein kinases. Ser16 is a substrate forprotein kinase A, Thr17 for Ca2+-calmodulin-dependentprotein kinase II. Domain IB forms a connection with do-main II (residues 31–52) that traverses the membrane,probably as an -helix. The transmembrane segment, inparticular, specifically placed leucines and isoleucinesforming a leucine zipper, are responsible for the forma-tion of pentamers. Multimers are in equilibrium withmonomers. The latter are the active forms that inter-act with the SERCA Ca2+ pump, preferentially in its E2state, thereby lowering the apparent affinity for Ca2+.The inhibition is releaved by phosphorylation of PLB.However, phosphorylated PLB remains associated withthe Ca2+ pump [25]. Pentamer formation is inhibited bymutations in the transmembrane sequence. In agreementwith the hypothesis that PLB interacts with the Ca2+

pump as a monomer, these mutants are superinhibitory[26,27].All three domains of PLB interact with three dis-

tinct sites of the SERCA protein. Domain IA binds to asurface site in the N-domain formed by the sequenceLys397–Asp–Asp–Lys–Pro–Val (Fig. 2). This sequence ispresent in SERCA1 and SERCA2 but not in SERCA3. ASERCA2 chimera containing a SERCA3 domain encom-passing this sequence did not functionally interact with

PLB [28]. The domain IA/SERCA interaction would not,by itself, be inhibitory, but would modulate the inter-actions of the other domains. Depending on the phos-phorylation status of its Ser16 and/or Thr17, domain IAregulates these effects via long-range interactions. All PLBresidues have been mutated to evaluate the role of eachamino acid. Alanine scanning and other substitutionshave shown that both electrostatic and hydrophobic in-teractions in domain IA are important. The net chargein residues 1–18 should be +1 or +2 for PLB/SERCA2ainteraction to occur. PLB function is also lost by re-placing Val4, Leu7 or Ile12 by the less hydrophobicalanine.Domain IB binds to the L67 loop of the Ca2+ pump. The

functional effect of PLB is reduced by mutations ofAsn810, Asp813 and Arg822 in SERCA1 to alanine, ofwhich Asp813, located close to M6, is the most criticalfor the physical interaction [29]. Surprisingly, some pointmutations in PLB domain IB enhance inhibitory function.Particularly the alanine mutant Asn27 but also Asn29and Asn30 are superinhibitory, whereas Arg25, Gln26 orLeu28 have diminished functional interactions. Possibly,it is the PLB–L67 interaction that is specifically disruptedby PLB phosphorylation, leaving intact the interaction ofthe N-terminus of PLB with the site in the N domain ofSERCA. Asn30 in domain IB of PLB may also interact witha site close to M4, as demonstrated by thiol cross-linkingof an Asn30 cysteine mutant to Cys318 in SERCA[30].The transmembrane domain of PLB interacts with the

M6 helix of SERCA [31]. Mutation of several hydrophobicresidues to alanine reduces both the inhibitory effect ofPLB and its physical association with the Ca2+ pump asdetermined in co-immunoprecipitation experiments. It ispossible that other not yet identified interaction sites withPLB exist in the transmembrane domain of SERCA. Possi-ble candidates are M2 and M9, which flank the access siteto M6.

THE SERCA-PUMP FAMILY

A phylogenetic analysis of the P-type ATPase superfam-ily based on the analysis of conserved core sequencesindicates that the SERCA pumps together with theSPCA1 pumps (see below) clearly belong to the sameP2A group [32]. As can be learned from inspecting thegenome of those invertebrates for which the genomeis largely known (Caenorhabditis elegans, Drosophilamelanogaster), many invertebrates apparently posses onlyone single SERCA gene [33–35]. A notable exception isthe parasitic flatworm Schistosoma mansoni that has atleast two SERCA-like genes SMA1 and SMA2 besides anSPCA1-related gene [36,37]. Invertebrate SERCA-type AT-Pases typically present about 70% amino acid identity



with the mammalian SERCA enzymes. Vertebrates haveevolved three different SERCA genes, each showing dif-ferent splice variants: ATP2A1–3 encoding respectivelythe SERCA1a/b, SERCA2a/b and SERCA3a/b/c/d/e pro-tein isoforms. The single SERCA gene of the invertebratescompares most to the vertebrate SERCA2 gene (ATP2A2)(see below). All animal SERCA isoforms are targeted to theER or to specialized ER-derived subcompartments suchas the muscle SR. Operationally, SERCA Ca2+-transportATPases can be distinguished from the plasma-membraneCa2+ pumps (PMCA) or even from the more closely re-lated Golgi SPCA1 pumps by the use of SERCA-selectiveinhibitors: thapsigargin [38], cyclopiazonic acid [39] and2,5-di-(tert-butyl)-1,4-benzohydroquinone [40].A comparison of the genomic exon–intron layout con-

firms the close phylogenetic relationship amongst the dif-ferent SERCAmembers. Indeed, the position of the intronsin the ATP2A1–3 genes is highly conserved, except for theexons downstream of exon 21, where the primary genetranscripts are subject to alternative processing (see Fig. 5).There is only one exception: exons 8 and 9 in ATP2A1 areseparated by an intron, but joined in the two other ver-tebrate SERCA genes [41]. Whereas alternative transcriptprocessing affects homologous regions of the genes, themode of processing is clearly different between the genes(see section on SERCA2 for further comments). Even inthe SERCA gene of Artemia franciscana, 12 out of 17 in-trons are inserted in the same position as in the rabbitSERCA1 gene [42]. Remarkably, the vertebrates show amore elaborated intron layout compared to C. elegans orD. melanogaster. The nematode SERCA gene (Sca-1) com-prises only eight exons and seven introns, with four of theintron positions conserved with the vertebrates [35]. Thefly gene (Ca-P60A) counts nine exons and eight introns,five of which are in identical positions as in the vertebrates.Such a conservation of intron positions is not seen be-tween the SERCA genes and either the PMCA genes or theSPCA/PMR1 genes, nor between PMCA and SPCA/PMR1.Plants appear to have the most complex Ca2+-transport

ATPase system. Arabidopsis has four P2A genes (ECA1–4),all related to the animal SERCA genes. However, SPCA1/PMR1 homologues seem to be absent from this plant.In addition the plant P2B ATPases, which contain acalmodulin-binding domain at their N terminus (not atthe C-terminus!) are more related to the animal PMCAand also transport Ca2+. Arabidopsis has 10 different P2Bgenes and for some of those, their proteins are targetedto the ER and possibly also to other intracellular com-partments [43]. The reader is referred to the review ofAxelsen and Palmgren [43] and the references therein forfurther information on this exciting world of plant Ca2+

pumps.SERCA genes appear to be absent from the yeast

genomes and possibly from other fungi as well.

SERCA1 proteins: products of the ATP2A1 gene

SERCA1a/b represent the most specialized isoforms ofCa2+ pumps and are found in high amounts in the SRof fast-twitch skeletal-muscle fibers of different animalphyla. Two SERCA1 splice variants have been described infish [44], amphibia [45], birds [46] and mammals [47,48]:an adult SERCA1a (994 amino acids in human) and aneonatal SERCA1b (1001 amino acids in human). Thedifferences between both isoforms result from the alterna-tive splicing of the ATP2A1 gene transcript and affect onlythe C-terminal amino acids positioned downstream fromamino acid 993. In the neonatal SERCA1b form, a highlycharged octapeptide tail replaces the single C-terminalresidue Glu994 of the adult form. The functional meaningof this difference remains elusive.Because it is relatively easily purified, SERCA1a was the

first pump of the family to be studied in detail and thesestudies resulted in an impressive number of landmarkpublications. SERCA1 was also the first member of thefamily to be molecularly cloned [47] and also the onlyP-type ion motive ATPase for which at present a detailed3D structure is available [6,7]. The cDNA of this pump wasalso subject to an extensive mutation analysis which al-lowed to explore the structure–function relationship (seeabove) of the corresponding pump so that we now for thefirst time begin to understand how a Ca2+ pump, and infact also other related ion pumps work [12]. Furthermoreit was also the first pump of the SERCA family that waslinked to a disease (Brody’s disease, see OMIM 108730 forfurther details) [49].


Whereas SERCA1 is the best understood member of theSERCA group in terms of structure–function relationship,SERCA2 appears by far the most widespread of all SERCAisoforms and it appears phylogenetically the oldest. Likefor SERCA1, mammals [50,51] and birds [46] also expresstwo protein isoforms of SERCA2: SERCA2a (997 aminoacids) and SERCA2b (1042 amino acids). Transcripts of themammalian ATP2A2 gene are alternatively processed toyield four different mRNAs, one of which is translated intothe SERCA2a protein, while the other three differ onlyin their 3′-untranslated region and all encode SERCA2b.SERCA2a is the main Ca2+-pump isoform in the SR ofcardiac muscle and of slow-twitch skeletal-muscle fibers.Also in smooth muscle some 20–25% of the SERCA2mRNA and a corresponding fraction of the protein are ofthe SERCA2a form. The remainder is SERCA2b. Further,some neuronal cells including the cerebellar Purkinjeneurons also express SERCA2a [52]. In view of its phylo-genetic primitive and widespread character SERCA2b hasbeen considered as the house-keeping isoform, although



Fig. 5 Exon/intron layout of the three vertebrate SERCA encoding genes. For each of the genes a partial exon/intron is given. Exons arerepresented by boxes. Wide boxes are translated segments, less wide boxes are untranslated. Introns and downstream flanking regions arerepresented by thin horizontal lines. Lines with letters represent the different splice modes. For SERCA2 bn indicates the removal of theoptional untranslated exon specific for neuronal tissue. The different classes of mRNAs are represented below the corresponding DNAs. Theprotein products are shown on the right. Sa–Se: position of stop codons for the corresponding protein isoforms; pA, pAu, pAd: position ofpolyadenylation site (with specification of upstream or downstream in case of SERCA2); 5′D1, 5′D2: two optional splice donor sites; 3′A:splice acceptor site. Note that for each of the genes the introns upstream of exon 21 are constitutively spliced out. The fate of the downstreamexons depends on the tissue (SERCA2 and SERCA3) or the developmental stage (SERCA1).



some tissues express higher levels than others. Expressionof the SERCA2a protein variant results from the activa-tion in a tissue-specific manner of an otherwise inefficientsplicing process at the 3′-end of the transcript wherebyexons 22–24 are spliced out and exon 21 is joined toexon 25 [53,54] (see Fig. 5). The trans-acting factor(s) re-sponsible for this tissue-specific splicing remain hithertounknown, but it is clear that the execution of this formof splicing is dependent on ongoing protein synthesisand is tightly coupled to the myogenic differentiation[55]. When proliferating myoblast-like BC3H1 cells are ex-posed to a mitogen-deficient medium, the muscle-specificalternative splicing pattern of SERCA2 transcripts is ac-tivated [55,56]. The same mode of splicing is induced inthe RNA transcribed from an artificial minigene compris-ing only the downstream elements of the ATP2A2 gene[53,54]. This indicates that all information required to ob-tain muscle-specific alternative splicing of the transcriptis found in this region of the gene. Furthermore a similarmuscle-like alternative splicing of the minigene transcriptcan be induced by growth factor depletion in non-muscle10T1/2 fibroblasts that were previously transfected withmyogenin. These results mean that the trans-acting fac-tor(s) responsible for muscle-specific SERCA2 processingcan be induced by one of the master regulatory genes ofmuscle differentiation and that growth factors antagonizethis induction [53]. The 3′-end of the ATP2A2 genes ofmouse and pig are highly conserved even in the untrans-lated regions [57]. Part of this conservation is possiblyrelated to alternative splicing, but recent observationsalso support the possibility that the 3′-untranslated regionof SERCA2a mRNA confers a greater stability to its mes-senger than that of the SERCA2b mRNAs, a mechanismexplaining in part the higher expression of SERCA2a thanof SERCA2b [58].

Functional differences between SERCA2a andSERCA2b

Upon expression of SERCA2a and SERCA2b in COS cells,SERCA2b showed a two-fold higher apparent affinityfor Ca2+ but at the same time a two-fold lower catalyticturnover rate [59–61]. Both enzyme isoforms present,however, the same sensitivity towards PLB and thapsigar-gin [62].Interestingly, like for their vertebrate homologues, also

the transcripts of many invertebrate SERCA genes showthe potentiality to be alternatively spliced whereby theymake use of the same mode of alternative splicing as forthe vertebrate SERCA2 (but which differs from SERCA1and SERCA3) to execute splicing at a homologous posi-tion. Again two proteins can be formed: a short SERCAaprotein structurally related to the vertebrate SERCA2aand a longer SERCAb protein related to the vertebrate

SERCA2b. This is well documented for the primitive nema-tode C. elegans, in which both isoforms are in addition alsofunctionally conserved with respect to their vertebratecounterparts [35], i.e. the shorter SERCAa form showsapproximately half the apparent Ca2+ affinity of SERCAb.Also the crustacean Artemia appears to express only oneSERCA gene, the transcript of which is also alternativelyspliced according to the vertebrate SERCA2mode yieldinga 1003 amino acid long transcript expressed inmuscle anda 1027 amino acid long transcript in other tissues. Similarto vertebrate SERCA2b, this longer isoform contains ahydrophobic segment with the propensity of forming anadditional (11th) transmembrane segment [63,64].

Does SERCA2b interact with calreticulin or calnexinto acquire its typical functional properties?

The characteristic two-fold higher apparent affinity forCa2+ of the vertebrate SERCA2b compared to SERCA2aand of SERCAb relative to the SERCAa splice variant of C.elegans as originally deduced from overexpression studiesin COS cells [35,59,60] was recently also confirmed in vivoby comparing the Ca2+ uptake in fragmented cardiac SRprepared from mice from which the ATP2A2 gene was al-tered in such a way that the animals expressed SERCA2binstead of the normal SERCA2a in their myocardium [61].The vertebrate SERCA2a and 2b splice variants differ onlyin their C terminus. SERCA2b has, compared to SERCA2a,an additional membrane-spanning segment that is fol-lowed by a 12 amino acid luminal extension. From thisit follows that the two putative Ca2+-binding/transportsites, which, as already mentioned, are localized in thetransmembrane segment of both pumps, are the same andthus cannot be responsible for the observed difference inaffinity. This apparent difference in affinity for cytosolicCa2+ could possibly be ascribed to a kinetic effect. It canbe hypothesized that the presence of the SERCA2b tailwould slow down the catalytic turnover rate whereby thedwelling time in the (high Ca2+ affinity) E1 conformationis shortened at the expense of that in the (lower affinity)E2 conformation [65]. Mutation experiments showed thatupon removal of the last 12 amino acids from SERCA2b, itacquired the same properties as SERCA2a [65]. Camachoand coworkers [66] presented a provocative explanationfor this difference in properties of the two SERCA2 iso-forms. According to these authors the characteristic highaffinity (and corresponding lower catalytic turnover rate)of the SERCA2b form required the interaction of the lu-minal SERCA2b tail with calreticulin, or as proposed inlater experiments of the same group, with calnexin [67].Calreticulin is a multifunctional Ca2+-binding solubleprotein found in the lumen of the ER. Calnexin can beconsidered as its membrane-bound homologue. The latteris a type-I integral membrane protein that has a lumenal



Ca2+-binding site and a cytosolic domain containing twoconsensus motifs (Ser485 and Ser562) for protein kinaseC/proline-directed kinase (PKD) phosphorylation [67].Calreticulin and calnexin both act as lectin-like molecularchaperones binding to unfolded and newly synthesizedglycoproteins, which they recognize via their monoglu-cosylated carbohydrate moiety [68,69]. Of both SERCA2isoforms, only the SERCA2b form, but not the SERCA2aone, has a lumenal C-terminal tail with a consensus sitefor N-linked glysosylation (Asn1036). Mutation of this siteto alanine confers SERCA2a-like properties to SERCA2b.This finding appears to support the view that glycosyla-tion of this Asn residue in SERCA2 is a prerequisite forcalreticulin or calnexin binding. However, some recentevidence speaks against this hypothesis. First, in spite ofserious efforts, glycosylation of Asn1036 could not bedemonstrated [67]. Presumably the asparagine is posi-tioned too close to the membrane to be a good substratefor glycosylation. It should however be taken into accountthat, according to recent evidence, calnexin can also exertits chaperone function independently of the state of gly-cosylation of the target protein [70]. Second, although inthe SERCAb form of C. elegans this glycosylation site is notconserved, it does still present a two-fold higher affinityfor Ca2+ compared to the SERCAa form in this animal [35].Third, the Ca2+ affinity for Ca2+ uptake in the SR preparedfrom gene targeted mice expressing SERCA2b in their car-diomyocytes instead of SERCA2a, shows also a two-foldhigher affinity compared to the situation in the wild-typeSERCA2a controls [61] but cardiomyocytes have, at leastin the adult hearts, very low levels of calreticulin.The interaction of calreticulin with SERCA2b only oc-

curs in the presence of sufficiently high lumenal Ca2+ [68].For calnexin the interaction appears to be additionally de-pendent on the phosphorylation of Ser562, thus creatingan additional level of complexity. A model was proposedrecently to predict the functional consequences of cy-tosolic phosphorylation of calnexin at Ser562 [67]. Underresting conditions, calnexin is phosphorylated on Ser562and in the presence of lumenal Ca2+, its lumenal tail inter-acts with the SERCA2b tail. Thereby it would decrease thecatalytic turnover rate of the pump but at the same timegearshift it to a higher Ca2+ sensitivity. Upon cell activa-tion, Ca2+ is released from the ER and the lumenal [Ca2+]decreases. The local increase in [Ca2+]c would induce aCa2+-dependent dephosphorylation (by calcineurin?) ofSer562 thereby shifting the pump temporarily back to ahigh-turnover/low-affinity state as long as [Ca2+]c is high.

Darier’s disease

Darier’s disease (DD) is caused by mutations in theATP2A2 gene (see OMIM 108740 for further details). Theobservation, at the end of 1999 [71], that DD was linked

to a variety of mutations in the ATP2A2 gene came as asurprise and posed immediately a paradox. Indeed the dis-ease manifests itself almost exclusively as an autosomaldominant keratinocyte disorder. Surprisingly, there is atpresent no consistent evidence for extracutaneous mani-festations of DD with the possible exception of a slightlyincreased incidence of neuropsychiatric disorders in someof the patients [72]. The presence of a neuronal phenotypemay not be too surprising because SERCA2b is expressedat relatively high levels in some neurons in the brain [52].Cerebellar Purkinje neurons express besides SERCA2balso SERCA2a and even SERCA3. The presence of a sep-arate bipolar disorder susceptibility gene in the Darierregion may be a complicating factor [73]. A recent studyof cardiac systolic and diastolic function using echocar-diography in 10 Darier patients did not show any defects,and also bleeding time and platelet aggregation were nor-mal in these patients [74]. Mice that lack one functionalATP2A2 allele, however, do show impaired cardiac con-tractility and relaxation [75] and develop squamous celltumors at older age [76].

Phospholamban and sarcolipin are the primaryregulators of SERCA2

It is beyond the scope of the present review to treat thevast literature on PLB. For further information the readeris referred to a number of recent reviews [10,77,78]. PLBis a 52 amino acid intrinsic membrane protein (monomerMW = 6080Da) found in the SR or ER of mainly musclecells where it acts as a reversible endogenous inhibitorof SERCA. The inhibition is manifested by an apparentdecrease in Ca2+ affinity of the Ca2+-ATPase and trans-port activity with no effect on the Vmax. PLB represents,together with the homologously related sarcolipin (seebelow), the main regulator of SERCA pumps. Althoughfirst described in cardiac muscle, where it is particularlyabundant in the ventricle and about 10-fold less repre-sented in the atria, PLB was later found to be expressed inslow-twitch skeletal muscle of large animals [79], in sometypes of smooth muscle [80] and according to a recent re-port also in endothelial cells [81]. Remarkably, PLB is notexpressed in fast-twitch skeletal-muscle fibers in whichSERCA1 is the major Ca2+-pump isoform, but when ec-topically co-expressed together with this pump isoformin COS or HEK cells it is perfectly able to regulate its ac-tivity. Ventricular SR is estimated to contain roughly theequivalent of two PLB monomers per SERCA2a molecule[82]. This concentration is below that needed for maximalinhibition of the pump so that increasing its concen-tration further inhibits the pump until a maximum isreached at about three PLB/SERCA monomers. PLB showsa strong tendency to form homopentameric complexes inthe membrane. It is estimated that about 25% of the PLB



is monomeric under normal conditions [83]. This pen-tamerization is due to the formation of a zipper structureformed by interdigitating leucine and isoleucine groupsin two adjacent tiers of the -helix in the transmembranedomain of PLB [83]. It is the binding of the monomer,but apparently not of the pentamer of PLB to SERCA1 orSERCA2 molecules, that lowers the apparent affinity ofthese pumps for Ca2+. Mutations in the transmembranedomain of PLB that interfere with pentamerization there-fore increase the activity of PLB as an inhibitor [84,85].Phosphorylation by protein kinases shifts the equilibriumfrom monomers to pentamers [86]. Binding of Ca2+ tothe ATPase prevents the binding of PLB to the pump [87].The interaction of phospholamban with SERCA involvesmultiple sites, as indicated earlier.Phosphorylation of PLB by cyclicAMP- or cyclicGMP-

dependent protein kinase at Ser16 or by Ca2+/calmodulin-dependent protein kinase II at Thr17 lowers the netelectrical charge of the cytosolic domain of PLB and low-ers the affinity of SERCA for cytosolic Ca2+. It is thoughtthat long-range interactions within both PLB and SERCAmediate this effect. However, the phosphorylated form ofPLB remains associated with SERCA [87,88].There exists some controversy on the physiological ef-

fect of a possible direct phosphorylation of SERCA2. Itwas claimed that phosphorylation of SERCA2a/b on Ser38(but not SERCA1 or SERCA3, which lack the correspond-ing site), by Ca2+/calmodulin-dependent protein kinase IIdoubles the Vmax for Ca2+ uptake [89,90] but two othergroups did not observe this [91,92].Sarcolipin (SLN) is a 31 amino acid predominantly

-helical transmembrane protein (MW = 3733Da for therabbit; see 1JDMA for its 3D structure). SLN is a regu-lator of fast-twitch skeletal muscle, but is also found inslow-twitch and in cardiac muscle. It can be consideredas a shorter homologue of PLB [93]. The fact that morethan half of its sequence (19 residues; from residue 8 to26) consists of hydrophobic amino acids explains its highsolubility in acid chloroform/methanol, but this does notjustify its designation as a proteolipid. Indeed there areno attached fatty acyl chains or other prosthetic groupsin SLN [94]. SLN has a lower tendency to form oligomersin detergents and in phospholipid liposomes, comparedto PLB. Unlike for PLB, SLN pentamers are not specificallyformed and the SLN oligomers are not SDS-stable [93].The effect of SLN on SERCA1a and SERCA2a has beeninvestigated by co-reconstitution in membranes and byco-expression in HEK cells. SLN shifts the Km for Ca2+

to a higher value and thus behaves in this respect likePLB. Remarkably, however, the effects of PLB and SLN aresynergistic. There exists still some controversy concern-ing the effect of SLN on the Vmax of SERCA. Some reportsclaim that SLN increases the Vmax of the ATPase [93,95]whereas others describe on the contrary a slight decrease

and blame the reported increasing effect on the ATPaseto an artefact resulting from an underestimation of theSERCA by the ELISA quantification in the presence of SLN[96]. This issue clearly needs further clarification. How-ever, co-immunoprecipitation of SERCA1a or SERCA2awith antibodies against PLB or (epitope-tagged) SLN atdifferent free [Ca2+] showed that the physical interactionof either inhibitor with SERCA is interrupted at free [Ca2+]used to measure the Vmax [96]. One important observa-tion is that SLN directly binds to PLB and thereby shiftsthe equilibrium from the pentameric storage form to themonomeric inhibitory form of PLB.A major point of difference between both inhibitors

is that unlike for PLB, the interaction of SLN with theATPase is not modulated by phosphorylation of the in-hibitor. Indeed, SLN contains in its cytosolic domain onlyone conserved potential phosphorylation site Thr5, butthis is apparently not phosphorylated. Thus, the effect ofSLN on the ATPase is believed to be mainly controlled bychanging SLN’s expression level.

SERCA2 and ER stress

Intrinsic membrane proteins, lumenal resident proteinsof the endocytic and exocytic organelles including theER, Golgi apparatus and lysosomes, as well as secretedproteins are all synthesized in the rough ER. The lumenalresidents and the secreted proteins are cotranslationallytranslocated into the ER lumen where they, at least par-tially, mature. The post-translational modifications, fold-ing, and oligomerization of the nascent proteins requirea unique Ca2+-rich (and oxidizing) environment. Distur-bances of the Ca2+ homeostasis and accumulation of un-folded proteins in the ER cause ER stress, and when theseinsults persist, lead to cell death [97]. Molecules that actas ER-stress inducers belong to many diverse categoriesincluding Ca2+ ionophores like A23187 and the SERCAinhibitor thapsigargin, which both deplete the ER Ca2+

stores, inhibitors of glycosylation like tunicamycin, andbrefeldin-A, an inhibitor of ER–Golgi transport [98,99].When incorrectly folded proteins accumulate in the ER,the unfolded protein response (UPR) pathway is activated(see, for recent reviews [100–102]). This conserved UPRpathway leads to a decrease in general protein transla-tion activity. The accumulation of unfolded proteins inthe ER causes the oligomerization and autophospho-rylation of PERK, a type-I transmembrane ER protein.This results in the phosphorylation of translation initi-ation factor 2 (eIF2-), which thereby prevents the as-sociation of the ribosome subunits with the mRNA andthus leads to translational attenuation. Furthermore, mis-folded proteins are dislocated from the ER lumen backinto the cytoplasm via the translocon. In the cytoplasm,they are ubiquitinated and degraded by the action of the



proteasome, a process known as ER-associated degrada-tion (ERAD).Remarkably, the gene transcription of BiP/GRP78, GRP-

94 and other molecular chaperones, which could help torefold the misfolded proteins, is increased in conditions ofER stress. SERCA2 appears to be also one of these tran-scriptionally upregulated proteins [103,104], but its rate oftranscription can be controlled by forms of ER stress inde-pendently of ER Ca2+ depletion [103].The genes that are transcriptionally activated by the

UPR, including SERCA2 (note that SERCA3 is not upreg-ulated under these conditions) typically contain ER stressresponse elements (ERSE) in their promoter. It was re-cently shown that transcription factor ATF6 is pivotal forthis ERSE-dependent upregulation of SERCA2 transcrip-tion [105]. ATF6 is an unusual transcription factor foundin the ER and comprising an N-terminal cytosolic basicleucine zipper factor domain, a single central transmem-brane domain and a C-terminal lumenal ER stress sensingdomain [106]. Upon ER stress, the protein is escorted tothe Golgi where it is cleaved twice: first within the Golgilumen by site I protease (S1P) and then by site II protease(S2P), which cleaves at a site within the membrane [106].The latter cut releases the actual transcription factor thatthen moves to the nucleus where it stimulates amongstothers the transcription of ATP2A2. The intramembranecleavage of ATF6 by S2P is only one example of a recentlydiscovered rapidly growing control mechanism: regulatedintramembrane proteolysis (Rip) that is also involved inthe cleaving of sterol regulatory element-binding protein,amyloid precursor protein and Notch (see [107,108]).Notch receptors fail to leave the trans-Golgi apparatus

on the way to their normal destination in the plasmamembrane in Drosophila mutants with a defect in theCa-P60A gene encoding SERCA in the flies, or in S2 in-sect cells that were treated with SERCA inhibitors [109].Several other transmembrane proteins were mislocalizedin the Ca-P60A mutant cells, pointing to the importanceof SERCA for protein trafficking.It is of interest to note that according to a recent sug-

gestion BiP also functions as a gate keeper that preventsCa2+ loss from the ER by closing the aqueous transloconpore during protein integration into the ER membrane[110,111].

SERCA2 and apoptosis

When ER stress persists, apoptosis is induced. This allowsthe damaged cell to be eliminated without causing in-flammation or tissue damage [1]. Some controversy existson exactly how ER stress leads to apoptosis. In particular,the role played by ER lumenal Ca2+ in this process causedconfusion. Part of this controversy may be explained bypleiotropic often cell-specific differences in the apoptotic

machinery and/or in its controlling pathways. The relativecontribution of mitochondrial-, plasma-membrane- orER-induced apoptosis might differ amongst the differentcells. This may be related to the fact that some cells havemore active capacitative Ca2+-entry pathways than others.Also differences in the cytotoxic apoptotic stimuli usedin these studies (unnatural ones like Ca2+ ionophores,thapsigargin, or more natural ones like glucocorticoids,ceramide, etc.) may also have contributed to the contro-versy. According to an early view, it is the decrease inlumenal [Ca2+] in the ER that is more directly linked toapoptosis [112,113]. In some lymphoid cells, depletion ofER Ca2+ by ionophores or thapsigargin triggers apoptosis.The observation that apoptosis was even augmented bythe removal of extracellular Ca2+, suggested that an in-crease in [Ca2+]c could not be the mediator of apoptosis[112] and it rather pointed to ER lumenal Ca2+ as the cul-prit. As mentioned above, lumenal ER Ca2+ is of such avital importance for a cell, that its depletion by such harshinterventions like the application of ionophores or thapsi-gargin is likely to cause a deadly blow to the cell. Themoregenerally accepted view on apoptosis, however, holds thatit is the rise in [Ca2+]c that causes cell death since it canbe prevented by chelating cytosolic Ca2+ by intracellu-lar chelators or overexpression of calbindin [114–116].In a model inspired on this latter view, procaspase-12bound to the cytoplasmic side of the ER would becleaved and activated after Ca2+ release from the ERand Ca2+-dependent translocation of cytosolic caspase-7to the ER surface [99]. Caspase-12 appears to be typi-cally involved in ER-stress-induced apoptosis but not inplasma-membrane- or mitochondrial-induced apoptosis.The latter forms of apoptosis make use of other caspases[98]. A recent report described the expression of SERCA1truncated proteins unable to pump Ca2+, but instead ap-parently causing a Ca2+ leak through the ER membranesof several non-muscle human tissues [117]. The overex-pression of these SERCA1 mutants in liver cells causedapoptosis. Further information on the role of SERCA withrespect to apoptosis can be obtained from results of theanti-apoptotic effect of Bcl-2 as indicated below.

SERCA2 and cell growth

Published data concerning the role of SERCA2 in con-trolling cell growth and the cell cycle are contradictory.Most studies showed that increased growth rate is corre-lated with an increase in the filling state of the cellularCa2+ pool and that interfering with SERCA2 activity de-creases cell proliferation and brings the cells in a G0-likestate [118–122]. Indeed, in prostate cancer cells (LNCaP),SERCA2b expression is correlated with cell prolifera-tion. Moreover, in the presence of low thapsigargin con-centrations, Ca2+ pool load was decreased and EGF, a



physiological autocrine growth factor for prostate, was nolonger able to stimulate cell growth [122]. However, somestudies in other cells came to an opposite conclusion. InSwiss 3T3 cells, Ca2+-pump blockers were found to stimu-late cell proliferation [123] and SERCA2a overexpressionin COS cells resulted in a dose-related apoptotic effect,with less cells remaining in the G1 phase of the cell cycleand a cell-cycle arrest in the G2/M phase [116].

SERCA2, Bcl-2 and cellular Ca 2+ storage

Also the relationship between the oncoprotein Bcl-2 andintracellular Ca2+ storage is much debated. Bcl-2 is foundin multiple intracellular membranes including the ER[124]. It is by far the most studied anti-apoptotic protein[125], but its mode of action remains still largely un-known and its interaction with SERCA uncertain. It hasbeen reported that overexpression of Bcl-2 in HeLa cells[126] or LNCaP prostate cancer cells [125] reduces thelumenal total and free [Ca2+] in the ER, possibly in partby increasing the passive leak of Ca2+ from the store, andpartially also by reducing the expression of SERCA2b andof calreticulin [125]. This reduced Ca2+ load in the ERwould make less Ca2+ available for release into the cy-tosol and thus protect the cells from apoptosis. Kuo et al.[127], however, defend the opposite view. They showedthat overexpression of Bcl-2 in breast epithelial cells stim-ulates the production of SERCA2 mRNA and protein.This would lead to an increased Ca2+ load in the ER andthus create conditions promoting cell proliferation, whichthereby counteract apoptosis. Also He et al. [113] reportedthat Bcl-2 partially counteracts the Ca2+-depleting effectof thapsigargin on the ER and thus prevents apoptosis,but the underlying mechanism is unclear.

Other factors interacting with SERCA2

With some 20 members known so far, S100 proteins rep-resent the largest subgroup of EF-hand Ca2+-binding pro-teins that regulate a wide variety of intracellular processes[128]. Amongst them S100A1 is the most abundant S100protein in striated muscle and, in particular, in cardiacmuscle where it colocalizes with the SR [128,129]. Whenadded to permeabilized cardiomyocytes at a calculated ra-tio of 3:1 over the SERCA2a, it significantly stimulatedSR Ca2+ uptake at all Ca2+ concentrations in the rangebetween 0.2 and 1M [129]. This is in agreement withthe observed co-immunoprecipitation of SERCA2a withS100A1 antibodies [130].It should furthermore be noted that the C-terminus

of SERCA2 and SERCA1 can directly interact with thetyrosine-phosphorylated insulin receptor substrates IRS-1and IRS-2 [131]. The physiological meaning of this inter-action is not yet clear but it creates the possibility for a

direct link between the insulin- and the Ca2+-signalingpathways. It was also reported that overexpression ofIRS-1 in cells inhibits Ca2+ loading of the ER [132] but towhat extent a direct interaction of IRS-1 with the SERCA2or SERCA3 pumps is involved remains unresolved. A linkbetween IRS-1 and inhibition of SERCA3 expression hasbeen observed [133] (see below).


Among the vertebrate SERCA pumps, the SERCA3 pumpwas the most recently discovered member [134] and it re-mains the least well understood (see also [135] for an earlyreview and OMIM 601929). SERCA3 deviates both struc-turally and functionally more from SERCA1 and SERCA2than the two latter from each other. The human SERCA3aprotein shows 76 and 77% sequence identity with humanSERCA1a and human SERCA2a, respectively, whereas hu-man SERCA1a and human SERCA2a are for 84% identi-cal. Certainly the most obvious functional characteristic,shown by all SERCA3 isoforms studied so far (see below),is their about five-fold lower (supramicromolar) apparentaffinity for cytosolic Ca2+, their slightly higher pH opti-mum compared to SERCA1 or SERCA2, and their insen-sitivity to PLB [59,136,137]. Work with SERCA1/SERCA3chimera shows that not the Ca2+ sites in the transmem-brane segments (which are conserved between all SERCAs)are responsible for this altered Ca2+ affinity, but that in-teractions involving the large cytosolic domain of SERCA3[138] shift the equilibrium between the conformationalstates E1 and E2 in favor of the low-affinity E2 state. Thislower affinity for cytosolic Ca2+ would mean that SERCA3becomes only active when Ca2+ reaches high levels, e.g.after massive stimulation or during the peak of cytosolicCa2+ oscillations. This latter view is experimentally sup-ported by recent data [139].Five different splice variants (SERCA3a–e) of the pump

have been described in human cells (Fig. 5) [41,137,140].The alternative splicing can follow slightly different pat-terns in the different mammalian species, which entails asomewhat confusing picture. Indeed for the rat an isoformdesignated as rSERCA3b/c has been described which, dueto the lack of a sequence at the 5′-end of exon 21, ter-minates in exon 21 whereas all other known human androdent isoforms terminate at various places in exon 22[137,141]. Avian SERCA3a and 3b cDNAs were recentlycloned from chicken macrophages [142]. Up till nowSERCA3 has been detected in many mammalian cells in-cluding secretory epithelial cells of the intestine, tracheaand salivary glands [143–146], endocrine pancreatic cells [147], endothelial cells [148], different types of whiteblood cells [137,149] and in cerebellar Purkinje neurons[150]. In spite of the relatively wide tissue distribution,no overt phenotype became apparent in the SERCA3



ablation studies [151] although minor disturbances inendothelium- and epithelium-dependent relaxation ofrespectively vascular [151] and tracheal [152] smoothmuscle were observed.SERCA3 seems to represent a specialized isoform that

is (almost) always co-expressed with the house-keepingSERCA2b (see [148] for a possible exception in human um-bilical vein endothelium) but that may be targeted to adifferent subcellular compartment [145].It is therefore not surprising that in several cell types

the expression of SERCA3 behaves as a differentiationmarker. SERCA3 expression is decreased or even lostwhen endothelial cells are brought into culture, exceptunder conditions where the differentiation status is main-tained or enhanced by culturing the cells on matrigel[148]. Likewise, in colonic crypt cells, SERCA3 expres-sion increases with the maturation of the cells, but itis strongly decreased or completely lost in colon carci-noma cells [146]. In the human myeloid/promyelocyticcell lines HL-60 and NB4 and in freshly isolated acutepromyelocytic leukaemia cells, in vitro differentiation ei-ther along the neutrophil granulocytic lineage by all-transretinoic acid or cyclicAMP analogs, or along the mono-cyte/macrophage lineage by phorbol esters, resulted inan increased expression of SERCA3 [153]. Expression ofSERCA3 in endothelial cells is increased in spontaneouslyhypertensive (SHR) rats [154].It is as yet poorly understood how ATP2A3 expression

is controlled. The upstream regulatory elements of thehuman ATP2A3 and mouse ATP2A3 genes have beendocumented [41,155] and their promoters were found tolack TATA- and CAAT-boxes, but instead to present typicalGC-rich islands. Thus, in this respect the ATP2A3 pro-moters differ from the ATP2A1 and ATP2A2 promoters,which both possess consensus TATA- and CAAT-boxes[57,156,157]. There are also no reports that SERCA3 isstimulated by thyroid hormone and its promoter regionappears to lack thyroid hormone responsive elements(TRE) in contrast to the two other SERCA members whosepromoters do contain TREs [158,159]. The conserved sin-gle ETS-1-binding site (EBS) and two Sp1 sites, positionedimmediately downstream from it in the ATP2A3 promoter,were found to mediate SERCA3 expression through thepresence of endothelial nuclear factors Ets-1 and Sp1.Ets-1 overexpression induced the expression of SERCA3in endothelial cells and in fibroblasts [155].

SERCA3 and insulin secretion in pancreatic cells

It is widely recognized that cytosolic Ca2+ is a key reg-ulator of insulin release from pancreatic cells, but therole of Ca2+ accumulation by the ER and by other in-tracellular Ca2+ stores in this process is complex and asyet poorly understood. Remarkably, glucose, which is

the main physiological insulin secretagogue, efficientlystimulates Ca2+ uptake into the ER of cells. This re-sults from an increase in cellular ATP concentration, i.e.one of SERCA’s substrates, and in part also by an in-creasing [Ca2+]c, the other substrate of the pump, sub-sequent to the closure of KATP channels [160]. In type-2diabetes or non-insulin-dependent-diabetes mellitus, theglucose-dependent cytosolic Ca2+ signaling is disturbedand changes in SERCA activity have been held respon-sible for this. Decreased Ca2+-ATPase is observed in theislets of rats made diabetic by injection of streptozo-tocin [161] or in spontaneously diabetic db/db rats [162].Roughly half of the SERCA pumps in cells are of thehousekeeping SERCA2b type, the other half are SERCA3.A number of observations suggested that it is specificallythe SERCA3 activity that is affected in diabetes. Thus,a selective decrease in the expression of SERCA3, butnot of SERCA2b was observed in Goto–Kakizaki diabeticrats [147] and in patially pancreatectomized rats [163].Furthermore, a two-fold overexpression of IRS-1 in insuli-noma cells, such as occurs after prolonged exposureof cells to insulin, inhibits SERCA3 expression by 42%but leaves the expression of SERCA2b unaffected [133].Hence, insulin appears to take part in an autocrine posi-tive feedback loop in cells stimulating its own secretion.Also in convergence with this presumed role of SERCA3 inthe aetiology of type-2 diabetes, is the reported increasedincidence of some rare missense mutations in the humanATP2A3 gene found in these patients [164]. However, thecausal link between the mutations and the phenotypeneeds further confirmation. Indeed, recent experimentscast some doubt on the idea that SERCA3 is necessarilydirectly involved in type-2 diabetes. First SERCA3 (−/−)mice do not develop diabetes [151], and they are normo-glycemic, have a normal insulinemia and react similarlyin a refeeding test as wild-type mice [139]. Second, thealmost 40% reduced expression of SERCA3 in MIN6 cellsby injection of antisense oligonucleotides did not affectthe apparent size of the ER Ca2+ store, whereas a roughlyequal suppression of SERCA2b markedly lowered theamount of Ca2+ releasable from the ER [165]. The role ofSERCA3 in cells may be more subtle than expected. Itchanges the cytosolic Ca2+ oscillation pattern by buffer-ing Ca2+ at a moment the ion approaches peak levelsand by subsequently slowly releasing Ca2+ when [Ca2+]cdecreases again [139]. The physiological consequences ofthis remain to be elucidated.

OTHER MORE PRIMITIVE Ca2+ PUMPS?

In most eukaryotes the lumenal Ca2+ concentrations inthe ER are maintained at or above 0.1mM through theaction of SERCA pumps. However, SERCA pumps appearto be absent from yeast, both from budding yeast like



Saccharomyces cerevisiae and from fission yeast likeSchizosaccharomyces pombe. The question then comes up,which Ca2+ pump fulfills this role in these latter organ-isms? There were suggestions that SPCA1/Pmr1 pumps,which are targeted to the Golgi (see below), could do thejob but how exactly Ca2+ would then be redistributedbetween Golgi and ER remained unclear. Very recently,however, two new putative Ca2+ pumps belonging to theas yet largely uncharacterized type-V branch of P-typeATPases were suggested as possible candidates for thisfunction: the COD1p/Spf1p in S. cerevisiae [166], and theCta4p in S. pombe [167].

SPCA Ca2+/Mn2+ ATPases

In recent years, a new class of Ca2+ pumps has emerged,the first member of which was found in the yeast S. cere-visiae and named Pmr1 (for plasma membrane ATPase-related) [168]. The first mammalian homologue wascloned from rat [169]. At present, Pmr1-related sequenceshave been identified in many distant species, e.g. severalspecies of yeast, C. elegans [170], D. melanogaster (PIDaccession number 7296577) and human [171,172]. Themammalian homologues were called SPCA for secretorypathway Ca2+-ATPase [4] and this nomenclature will beused throughout this review. The corresponding humangene is indicated as ATP2C1. In the human genome thereis a second related gene ATP2C2. The corresponding pro-teins will therefore be called SPCA1 and SPCA2. In yeast,the SPCA protein is localized to themedial-Golgi compart-ment, a hitherto unusual distribution for a Ca2+ pump,where it was found to be important for functioning of thesecretory pathway [173,174]. SPCA not only delivers Ca2+

but also Mn2+ into the secretory pathway [174,175].

The SPCA1 proteins: products of the ATP2C1 gene

The amino acid sequence of SPCA1 is shorter than that ofSERCA. However, the alignment indicates that all 10 trans-membrane segments are present (Fig. 6). The difference inlength is mainly caused by gaps in the SPCA1 sequence.The gaps predominantly occur in the lumenal loops con-necting the transmembrane helices and in regions of theN domain that in SERCA are localized at the surface. Thealignment shows that the residues critical for pump func-tion are conserved, such as the phosphorylation site, theThr–Gly–Glu loop in the A domain, the ATP-binding andFITC-binding regions, the Asp–Pro–Pro–Arg loop con-necting the N and P domain. However, in SPCA1, like inPMCA, only the Ca2+-coordinating residues in M4 and M6that form site II are conserved. Therefore, probably onlyone cation is transported for each ATP hydrolyzed [176].The transcript of the human ATP2C1 gene is alter-

natively spliced, giving rise to at least four different

3′-untranslated termini and two protein isoforms [172].The splice variant SPCA1a is longer than SPCA1b. Thelonger C-terminal sequence does not contain an 11th hy-drophobic region as in the SERCA2b splice variant. Thereis no evidence for the generation of C-terminal SPCA1protein variants in C. elegans [170].It is an interesting but unresolved question, which struc-

tural elements contribute to the capacity of SPCA pumpsto pump also Mn2+ with about the same affinity as Ca2+.The different selectivity of SPCA could reside in the ac-cess pathway or in the binding sites or both. Mandal et al.[177] have provided evidence that the species-invariantGln783 in M6 is a critical residue for Mn2+ transport bythe yeast protein. It is, however, not clear whether thisresidue is directly contacting the transported ion, be-cause in the SERCA1 structure the homologous residue(Thr805) is located away from the binding site, and theaccess pathway rather seems to be formed by residues inthe N-terminal part of M3 [20].An EF hand-like domain near the N-terminus of the

S. cerevisiae sequence seems to modulate ion transport[178]. The corresponding domain in SERCA indeed formsa helix–loop–helix structure, as in typical EF-hand do-mains. However, because its primary structure is poorlyconserved among the SPCA1 proteins, it remains to bedemonstrated whether a similar function occurs in otherspecies than yeast.It is difficult at this stage to speculate on the evolution-

ary relationship between SERCA and SPCA Ca2+ pumps.Both pumps are sufficiently similar, both with respect toprimary structure and substrate specificity, to be classi-fied as members of the 2A group of P-type ion transportATPases [32]. The picture is complicated, however, bythe non-universal expression of these pumps in eukary-otes. All animals express both SERCA and SPCA-relatedCa2+ pumps, whereas fungi like the yeast S. cerevisiae ex-press an SPCA-related pump (Pmr1), but do not possessSERCA-like sequences in their genome. In contrast, thegenome of the plant Arabidopsis thaliana does not con-tain genes that can be classified within the ATP2C (SPCApumps) group, whereas ATP2A-like (SERCA encoding)genes are present. Based on this distribution, it can thusnot be decided whether early eukaryotes used SERCA-or SPCA-like pumps or both for Ca2+ regulation. BecauseSERCA pumps possess two Ca2+-binding sites (site I andsite II) and SPCA pumps possess only the more ‘primitive’site II, SPCA-type of pumps could be considered as theoldest. However, it is also possible that SPCA pumps havelost one of two Ca2+-binding sites, if both sites were in-herited already from eukaryotic ancestors. This possibilitycannot be excluded because the recent sequencing ofbacterial genomes shows the presence of type 2A-like se-quences in several species. Analysis of these sequencesfor amino acids homologous to the Ca2+-coordinating



Fig. 6 Alignment of the amino acid sequences of rabbit SERCA1 and human SPCA1 and SPCA2. SPCA1b: protein product of the humanATP2C1 gene, indicated as splice variant ATP2C1b [171], GenBank accession number AF181121; SPCA2: predicted protein product of thehuman ATP2C2 gene (GenBank accession number AB014603, with omission of the first 55 amino acids); SERCA1a: rabbit SERCA1, adultisoform (GenBank accession number M12898, i.e. the neonatal isoform with its eight C-terminal amino acids (DPEDERRK) replaced by asingle G). Alignment made with the GCG program Pileup, with gap creation penalty = 18 and gap extension penalty = 2. Conserved residuesare indicated in bold face. The -helices forming the membrane domain in SERCA are underlined. The phosphorylation site (P), residuesinvolved in ATP binding and FITC binding are indicated above the alignment. Ca2+-coordinating residues on SERCA are indicated bytriangles (: site I; : site II) below the alignment. “Mn2+” indicates the Gln residue contributing to Mn2+ selectivity in the SPCA pumps.



residues in SERCA reveals a spectrum of degree of con-servation. While most of these putative pumps appear toposses only the more ‘primitive’ site II, some of themalso present a full or almost full conservation of site I,e.g. the product of the yloB gene of Bacillus subtilis (pro-tein accession NP 389448) and the MgtA2 gene productof Thermoanaerobacter tengcongensis (protein accessionAAM24443) [179–181]. Therefore, the possibility remainsopen that both SERCA- and SPCA-type Ca2+ pumpsof eukaryotes have been inherited independently fromprokaryotes.

Functional properties of SPCA1

Until recently, SPCA was partially characterized only inthe yeast S. cerevisiae, an organism that as far as its Ca2+

housekeeping concerns differs from animal cells becausein this organism SPCA is the major Ca2+ pump [182] andbecause yeast does not express SERCAs, inositol trispho-sphate (IP3) or ryanodine receptors. Similar to the SPCApump from S. cerevisiae [183], the corresponding enzymesfrom C. elegans [170] and man [184] transport Ca2+ withan affinity comparable to that of the ER-based SERCA2pump. SPCA1 also transports Mn2+ ions with high affinity[170,184]. The SERCAs in contrast do not catalyze activeMn2+ transport into the SR and ER [185]. It is unknownwhether there are functional differences between SPCA1aand SPCA1b.Like all P-type ATPases also the SPCA pumps are in-

hibited by vanadate [183]. Thapsigargin, the potent andspecific inhibitor of the SERCA family of Ca2+ pumps[186], does not inhibit SPCA1 activity at concentrationsof up to 5M [183]. SPCA1 is at least 2 orders of magni-tude less sensitive to the inhibition by cyclopiazonic acidor 2,5-di-(tert-butyl)-1,4-benzohydroquinone than SERCA[187].

Subcellular localization of SPCA1

The heterologously expressed C. elegans SPCA in COS-1cells [170,188] and the human SPCA1 in Chinese hamsterovary cells [184] seem to localize to the Golgi area. Suchlight-microscopical analyses do not allow to discriminatebetween a localization of SPCA1 in one of the classicalGolgi subcompartments (cis-,medial- or trans-Golgi), in thetrans-Golgi network (TGN) or in both. The TGN, character-ized by its relatively lower lumenal pH is involved in dy-namic exchanges with other acid compartments, amongstwhich are the lysosomes, the endosomes and the secretoryvesicles. When present in the TGN, recycling of SPCA1through the downstream compartments could be possible.The thapsigargin-insensitive Ca2+ storage in dense coresecretory vesicles [189] would be compatible with thispossibility.

Thapsigargin is a useful compound to assess how muchof the Ca2+ uptake into the Golgi apparatus dependson SERCA and how much on SPCA1 Ca2+-pump activ-ity, since only the SERCAs are blocked by thapsigargin.Ca2+ uptake in a Golgi-enriched fraction of rat liver wasalmost completely inhibited by thapsigargin [190], indi-cating that it totally depended on a SERCA Ca2+ pump. Incontrast, Ca2+ uptake into a stacked Golgi fraction of ratliver was only inhibited 50% by thapsigargin [191]. Ca2+

uptake by the Golgi in aequorin-expressing HeLa cellswas only for 50% mediated by SERCA Ca2+ pumps [192],while that in HeLa and Chinese hamster ovary cells over-expressing the Ca2+-binding protein CALNUC dependedfor about 70% on SERCA Ca2+ pumps [193]. Although thisthapsigargin-independent Ca2+ uptake has been ascribedto PMCA Ca2+ pumps in transit to the plasma membrane[191], it is more likely that it depends on SPCA1. Prelim-inary experiments from our group indicate that the rela-tive contribution of both Ca2+ pumps to the Golgi Ca2+

uptake depends on the cell type. In some cells, more than80% of the Ca2+ uptake into the Golgi complex is not af-fected by thapsigargin and therefore depends on SPCA1.Furthermore, the level of SPCA1 expression is dynami-cally regulated, e.g. its expression level in the mammarytissue dramatically increases before parturition in rats[194].In yeast, where SERCA-type Ca2+-ATPases are not ex-

pressed, the SPCA Ca2+ pump is the major pump that con-tributes to the steady-state free [Ca2+] in the ER [182]; thislevel of Ca2+ decreases by 50% in pmr1-nullmutants [195].The situation is different in mammalian cells since theydo express SERCA Ca2+ pumps. Some of the SPCA1 Ca2+

pumps may, however, still be present in the ER, since thisorganelle can still accumulate some Ca2+ in the presenceof thapsigargin.

Role of the Golgi complex in Ca 2+ and Mn 2+

homeostasis

The Golgi apparatus seems to be important for shaping cy-tosolic Ca2+ signals. [Ca2+]c increases up to 16-fold in thepmr1 mutant in yeast [196]. COS-1 cells overexpressingSPCA1, but not SERCA1 or SERCA2b, respond to extra-cellular ATP with typical baseline Ca2+ oscillations, bothin the absence [197] or in the presence of a functional ER[188]. These oscillations have all the typical characteristicsof baseline cytosolic Ca2+ oscillations:

(i) The spikes have a cell-specific shape.(ii) They are sometimes abortive.(iii) The frequency of the spikes increases with increasing

ATP concentrations.(iv) There is often a long latency before the first [Ca2+]c

rise.



(v) The oscillation occurs for a limited period of timein Ca2+-free solution indicating that Ca2+ is releasedfrom an internal Ca2+ store.

(vi) The oscillation needs extracellular Ca2+ to be sus-tained.

COS-1 cells overexpressing SPCA1, but not SERCA1 orSERCA2b, show a delayed [Ca2+]c rise during capacitativeCa2+ entry in the presence of ATP, both in the absence[197] or in the presence of a functional ER [188]. The ideais that the Golgi apparatus may for a while accumulate theCa2+ that flows into the cell thereby keeping the cytosolic[Ca2+] low.SPCA1 in yeast appears to be the principal route for re-

moving excess Mn2+ from the cytoplasm. Cytosolic Mn2+

accumulates in pmr1 mutants and can serve as an inor-ganic scavenger of superoxide radicals, thereby bypassingthe requirement for cytosolic superoxide dismutase in aer-obic growth [175]. Pmr1 mutants are very sensitive to thegrowth toxicity of millimolar concentrations of extracellu-lar Mn2+, indicating that delivery into the secretory path-way by SPCA, and subsequent exocytosis, must be a majorroute for removing Mn2+ from the cytoplasm.Ca2+ in the lumen of the Golgi apparatus controls impor-

tant functions, including lumenal and membrane proteintraffic [198], cargo condensation, and precursor process-ing [199,200]. A major fraction of the Ca2+ does not seemto be free in the Golgi lumen but is probably sequestered.In fact, several Ca2+-binding proteins have been identifiedin the Golgi apparatus: CALNUC (nucleobindin) [201] andCab45 [202] are restricted to the Golgi complex, while typ-ical ER proteins like calumenin and GRP94 (endoplasmin)have also been detected in the Golgi apparatus [203,204].Mn2+ in the Golgi complex is needed for N- and O-linked

glycosylation of cellular proteins [205,206] and for optimalactivity of the Golgi-localized casein kinase. The latter en-zyme is abundantly expressed in the lactating mammarygland but it is also present in other cell types [207,208].

Properties of SPCA1-containing Ca 2+ stores

Those cellular organelles in COS-1 cells that accumulateCa2+ through the transfected C. elegans SPCA present alower passive Ca2+ permeability than the ER and onlya small amount of Ca2+ can be released by stimulatingthe IP3 receptors. The EC50 dose of IP3 is furthermorefive times higher than for releasing Ca2+ from the ER[197]. Similar findings were done for the endogenouslyexpressed SPCA1 in A7r5 smooth-muscle cells and in16HBE14o-bronchial mucosal cells [187]. Inositol 1,3,4,5-tetrakisphosphate, cyclic ADP-ribose, caffeine and nico-tinic acid adenine dinucleotide phosphate do not releaseCa2+ from the thapsigargin-insensitive non-mitochondrialCa2+ stores in the latter two cell types [187].

Some of the functional properties observed for theSPCA1-containing compartment are in agreement withwhat has been reported for the Golgi apparatus in in-tact cells. The reduced passive Ca2+ leak of the SPCA1-containing Ca2+ store corresponds with observations inLLC-PK1 epithelial cells, Swiss 3T3 fibroblasts and L5 ratmyoblasts, where the Golgi apparatus is substantiallymore resistant to Ca2+ depletion than the other regions ofthe cell [209]. The difference in EC50 for IP3-induced Ca2+

release between the ER and the SPCA1-containing Ca2+

store is in agreement with measurements of the lumenalfree [Ca2+] within organelles in permeabilized BHK-21cells, which showed a lower responsiveness to IP3 in theperinuclear Golgi region than in the other regions of thecell [210]. The relatively small IP3-induced Ca2+ release iscompatible with the observed small Ca2+ release from theGolgi apparatus by a supramaximal histamine concentra-tion in HeLa cells [211].

Hailey–Hailey disease

Hailey–Hailey disease is an autosomal dominant skin dis-ease with recurrent eruption of vesicles and bullae involv-ing predominantly the neck, groin and axillary regions.Histopathology shows suprabasal cleavage in epidermalcells. There may also be extracutaneous manifestationsof the disease. The disease is caused by nonsense andmissense mutations inactivating one allele of ATP2C1[171,172,212–215]. These findings support a mechanismthrough haploinsufficiency, suggesting that epidermalcells are sensitive to the dosage of SPCA1.It is not clear how the mutations in ATP2C1 lead to the

observed defects. The expected decrease in [Ca2+] and/or[Mn2+] in the Golgi complex could lead to a decreasein glycosylation, proteolytic processing, folding, traffick-ing, or sorting of key molecules involved in cell-to-celladhesion, such as desmosomal proteins. This may causean inability to maintain structurally intact desmosomes,leading to the cleavage of the epidermal cells. The exper-imentally observed increase in the [Ca2+]c [171] could,amongst others, lead to changes in gene expression orpost-translational modification of proteins (e.g. by proteinkinase C).It is also not clear why clinical symptoms of Hailey–

Hailey disease are restricted to the epidermis and, more inparticular, to certain areas of the skin despite the expres-sion of SPCA1 in several tissues. It is known that thereare cell-type dependent differences in the extent of glyco-sylation of desmosomal glycoproteins [216]. It is possiblethat the particular glycosylation state of the desmosomalglycoproteins in the epidermis makes themmore vulnera-ble to subtle [Ca2+] changes caused by haploinsufficiencyof ATP2C1. Alternatively, noncutaneous tissues may havecompensatory mechanisms that are lacking in the skin.



This could also explain why lesions are exacerbated by ex-ternal factors such as sweating, friction and infection ofthe skin. Haploinsufficiency of ATP2C1 could be compen-sated by other Ca2+-regulatorymechanisms in the cell, butwhen the cell is placed under stress, e.g. mechanical stressfrom friction, or heat, the subtle deficiency is exposed.

The SPCA2 protein: product of the ATP2C2 gene

In the human genome, the predicted gene designatedKIAA0703was recognized as encoding a P-type ion-motiveATPase but its putative product has never been character-ized. The sequence is sufficiently similar to that of ATP2C1to warrant its inclusion in the SPCA family. Therefore, thegene is indicated as ATP2C2 and the protein as SPCA2[184].Sequence analysis of the cDNA and the genomic DNA

revealed the presence of 25 exons, spanning a genomicregion of 59.6 kb on chromosome 16 (l6q22). All theintron–exon boundaries are conserved between ATP2C1and ATP2C2. Translation of the 2892bp coding sequenceresults in a protein of 963 amino acids (105kDa). Theresulting protein exhibits about 60% sequence identitycompared to the human SPCA1. The expression patternof ATP2C2 is somewhat different from that of ATP2C1.While ATP2C1 is considered to be a housekeeping gene,ATP2C2 has a more restricted expression pattern. ThemRNA is found in the human gastrointestinal tract, fromthe stomach to the rectum, but not in the oesophagus. Wecould also show its occurrence in bone marrow, tracheaand prostate tissue. The relative amount of the ATP2C1and ATP2C2 transcripts is cell-type dependent.

CONCLUSIONS

Vertebrates have evolved an impressive number of differ-ent intracellular Ca2+-transport ATPases by a combinationof gene multiplications and of alternative gene transcriptprocessing. Together these multiple Ca2+-pump isoformsare responsible for creating Ca2+ gradients across themembranes of the ER/SR (sub)compartments and of othermembranous components of the secretory pathway in-cluding the Golgi complex. Whereas in the past, emphasiswas placed on the role of the various intracellular compart-ments, as stores for Ca2+ activating a plethora of cellularprocesses in the cytoplasm, it became recently apparentthat lumenal Ca2+ in these compartments also serves anumber of functions. Interfering with Ca2+ accumulationthus causes severe cellular stress. Deciphering the exactrole of each of the different Ca2+-pump isoforms remainsa daunting task. Interfering with the production or thefunction of one isoform by gene targeting or antisenseoligonucleotides often causes disappointingly little overtphenotype, so that the exact function of the isoform must

be subtle and remains hitherto largely unknown. Progressin RNA interference technology and its application tovertebrate cells may open new possibilities in this respect.Recent determinations of the crystallographic structure

of some of the catalytic intermediates of the SERCA1apump caused a real breakthrough and paved the way tounderstand the function not only of SERCA pumps butalso of ATPases transporting other ions.It finally became clear that the thapsigargin-sensitive

SERCA family is supplemented by a growing num-ber of thapsigargin-insensitive pumps: the Golgi basedSPCA1 (possibly also SPCA2 pumps) and may be alsothe COD1p/Spf1 homologues. For the latter pumps, theirdirect involvement in Ca2+ transport still needs to beexperimentally demonstrated. Novel views about thesubcellular organization of Ca2+ signaling may thereforeemerge in the coming years.

REFERENCES

1. Berridge MJ, Lipp P, Bootman MD. The versatility anduniversatility of calcium signalling. Nat Rev Mol Cell Biol2000; 1: 11–21.

2. Møller JV, Juul B, le Maire M. Structural organization, iontransport, and energy transduction of P-type ATPases.Biochim Biophys Acta 1996; 1286: 1–51.

3. Wuytack F, Dode L, Baba-Aıssa F, Raeymaekers L. TheSERCA3-type of organellar Ca2+ pumps. Biosci Rep 1995;15: 299–306.

4. Shull GE. Gene knockout studies of Ca2+-transportingATPases. Eur J Biochem 2000; 267: 5284–5290.

5. East JM. Sarco(endo)plasmic reticulum calcium pumps:recent advances in our understanding of structure/functionand biology (Review). Mol Membr Biol 2000; 17: 189–200.

6. Toyoshima C, Nakasako M, Nomura H, Ogawa H. Crystalstructure of the calcium pump of sarcoplasmic reticulum at2.6 Å resolution. Nature 2000; 405: 647–655.

7. Toyoshima C, Nomura H. Structural changes in the calciumpump accompanying the dissociation of calcium. Nature2002; 418: 605–611.

8. Andersen JP. Dissection of the functional domains of thesarcoplasmic reticulum Ca2+-ATPase by site-directedmutagenesis. Biosci Rep 1995; 15: 243–261.

9. Martonosi AN. The structure and interactions ofCa2+-ATPase. Biosci Rep 1995; 15: 263–281.

10. MacLennan DH, Rice WJ, Green NM. The mechanism ofCa2+ transport by sarco(endo)plasmic reticulumCa2+-ATPases. J Biol Chem 1997; 272: 28815–28818.

11. Lee AG, East JM. What the structure of a calcium pump tellsus about its mechanism. Biochem J 2001; 356: 665–683.

12. Sweadner KJ, Donnet C. Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmicreticulum. Biochem J 2001; 356: 685–704.

13. Xu C, Rice WJ, He W, Stokes DL. A structural model for thecatalytic cycle of Ca2+-ATPase. J Mol Biol 2002; 316:201–211.

14. Aravind L, Galperin MY, Koonin EV. The catalytic domainof the P-type ATPase has the haloacid dehalogenase fold.Trends Biochem Sci 1998; 23: 127–129.



15. Clausen JD, McIntosh DB, Woolley DG, Andersen JP.Importance of Thr-353 of the conserved phosphorylationloop of the sarcoplasmic reticulum Ca2+-ATPase in MgATPbinding and catalytic activity. J Biol Chem 2001; 276:35741–35750.

16. Sørensen TL, Andersen JP. Importance of stalk segment S5for intramolecular communication in the sarcoplasmicreticulum Ca2+-ATPase. J Biol Chem 2000; 275:28954–28961.

17. Pick U. Interaction of fluorescein isothiocyanate withnucleotide-binding sites of the Ca-ATPase fromsarcoplasmic reticulum. Eur J Biochem 1981; 121: 187–195.

18. Nørregaard A, Vilsen B, Andersen JP. Transmembranesegment M3 is essential to thapsigargin sensitivity of thesarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 1994;269: 26598–26601.

19. Yu M, Zhang L, Rishi AK, Khadeer M, Inesi G, Hussain A.Specific substitutions at amino acid 256 of thesarcoplasmic/endoplasmic reticulum Ca2+ transportATPase mediate resistance to thapsigargin inthapsigargin-resistant hamster cells. J Biol Chem 1998; 273:3542–3546.

20. Andersen JP, Sørensen TL, Povlsen K, Vilsen B. Importanceof transmembrane segment M3 of the sarcoplasmicreticulum Ca2+-ATPase for control of the gateway to theCa2+ sites. J Biol Chem 2001; 276: 23312–23321.

21. Falson P, Menguy T, Corre F et al. The cytoplasmic loopbetween putative transmembrane segments 6 and 7 insarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and isfunctionally important. J Biol Chem 1997; 272:17258–17262.

22. Zhang Z, Lewis D, Sumbilla C, Inesi G, Toyoshima C. Therole of the M6–M7 loop (L67) in stabilization of thephosphorylation and Ca2+ binding domains of thesarcoplasmic reticulum Ca2+-ATPase (SERCA). J Biol Chem2001; 276: 15232–15239.

23. Hua S, Ma H, Lewis D, Inesi G, Toyoshima C. Functionalrole of N (nucleotide) and P (phosphorylation) domaininteractions in the sarcoplasmic reticulum (SERCA) ATPase.Biochemistry 2002; 41: 2264–2272.

24. Pollesello P, Annila A, Ovaska M. Structure of the 1–36amino-terminal fragment of human phospholamban bynuclear magnetic resonance and modelling of thephospholamban pentamer. Biophys J 1999; 76: 1784–1795.

25. Negash S, Yao Q, Sun H, Li J, Bigelow DJ, Squier TC.Phospholamban remains associated with the Ca2+- andMg2+-dependent ATPase following phosphorylation bycAMP-dependent protein kinase. Biochem J 2000; 351:195–205.

26. Autry JM, Jones LR. Functional co-expression of the caninecardiac Ca2+ pump and phospholamban in Spodopterafrugiperda (Sf21) cells reveals new insights on ATPaseregulation. J Biol Chem 1997; 272: 15872–15880.

27. Kimura Y, Kurzydlowski K, Tada M, MacLennan DH.Phospholamban inhibitory function is activated bydepolymerization. J Biol Chem 1997; 272: 15061–15064.

28. Toyofuku T, Kurzydlowski K, Tada M, MacLennan DH.Amino acids Lys–Asp–Asp–Lys–Pro–Val402 in theCa2+-ATPase of cardiac sarcoplasmic reticulum are criticalfor functional association with phospholamban. J BiolChem 1994; 269: 22929–22932.

29. Asahi M, Green NM, Kurzydlowski K, Tada M, MacLennanDH. Phospholamban domain IB forms an interaction sitewith the loop between transmembrane helices M6 and M7

of sarco(endo)plasmic reticulum Ca2+ ATPases. Proc NatlAcad Sci USA 2001; 98: 10061–10066.

30. Jones LR, Cornea RL, Chen Z. Close proximity betweenresidue 30 of phospholamban and cysteine 318 of thecardiac Ca2+ pump revealed by intermolecular thiolcross-linking. J Biol Chem 2002; 277: 28319–28329.

31. Asahi M, Kimura Y, Kurzydlowski K, Tada M, MacLennanDH. Transmembrane helix M6 in sarco(endo)plasmicreticulum Ca2+-ATPase forms a functional interaction sitewith phospholamban. Evidence for physical interactions atother sites. J Biol Chem 1999; 274: 32855–32862.

32. Axelsen KB, Palmgren MG. Evolution of substratespecificities in the P-type ATPase superfamily. J Mol Evol1998; 46: 84–101.

33. Magyar A, Bakos E, Varadi A. Structure and tissue-specificexpression of the Drosophila melanogaster organellar typeCa2+ ATPase gene. Biochem J 1995; 310: 872–877.

34. Cho JH, Bandyopadhyay J, Lee J, Park CS, Ahnn J. Twoisoforms of sarco/endoplasmic reticulum calcium ATPase(SERCA) are essential in Caenorhabditis elegans. Gene 2000;261: 211–219.

35. Zwaal RR, Van Baelen K, Groenen JTM et al. Thesarco-endoplasmic reticulum Ca2+ ATPase is required fordevelopment and muscle function in Caenorhabditiselegans. J Biol Chem 2001; 276: 43557–43563.

36. de Mendonça RL, Beck E, Rumjanek FD, Goffeau A.Cloning and characterization of a putativecalcium-transporting ATPase gene from Schistosomamansoni. Mol Biochem Parasitol 1995; 72: 129–139.

37. Talla E, de Mendonça RL, Degand I, Goffeau A, Ghislain M.Schistosoma mansoni Ca2+-ATPase SMA2 restores viabilityto yeast Ca2+-ATPase-deficient strains and functions incalcineurin-mediated Ca2+ tolerance. J Biol Chem 1998;273: 27831–27840.

38. Thastrup O, Cullen PJ, Drobak BK, Hanley MK, Dawson AP.Thapsigargin, a tumor promoter, discharges intracellularCa2+ stores by specific inhibition of the endoplasmicreticulum Ca2+-ATPase. Proc Natl Acad Sci USA 1990; 87:2466–2470.

39. Seidler NW, Jona I, Vegh M, Martonosi A. Cyclopiazonic acidis a specific inhibitor of the Ca2+ ATPase of the sarcoplasmicreticulum. J Biol Chem 1989; 264: 17816–17823.

40. Oldershaw KA, Taylor CW.2,5-Di-(tert-butyl)-1,4-benzohydroquinone mobilizesinositol 1,4,5-trisphosphate sensitive and insensitive stores.FEBS Lett 1990; 274: 214–216.

41. Dode L, De Greef C, Mountian I et al. Structure of thehuman sarco/endoplasmic reticulum Ca2+-ATPase 3 gene.Promoter analysis and alternative splicing of the SERCA3pre-mRNA. J Biol Chem 1998; 273: 13982–13994.

42. Escalante R, Sastre L. Structure of the Artemia fransciscanasarco/endoplasmic reticulum Ca-ATPase gene. J Biol Chem1994; 269: 13005–13012.

43. Axelsen KB, Palmgren MG. Inventory of the superfamily ofP-type ion pumps in Arabidopsis. Plant Physiol 2001; 126:696–706.

44. Londraville RL, Cramer TD, Franck JP, Tullis A, Block BA.Cloning of a neonatal calcium ATPase isoform (SERCA 1B)from extraocular muscle of adult blue marlin (Makairanigricans). Comp Biochem Physiol B Biochem Mol Biol 2000;127: 223–233.

45. Dode L, Van Baelen K, Wuytack F, Dean WL. Lowtemperature molecular adaptation of the skeletal musclesarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1) in



the wood frog (Rana sylvatica). J Biol Chem 2001; 276:3911–3919.

46. Campbell AM, Kessler PD, Sagara Y, Inesi G, FambroughDM. Nucleotide sequences of avian cardiac and brainSR/ER Ca2+-ATPases and functional comparisons with fasttwitch Ca2+ ATPase. Calcium affinities and inhibitoreffects. J Biol Chem 1991; 266: 16050–16055.

47. Brandl CJ, Green MN, Korckzak B, MacLennan DH. TwoCa2+-ATPase genes: homologies and mechanisticimplications of deduced amino acid sequences. Cell 1986;44: 597–607.

48. Brandl CJ, deLeon S, Martin DR, MacLennan DH. Adultforms of the Ca2+-ATPase of sarcoplasmic reticulum:expression in developing skeletal muscle. J Biol Chem 1987;262: 3768–3774.

49. Odermatt A, Taschner PE, Khanna VK et al. Mutations inthe gene encoding SERCA1, the fast twitch skeletal musclesarcoplasmic reticulum Ca2+-ATPase, are associated withBrody disease. Nat Genet 1996; 14: 191–194.

50. Lytton J, MacLennan DH. Molecular cloning of cDNAs fromhuman kidney coding for two alternatively splicedproducts of the cardiac Ca2+-ATPase gene. J Biol Chem1988; 263: 15024–15031.

51. Eggermont JA, Wuytack F, De Jaegere S, Nelles L, Casteels R.Evidence for two isoforms of the endoplasmic–reticulumCa2+ pump in pig smooth muscle. Biochem J 1989; 260:757–761.

52. Baba-Aıssa F, Raeymaekers L, Wuytack F, De Greef C,Missiaen L, Casteels R. Distribution of the organellar Ca2+transport ATPase SERCA2 isoforms in the cat brain. BrainRes 1996; 743: 141–153.

53. Van Den Bosch L, Eggermont J, De Smedt H, Mertens L,Wuytack F, Casteels R. Regulation of splicing is responsiblefor the expression of the muscle-specific 2a isoform of thesarco/endoplasmic–reticulum Ca2+-ATPase. Biochem J1994; 302: 559–566.

54. Mertens L, Van Den Bosch L, Verboomen H, Wuytack F, DeSmedt H, Eggermont J. Sequence and spatial requirementsfor regulated muscle-specific processing of thesarco/endoplasmic reticulum Ca2+-ATPase 2 gene(SERCA2) transcript. J Biol Chem 1995; 270: 11004–11011.

55. De Jaegere S, Wuytack F, De Smedt H, Van Den Bosch L,Casteels R. Alternative processing of the gene transcriptsencoding a plasma-membrane and a sarco/endoplasmicreticulum Ca2+ pump during differentiation of BC3H1muscle cells. Biochim Biophys Acta 1993; 1173: 188–194.

56. De Smedt H, Eggermont JA, Wuytack F et al. Isoformswitching of the sarco(endo)plasmic reticulum Ca2+ pumpduring differentiation of BC3H1 myoblasts. J Biol Chem1991; 266: 7092–7095.

57. Ver Heyen M, Reed TD, Blough RI et al. Structure andorganization of the mouse Atp2a2 gene encoding thesarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2)isoforms. Mamm Genome 2000; 11: 159–163.

58. Misquitta CM, Mwanjewe J, Nie L, Grover AK. Sarcoplasmicreticulum Ca2+ pump mRNA stability in cardiac andsmooth muscle: role of the 3′-untranslated region. Am JPhysiol 2002; 283: C560–C568.

59. Lytton J, Westlin M, Burk SE, Shull GE, MacLennan DH.Functional comparisons between isoforms of thesarcoplasmic or endoplasmic reticulum family of calciumpumps. J Biol Chem 1992; 267: 14483–14489.

60. Verboomen H, Wuytack F, De Smedt H, Himpens B,Casteels R. Functional difference between SERCA2a and

SERCA2b Ca2+ pumps and their modulation byphospholamban. Biochem J 1992; 286: 591–596.

61. Ver Heyen M, Heymans S, Antoons G et al. Replacement ofthe muscle-specific sarcoplasmic reticulum Ca2+-ATPaseisoform SERCA2a by the nonmuscle SERCA2b homologuecauses mild concentric hypertrophy and impairscontraction–relaxation of the heart. Circ Res 2001; 89:838–846.

62. Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits thesarcoplasmic or endoplasmic reticulum Ca-ATPase familyof calcium pumps. J Biol Chem 1991; 266: 22912–22918.

63. Escalante R, Sastre L. Similar alternative splicing eventsgenerate two sarcoplasmic reticulum Ca-ATPase isoformsin the crustacean Artemia franciscana and in vertebrates.J Biol Chem 1993; 268: 14090–14095.

64. Escalante R, Sastre L. Tissue-specific alternative promotersregulate the expression of the two sarco/endoplasmicreticulum Ca-ATPase isoforms from Artemia franciscana.DNA Cell Biol 1995; 14: 893–900.

65. Verboomen H, Wuytack F, Van Den Bosch L, Mertens L,Casteels R. The functional importance of the extremeC-terminal tail in the gene 2 organellar Ca2+-transportATPase (SERCA2a/b). Biochem J 1994; 303: 979–984.

66. John LM, Lechleiter JD, Camacho P. Differential modulationof SERCA2 isoforms by calreticulin. J Cell Biol 1998; 142:963–973.

67. Roderick HL, Lechleiter JD, Camacho P. Cytosolicphosphorylation of calnexin controls intracellular Ca2+oscillations via an interaction with SERCA2b. J Cell Biol2000; 149: 1235–1247.

68. Michalak M, Corbett EF, Mesaeli N, Nakamura K, Opas M.Calreticulin: one protein, one gene, many functions.Biochem J 1999; 344: 281–292.

69. Johnson S, Michalak M, Opas M, Eggleton P. The ins andout of calreticulin: from the ER lumen to the extracellularspace. Trends Cell Biol 2001; 11: 122–129.

70. Ihara Y, Cohen-Doyle MF, Saito Y, Williams DB. Calnexindiscriminates between protein conformational states andfunctions as a molecular chaperone in vitro. Mol Cell 1999;4: 331–341.

71. Sakuntabhai A, Ruiz-Perez V, Carter S et al. Mutations inATP2A2, encoding a Ca2+ pump, cause Darier disease. NatGenet 1999; 21: 271–277.

72. Munro CS. The phenotype of Darier’s disease: penetranceand expressivity in adults and children. Br J Dermatol 1992;127: 126–130.

73. Jones I, Jacobsen N, Green EK, Elvidge GP, Owen MJ,Craddock N. Evidence for familial cosegregation of majoraffective disorder and genetic markers flanking the genefor Darier’s disease. Mol Psychiatry 2002; 7: 424–427.

74. Tavadia S, Tait RC, McDonagh TA, Munro CS. Platelet andcardiac function in Darier’s disease. Clin Exp Dermatol2001; 26: 696–699.

75. Periasamy M, Reed TD, Liu LH et al. Impaired cardiacperformance in heterozygous mice with a null mutation inthe sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2(SERCA2) gene. J Biol Chem 1999; 274: 2556–2562.

76. Liu LH, Boivin GP, Prasad V, Periasamy M, Shull GE.Squamous cell tumors in mice heterozygous for a nullallele of Atp2a2, encoding the sarco(endo)plasmicreticulum Ca2+-ATPase isoform 2 Ca2+ pump. J Biol Chem2001; 276: 26737–26740.

77. Koss KL, Kranias EG. Phospholamban: a prominentregulator of myocardial contractility. Circ Res 1996; 79:1059–1063.



78. Simmerman HK, Jones LR. Phospholamban: proteinstructure mechanism of action and role in cardiac function.Physiol Rev 1998; 78: 921–947.

79. Briggs FN, Lee KF, Wechsler AW, Jones LR. Phospholambanexpressed in slow-twitch and chronically stimulatedfast-twitch muscles minimally affects calcium affinity ofsarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 1992;267: 26056–26061.

80. Raeymaekers L, Eggermont JA, Wuytack F, Casteels R.Effects of cyclic nucleotide dependent protein kinases onthe endoplasmic reticulum Ca2+ pump of bovinepulmonary artery. Cell Calcium 1990; 11: 261–268.

81. Sutliff RL, Hoying JB, Kadami VJ, Kranias EG, Paul RJ.Phospholamban is present in endothelial cells andmodulates endothelium-dependent relaxation Evidencefrom phospholamban gene-ablated mice. Circ Res 1999; 84:360–364.

82. Colyer J, Wang JH. Dependence of cardiac sarcoplasmicreticulum calcium pump activity on the phosphorylationstatus of phospholamban. J Biol Chem 1991; 266:17486–17493.

83. Simmerman HK, Kobayashi YM, Autry JM, Jones LR. Aleucine zipper stabilizes the pentameric membrane domainof phospholamban and forms a coiled-coil pore structure. JBiol Chem 1996; 271: 5941–5946.

84. Reddy LG, Autry JM, Jones LR, Thomas DD.Co-reconstitution of phospholamban mutants with theCa-ATPase reveals dependence of inhibitory function onphospholamban structure. J Biol Chem 1999; 274:7649–7655.

85. Cornea RL, Autry JM, Chen Z, Jones LR. Reexamination ofthe role of the leucine/isoleucine zipper residues ofphospholamban in inhibition of the Ca2+ pump of cardiacsarcoplasmic reticulum. J Biol Chem 2000; 275:41487–41494.

86. Cornea RL, Jones LR, Autry JM, Thomas DD. Mutation andphosphorylation change the oligomeric structure ofphospholamban in lipid bilayers. Biochemistry 1997; 36:2960–2967.

87. Asahi M, McKenna E, Kurzydlowski K, Tada M, MacLennanDH. Physical interactions between phospholamban andsarco(endo)plasmic reticulum Ca2+-ATPases aredissociated by elevated Ca2+, but not by phospholambanphosphorylation, vanadate or thapsigargin and areenhanced by ATP. J Biol Chem 2000; 275: 15034–15038.

88. Sharma P, Patchell VB, Gao Y, Evans JS, Levine BA.Cytoplasmic interactions between phospholambanresidues 1–20 and the calcium-activated ATPaseof the sarcoplasmic reticulum. Biochem J 2001; 355:699–706.

89. Xu A, Hawkins C, Narayanan N. Phosphorylation andactivation of the Ca2+-pumping ATPase of cardiacsarcoplasmic reticulum by Ca2+/calmodulin-dependentprotein kinase. J Biol Chem 1993; 268: 8394–8397.

90. Xu AD, Narayanan N. Reversible inhibition of thecalcium-pumping ATPase in native cardiac sarcoplasmicreticulum by a calmodulin-binding peptide. Evidence forcalmodulin-dependent regulation of the Vmax of calciumtransport. J Biol Chem 2000; 275: 4407–4416.

91. Odermatt A, Kurzydlowski K, MacLennan DH. The Vmax ofthe Ca2+-ATPase of cardiac sarcoplasmic reticulum(SERCA2a) is not altered by Ca2+/calmodulin-dependentphosphorylation or by interaction with phospholamban. JBiol Chem 1996; 271: 14206–14213.

92. Reddy LG, Jones LR, Pace RC, Stokes DL. Purified,reconstituted cardiac Ca2+-ATPase is regulated byphospholamban but not by direct phosphorylation withCa2+/calmodulin-dependent protein kinase. J Biol Chem1996; 271: 14964–14970.

93. Hellstern S, Pegoraro S, Karim CB. Sarcolipin thehomologue of phospholamban, forms oligomericstructures in detergent micelles and in liposomes. J BiolChem 2001; 276: 30845–30852.

94. Wawrzynow A, Theibert JL, Murphy C, Jona I, Martonosi A,Collins J. Sarcolipin, the proteolipid of skeletal musclesarcoplasmic reticulum, is a unique, amphipathic,31-residue peptide. Arch Biochem Biophys 1992; 298:620–623.

95. Odermatt A, Becker S, Khanna VK et al. Sarcolipin regulatesthe activity of SERCA1, the fast-twitch skeletal musclesarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 1998;273: 12360–12369.

96. Asahi M, Kurzydlowski K, Tada M, MacLennan DH.Sarcolipin inhibits polymerisation of phospholamban toinduce superinhibition of sarco(endo)plasmic reticulumCa2+-ATPases (SERCAs). J Biol Chem 2002; 277:26725–26728.

97. Ellgaard L, Molinari M, Helenius A. Setting the standards:quality control in the secretory pathway. Science 1999; 286:1882–1888.

98. Nakagawa T, Zhu H, Morishima N et al. Caspase-12mediates endoplasmic-reticulum-specific apoptosis andcytotoxicity by amyloid-. Nature 2000; 403: 98–103.

99. Rao RV, Hermel E, Castro-Obregon S et al. Couplingendoplasmic reticulum stress to the cell death program.Mechanism of caspase activation. J Biol Chem 2001; 276:33869–33874.

100. Kaufman RJ. Stress signaling from the lumen of theendoplasmic reticulum: coordination of genetranscriptional and translational controls. Genes Dev 1999;13: 1211–1233.

101. Mori K. Tripartite management of unfolded proteins in theendoplasmic reticulum. Cell 2000; 101: 451–454.

102. Ma Y, Hendershot LM. The unfolding tale of the unfoldedprotein response. Cell 2001; 107: 827–830.

103. Caspersen C, Pedersen PS, Treiman M. Thesarco/endoplasmic reticulum calcium-ATPase is anendoplasmic reticulum stress-inducible protein. J BiolChem 2000; 275: 22363–22372.

104. Højmann Larsen A, Frandsen A, Treiman M. Upregulationof the SERCA-type Ca2+ pump activity in response toendoplasmic reticulum stress in PC12 cells. BCM Biochem2001; 2: 4.

105. Thuerauf DJ, Hoover H, Meller J et al. Sarco/endoplasmicreticulum calcium ATPase-2 expression is regulatedby ATF6 during endoplasmic reticulum stress response.Intracellular signalling of calcium stress in a cardiacmyocyte model system. J Biol Chem 2001; 276:48309–48317.

106. Chen X, Shen J, Prywes R. The luminal domain of ATF6senses endoplasmic reticulum (ER) stress and causestranslocation of ATF6 from the ER to the Golgi. J Biol Chem2002; 277: 13045–13052.

107. Brown MS, Ye J, Rawson RB, Goldstein JL. Regulatedintramembrane proteolysis: a control mechanism conservedfrom bacteria to humans. Cell 2000; 100: 391–398.

108. Fortini ME. Notch and presenilin: a proteolytic mechanismemerges. Curr Opin Cell Biol 2001; 13: 627–634.



109. Periz G, Fortini ME. Ca2+-ATPase function is required forintracellular trafficking of the Notch receptor in Drosophila.EMBO J 1999; 18: 5983–5993.

110. Haigh NG, Johnson AE. A new role for BiP: closing theaqueous translocon pore during protein integration intothe ER membrane. J Cell Biol 2002; 156: 261–270.

111. Lomax RB, Camello C, Van Coppenolle F, Petersen OH,Tepikin AV. Basal and physiological Ca2+ leak from theendoplasmic reticulum of pancreatic acinar cells. Secondmessenger-activated channels and translocons. J Biol Chem2002; 277: 26479–26485.

112. Bian X, Hughes Jr FM, Huang Y, Cidlowsky JA, Putney JrJW. Roles of cytoplasmic Ca2+ and intracellular Ca2+ storesin induction and suppression of apoptosis in S49 cells. Am JPhysiol 1997; 272: C1241–C1249.

113. He H, Lam M, McCormick TS, Distelhorst CW. Maintenanceof calcium homeostasis in the endoplasmic reticulum byBcl-2. J Cell Biol 1997; 138: 1219–1228.

114. Dowd DR, MacDonald PN, Komm BS, Haussler MR,Miesfeld R. Stable expression of the calbindin-D28Kcomplementary DNA interferes with the apoptotic pathwayin lymphocytes. Mol Endocrinol 1992; 6: 1843–1848.

115. McConkey DJ, Orrenius S. The role of calcium in theregulation of apoptosis. Biochem Biophys Res Commun1997; 239: 357–366.

116. Ma TS, Mann DL, Lee JH, Gallinghouse GJ. SR compartmentcalcium and cell apoptosis in SERCA overexpression. CellCalcium 1999; 26: 25–36.

117. Chami M, Gozuacik D, Lagorce D et al. SERCA1 truncatedproteins unable to pump calcium reduce the endoplasmicreticulum calcium concentration and induce apoptosis. JCell Biol 2001; 153: 1301–1313.

118. Waldron RT, Short AD, Meadows JJ, Ghosh TK, Gill DL.Endoplasmic reticulum calcium pump expression andcontrol of cell growth. J Biol Chem 1994; 269: 11927–11933.

119. Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs JT. The roleof calcium, pH, and cell proliferation in the programmed(apoptotic) death of androgen-independent prostatic cancerinduced by thapsigargin. Cancer Res 1994; 54: 6167–6175.

120. Cheng G, Liu BF, Yu Y, Diglio C, Kuo TH. The exit from G0into the cell cycle requires and is controlled bysarco(endo)plasmic reticulum Ca2+ pump. Arch BiochemBiophys 1996; 329: 65–72.

121. Gill DL, Waldron RT, Rys-Sikora KE et al. Calcium poolscalcium entry and cell growth. Biosci Rep 1996; 16:139–157.

122. Legrand G, Humez S, Slomianny C et al. Ca2+ pools andcell growth: evidence for sarcoendoplasmic Ca2+-ATPases2B involvement in human prostate cancer cell growthcontrol. J Biol Chem 2001; 276: 47608–47614.

123. Charlesworth A, Rozengurt E. Thapsigargin anddi-tert-butylhydroquinone induce synergistic stimulationof DNA synthesis with phorbol ester and bombesin in Swiss3T3 cells. J Biol Chem 1994; 269: 32528–32535.

124. Lam M, Dubyak G, Chen L, Nunez G, Miesfeld RL,Distelhorst CW. Evidence that Bcl-2 represses apoptosis byregulating endoplasmic reticulum-associated calciumfluxes. Proc Natl Acad Sci USA 1994; 91: 6569–6573.

125. Vanden Abeele F, Skryma R, Shuba Y et al. Bcl-2-dependentmodulation of Ca2+ homeostasis and store-operatedchannels in prostate cancer cells. Cancer Cell 2002; 1:169–179.

126. Pinton P, Ferrari D, Rapizzi E, Di Virgilio F, Pozzan T,Rizzuto R. The Ca2+ concentration of the endoplasmicreticulum is a key determinant of ceramide-induced

apoptosis: significance for the molecular mechanism ofBcl-2 action. EMBO J 2001; 11: 2690–2701.

127. Kuo TH, Kim HR, Zhu L, Yu Y, Lin HM, Tsang W.Modulation of the endoplasmic reticulum calcium pumpby Bcl-2. Oncogene 1998; 17: 1903–1910.

128. Heizmann CW, Fritz G, Schäfer BW. S100 proteins:structure, functions and pathology. Frontiers Biosci 2002; 7:d1356–d1368.

129. Most P, Bernotat J, Ehlermann P et al. S100A1: a regulatorof myocardial contractility. Proc Natl Acad Sci USA 2001;98: 13889–13894.

130. Kiewitz R. Developmental expression and functionalcharacterization of the Ca2+-binding protein S100A1 in theheart. PhD thesis, University of Zurich, 2000.

131. Algenstaedt P, Antonetti DA, Yaffe MB, Kahn CR. Insulinreceptor substrate proteins create a link between thetyrosine phosphorylation cascade and the Ca2+-ATPases inmuscle and heart. J Biol Chem 1997; 272: 23696–23702.

132. Xu GG, Gao ZY, Borge Jr PD, Wolf BA. Insulin receptorsubstrate 1-induced inhibition of endoplasmic reticulumCa2+ uptake in cells. Autocrine regulation of intracellularCa2+ homeostasis and insulin secretion. J Biol Chem 1999;274: 18067–18074.

133. Xu GG, Gao ZY, Borge Jr PD, Jegier PA, Young RA, Wolf BA.Insulin regulation of beta-cell function involves a feedbackloop on SERCA gene expression Ca2+ homeostasis andinsulin expression and secretion. Biochemistry 2000; 39:14912–14919.

134. Burk SE, Lytton J, MacLennan DH, Shull GE. cDNA cloning,functional expression and mRNA tissue distribution of athird organellar Ca2+ pump. J Biol Chem 1989; 264:18561–18568.

135. Wuytack F, Dode L, Baba-Aıssa F, Raeymaekers L. TheSERCA3-type of organellar Ca2+ pumps. Biosci Rep 1995;15: 299–306.

136. Poch E, Leach S, Snape S, Cacic T, MacLennan DH, Lytton J.Functional characterization of alternatively spliced humanSERCA3 transcripts. Am J Physiol 1998; 275: C1449–C1458.

137. Martin V, Bredoux R, Corvazier E et al. Three novelsarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 3isoforms: expression regulation and function of themembers of the family. J Biol Chem 2002; 277:24442–24452.

138. MacLennan DH, Toyofuku T, Lytton J. Structure–functionrelationships in sarcoplasmic or endoplasmic reticulumtype Ca2+ pumps. Ann NY Acad Sci 1992; 671: 1–10.

139. Arredouani A, Guiot Y, Jonas J-C et al. SERCA3 ablationdoes not impair insulin secretion but suggests distinct rolesof different sarcoendoplasmic reticulum Ca2+ pumps forCa2+ homeostasis in pancreatic -cells. Diabetes 2002; 51:3245–3253.

140. Dode L, Wuytack F, Kools PFJ et al. cDNA cloning,expression and chromosomal localization of the humansarco/endoplasmic reticulum Ca-ATPase 3 gene. Biochem J1996; 318: 689–699.

141. Martin V, Bredoux R, Corvazier E, Papp B, Enouf J. PlateletCa2+-ATPases: a plural, species specific and multiplehypertension-regulated expression system. Hypertension2000; 35: 91–102.

142. Machuca I, Domenget C, Jurdic P. Identification of aviansarcoplasmic reticulum Ca2+-ATPase (SERCA3) as a novel1,25(OH)2D3 target gene in the monocytic lineage. Exp CellRes 1999; 250: 364–375.

143. Anger M, Samuel JL, Marotte F, Wuytack F, Rappaport L,Lompré AM. The sarco(endo)plasmic reticulum



Ca2+-ATPase mRNA isoform SERCA3 is expressed inendothelial and epithelial cells in various organs. FEBS Lett1993; 334: 45–48.

144. Wu KD, Lee WS, Wey J, Bungard D, Lytton J. Localizationand quantification of endoplasmic reticulum Ca2+-ATPaseisoform transcripts. Am J Physiol 1995; 269: C775–C784.

145. Lee MG, Xu X, Zeng W. Polarized expression of Ca2+pumps in pancreatic and salivary glands. Role in initiationand propagation of [Ca2+]i waves. J Biol Chem 1997; 272:15771–15776.

146. Gélébart P, Kovács T, Brouland JP et al. Expression ofendomembrane calcium pumps in colon and gastric cancercells. Induction of SERCA3 expression duringdifferentiation. J Biol Chem 2002; 277: 26310–26320.

147. Váradi A, Molnár E, Ostenson CG, Ashcroft SJH. Isoforms ofendoplasmic reticulum Ca2+-ATPase are differentiallyexpressed in normal and diabetic islets of Langerhans.Biochem J 1996; 319: 521–527.

148. Mountian I, Manolopoulos VG, De Smedt H, Parys JB,Missiaen L, Wuytack F. Expression patterns ofsarco/endoplasmic reticulum Ca2+-ATPase and inositol1,4,5-trisphosphate receptor isoforms in vascularendothelial cells. Cell Calcium 1999; 25: 371–380.

149. Wuytack F, Papp B, Verboomen H et al. Asarco/endoplasmic reticulum Ca2+ ATPase 3-type Ca2+pump is expressed in platelets, in lymphoid cells, and inmast cells. J Biol Chem 1994; 269: 1410–1416.

150. Baba-Aıssa F, Raeymaekers L, Wuytack F et al. Purkinjeneurons express the SERCA3 isoform of the organellar typeCa2+-transport ATPase. Mol Brain Res 1996; 41: 169–174.

151. Liu LH, Paul RJ, Sutliff RL et al. Defectiveendothelium-dependent relaxation of vascular smoothmuscle and endothelial cell Ca2+ signalling in mice lackingsarco(endo)plasmic reticulum Ca2+-ATPase isoform 3. J BiolChem 1997; 272: 30538–30545.

152. Kao J, Fortner CN, Liu LH, Shull GE, Paul RJ. Ablation of theSERCA3 gene alters epithelium-dependent relaxation inmouse tracheal smooth muscle. Am J Physiol 1999; 277:L264–L270.

153. Launay S, Gianni M, Kovács T et al. Lineage-specificmodulation of calcium pump expression during myeloiddifferentiation. Blood 1999; 93: 4395–4405.

154. Mountian I, Baba-Aıssa F, Jonas JC, De Smedt H, Wuytack F,Parys JB. Expression of Ca2+ transport genes in plateletsand endothelial cells in hypertension. Hypertension 2001;37: 135–141.

155. Hadri L, Ozog A, Soncin F, Lompré A-M. Basal transcriptionof the mouse sarco(endo)plasmic reticulum Ca2+-ATPasetype 3 gene in endothelial cells is controlled by Ets-1 andSp1. J Biol Chem 2002; 277: 36471–36478.

156. Korczak B, Zarain-Herzberg A, Brandl CJ, Ingles CJ, GreenNM, MacLennan DH. Structure of the rabbit fast-twitchskeletal muscle Ca2+-ATPase gene. J Biol Chem 1988; 263:4813–4819.

157. Zarain-Herzberg A, MacLennan DH, Periasamy M.Characterization of the rabbit cardiac sarco(endo)plasmicreticulum Ca2+-ATPase gene. J Biol Chem 1990; 265:4670–4677.

158. Moriscot AS, Sayen MR, Hartong R, Wu P, Dillmann WH.Transcription of the rat sarcoplasmic reticulum Ca2+adenosine triphosphatase gene is increased by3,5,3′-triiodothyronine receptor isoform-specificinteractions with the myocyte-specific enhancer factor-2.Endocrinology 1997; 138: 26–32.

159. Simonides WS, Brent GA, Thyelen MH, van der Linden CG,Larsen PR, van Hardeveld C. Characterization of thepromoter of the rat sarcoplasmic endoplasmic reticulumCa2+-ATPase 1 gene and analysis of thyroid hormoneresponsiveness. J Biol Chem 1996; 271: 32048–32056.

160. Tengholm A, Hellman B, Gylfe E. Glucose regulation of freeCa2+ in the endoplasmic reticulum of mouse pancreaticbeta cells. J Biol Chem 1999; 274: 36883–36890.

161. Levy J, Zhu ZX, Dunbar JC. The effect of glucose andcalcium on Ca2+-adenosine triphosphatase in pancreaticislets isolated from a normal and a non-insulin-dependentdiabetes mellitus rat model. Metabolism 1998; 47: 185–189.

162. Roe MW, Philipson LH, Frangakis CJ et al. Defectiveglucose-dependent endoplasmic reticulum Ca2+sequestration in diabetic mouse islets of Langerhans. J BiolChem 1994; 269: 18279–18282.

163. Jonas JC, Sharma A, Hasenkamp W et al. Chronichyperglycemia triggers loss of pancreatic celldifferentiation in an animal model of diabetes. J Biol Chem1999; 274: 14112–14121.

164. Varadi A, Lebel L, Hashim Y, Mehta Z, Ashcroft SJ, Turner R.Sequence variants of the sarco(endo)plasmic reticulumCa2+-transport ATPase 3 gene in caucasian type II diabeticpatients (UK prospective diabetes study 48). Diabetologia1999; 42: 1240–1243.

165. Varadi A, Rutter GA. Dynamic imaging of endoplasmicreticulum Ca2+ concentration in insulin-secreting MIN6cells using recombinant targeted cameleons. Roles ofsarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 andryanodine receptors. Diabetes 2002; 51 (Suppl 1):S190–S201.

166. Cronin SR, Rao RJ, Hampton RY. Cod1p/Spf1p is a P-typeATPase involved in ER function and Ca2+ homeostasis.J Cell Biol 2002; 157: 1017–1028.

167. Okorokova Façanha AL, Appelgren H, Tabish M, OkorokovL, Ekwall K. The endoplasmic reticulum cation P-typeATPase Cta4p is required for control of cell shape andmicrotubule dynamics. J Cell Biol 2002; 157: 1029–1039.

168. Rudolph HK, Antebi A, Fink GR et al. The yeast secretorypathway is perturbed by mutations in PMR1, a member ofa Ca2+ ATPase family. Cell 1989; 58: 133–145.

169. Gunteski-Hamblin AM, Clarke DM, Shull GE. Molecularcloning and tissue distribution of alternatively splicedmRNAs encoding possible mammalian homologues of theyeast secretory pathway calcium pump. Biochemistry 1992;31: 7600–7608.

170. Van Baelen K, Vanoevelen J, Missiaen L, Raeymaekers L,Wuytack F. The Golgi PMR1 P-type ATPase ofCaenorhabditis elegans. Identification of the gene anddemonstration of calcium and manganese transport. J BiolChem 2001; 276: 10683–10691.

171. Hu Z, Bonifas JM, Beech J et al. Mutations in ATP2C1,encoding a calcium pump, cause Hailey–Hailey disease.Nat Genet 2000; 24: 61–65.

172. Sudbrak R, Brown J, Dobson-Stone C et al. Hailey–Haileydisease is caused by mutations in ATP2C1 encoding anovel Ca2+ pump. Hum Mol Genet 2000; 9: 1131–1140.

173. Antebi A, Fink GR. The yeast Ca2+-ATPase homologue,PMR1, is required for normal Golgi function and localizesin a novel Golgi-like distribution. Mol Biol Cell 1992; 3:633–654.

174. Durr G, Strayle J, Plemper R et al. The medial-Golgi ionpump Pmr1 supplies the yeast secretory pathway withCa2+ and Mn2+ required for glycosylation, sorting, and



endoplasmic reticulum-associated protein degradation. MolBiol Cell 1998; 9: 1149–1162.

175. Lapinskas PJ, Cunningham KW, Liu XF, Fink GR, CulottaVC. Mutations in PMR1 suppress oxidative damage in yeastcells lacking superoxide dismutase. Mol Cell Biol 1995; 15:1382–1388.

176. Wei Y, Chen J, Rosas G, Tompkins DA, Holt PA, Rao R.Phenotypic screening of mutations in Pmr1, the yeastsecretory pathway Ca2+/Mn2+-ATPase, reveals residuescritical for ion selectivity and transport. J Biol Chem 2000;275: 23927–23932.

177. Mandal D, Woolf TB, Rao R. Manganese selectivity of Pmr1,the yeast secretory pathway ion pump, is defined byresidue Gln783 in transmembrane segment 6. ResidueAsp778 is essential for cation transport. J Biol Chem 2000;275: 23933–23938.

178. Wei Y, Marchi V, Wang R, Rao R. An N-terminal EFhand-like motif modulates ion transport by Pmr1, the yeastGolgi Ca2+/Mn2+-ATPase. Biochemistry 1999; 38:14534–14541.

179. Kunst F, Ogasawara N, Moszer I et al. The completegenome sequence of the Gram-positive bacterium Bacillussubtilis. Nature 1997; 390: 249–256.

180. Bao Q, Tian Y, Li W et al. A complete sequence of T.tengcongensis genome. Genome Res 2002; 12: 689–700.

181. Raeymaekers L, Wuytack EY, Willems I, Michiels CW,Wuytack F. Expression of a P-type Ca2+-transport ATPase inBacillus subtilis during sporulation. Cell Calcium 2002; 32:93–103.

182. Marchi V, Sorin A, Wei Y, Rao R. Induction of vacuolarCa2+-ATPase and H+/Ca2+ exchange activity in yeastmutants lacking Pmr1 the Golgi Ca2+-ATPase. FEBS Lett1999; 454: 181–186.

183. Sorin A, Rosas G, Rao R. PMR1, a Ca2+-ATPase in yeastGolgi, has properties distinct from sarco/endoplasmicreticulum and plasma membrane calcium pumps. J BiolChem 1997; 272: 9895–9901.

184. Ton VK, Mandal D, Vahadji C, Rao R. Functional expressionin yeast of the human secretory pathway Ca2+,Mn2+-ATPase defective in Hailey–Hailey disease. J BiolChem 2002; 277: 6422–6427.

185. Chiesi M, Inesi G. Adenosine 5′-triphosphate dependentfluxes of manganese and hydrogen ions in sarcoplasmicreticulum vesicles. Biochemistry 1980; 19: 2912–2918.

186. Sagara Y, Inesi G. Inhibition of the sarcoplasmic reticulumCa2+ transport ATPase by thapsigargin at subnanomolarconcentrations. J Biol Chem 1991; 266: 13503–13506.

187. Missiaen L, Vanoevelen J, Parys JB et al. Ca2+ uptake andrelease properties of a thapsigargin-insensitivenonmitochondrial Ca2+ store in A7r5 and 16HBE14o-cells.J Biol Chem 2002; 277: 6898–6902.

188. Missiaen L, Vanoevelen J, Van Acker K et al. Ca2+ signals inPmr1-GFP expressing COS-1 cells with functionalendoplasmic reticulum. Biochem Biophys Res Commun2002; 294: 249–253.

189. Mitchell KJ, Pinton P, Varadi A et al. Dense core secretoryvesicles revealed as a dynamic Ca2+ store inneuroendocrine cells with a vesicle-associated membraneprotein aequorin chimaera. J Cell Biol 2001; 155: 41–51.

190. Rojas P, Surroca A, Orellana A, Wolff D. Kineticcharacterization of calcium uptake by the rat liver Golgiapparatus. Cell Biol Int 2000; 24: 229–233.

191. Taylor RS, Jones SM, Dahl RH, Nordeen MH, Howell KE.Characterization of the Golgi complex cleared of proteins

in transit and examination of calcium uptake activities. MolBiol Cell 1997; 8: 1911–1931.

192. Pinton P, Pozzan T, Rizzuto R. The Golgi apparatus is aninositol 1,4,5-trisphosphate-sensitive Ca2+ store, withfunctional properties distinct from those of theendoplasmic reticulum. EMBO J 1998; 17: 5298–5308.

193. Lin P, Yao Y, Hofmeister R, Tsien RY, Farquhar MG.Overexpression of CALNUC (nucleobindin) increasesagonist and thapsigargin releasable Ca2+ storage in theGolgi. J Cell Biol 1999; 145: 279–289.

194. Reinhardt TA, Horst RL. Ca2+-ATPases and their expressionin the mammary gland of pregnant and lactating rats. Am JPhysiol 1999; 276: C796–C802.

195. Strayle J, Pozzan T, Rudolph HK. Steady-state free Ca2+ inthe yeast endoplasmic reticulum reaches only 10 M andis mainly controlled by the secretory pathway pump Pmr1.EMBO J 1999; 18: 4733–4743.

196. Halachmi D, Eilam Y. Elevated cytosolic free Ca2+concentrations and massive Ca2+ accumulation withinvacuoles, in yeast mutant lacking PMR1, a homolog ofCa2+-ATPase. FEBS Lett 1996; 392: 194–200.

197. Missiaen L, Van Acker K, Parys JB et al. Baseline cytosolicCa2+ oscillations derived from a non-endoplasmicreticulum Ca2+ store. J Biol Chem 2001; 276: 39161–39170.

198. Carnell L, Moore HP. Transport via the regulated secretorypathway in semi-intact PC12 cells: role of intra-cisternalcalcium and pH in the transport and sorting ofsecretogranin II. J Cell Biol 1994; 127: 693–705.

199. Chanat E, Huttner WB. Milieu-induced, selectiveaggregation of regulated secretory proteins in thetrans-Golgi network. J Cell Biol 1991; 115: 1505–1519.

200. Oda K. Calcium depletion blocks proteolytic cleavages ofplasma protein precursors which occur at the Golgi and/ortrans-Golgi network. Possible involvement ofCa2+-dependent Golgi endoproteases. J Biol Chem 1992;267: 17465–17471.

201. Lin P, Le-Niculescu H, Hofmeister R et al. The mammaliancalcium-binding protein, nucleobindin (CALNUC), is aGolgi resident protein. J Cell Biol 1998; 141: 1515–1527.

202. Scherer PE, Lederkremer GZ, Williams S, Fogliano M,Baldini G, Lodish HF. Cab45, a novel Ca2+-bindingprotein localized to the Golgi lumen. J Cell Biol 1996; 133:257–268.

203. Vorum H, Hager H, Christensen BM, Nielsen S, Honore B.Human calumenin localizes to the secretory pathway and issecreted to the medium. Exp Cell Res 1999; 248: 473–481.

204. Brunati AM, Contri A, Muenchbach M, James P, Marin O,Pinna LA. GRP94 (endoplasmin) co-purifies with and isphosphorylated by Golgi apparatus casein kinase. FEBS Lett2000; 471: 151–155.

205. Kaufman RJ, Swaroop M, Murtha-Riel P. Depletion ofmanganese within the secretory pathway inhibits O-linkedglycosylation in mammalian cells. Biochemistry 1994; 33:9813–9819.

206. Varki A. Factors controlling the glycosylation potential ofthe Golgi apparatus. Trends Cell Biol 1998; 8: 34–40.

207. West DW, Clegg RA. Casein kinase activity in rat mammarygland Golgi vesicles. Demonstration of latency andrequirement for a transmembrane ATP carrier. Biochem J1984; 219: 181–187.

208. Lasa M, Marin O, Pinna LA. Rat liver Golgi apparatuscontains a protein kinase similar to the casein kinase oflactating mammary gland. Eur J Biochem 1997; 243:719–725.



209. Chandra S, Kable EP, Morrison GH, Webb WW. Calciumsequestration in the Golgi apparatus of culturedmammaliancells revealed by laser scanning confocal microscopy andion microscopy. J Cell Sci 1991; 100: 747–752.

210. Hoffer AM, Schlue WR, Curci S, Machen TE. Spatialdistribution and quantitation of free luminal [Ca] withinthe InsP3-sensitive internal store of individual BHK-21cells: ion dependence of InsP3-induced Ca release andreloading. FASEB J 1995; 9: 788–798.

211. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, TsienRY. Reducing the environmental sensitivity of yellowfluorescent protein. Mechanism and applications. J BiolChem 2001; 276: 29188–29194.

212. Ikeda S, Shigihara T, Mayuzumi N, Yu X, Ogawa H.Mutations of ATP2C1 in Japanese patients withHailey–Hailey disease: intrafamilial and interfamilial

phenotype variations and lack of correlation with mutationpatterns. J Invest Dermatol 2001; 117: 1654–1656.

213. Dobson-Stone C, Fairclough R, Dunne E et al.Hailey–Hailey disease: molecular and clinicalcharacterization of novel mutations in the ATP2C1 gene.J Invest Dermatol 2002; 118: 338–343.

214. Yokota K, Yasukawa K, Shimizu H. Analysis of ATP2C1gene mutation in 10 unrelated Japanese families withHailey–Hailey disease. J Invest Dermatol 2002; 118:550–551.

215. Chao SC, Tsai YM, Yang MH. Mutation analysis of ATP2C1gene in Taiwanese patients with Hailey–Hailey disease.Br J Dermatol 2002; 146: 595–600.

216. Suhrbier A, Garrod D. An investigation of the molecularcomponents of desmosomes in epithelial cells of fivevertebrates. J Cell Sci 1986; 81: 223–242.


Molecular physiology of the SERCA and SPCA pumps

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