Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum...

Deficiency of ATP2C1, a Golgi Ion Pump, Induces SecretoryPathway Defects in Endoplasmic Reticulum (ER)-associatedDegradation and Sensitivity to ER Stress*

Jose Ramos-Castañeda‡,§, Young-nam Park‡, Ming Liu‡, Karin Hauser¶, Hans Rudolph¶,Gary E. Shull||, Marcel F. Jonkman**, Kazutoshi Mori‡‡, Shigaku Ikeda§§, HideokiOgawa§§, and Peter Arvan‡,¶¶

‡ Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann ArborMichigan 48109 § Centro de Investigaciones sobre Enfermedades Infecciosas, Cuernavaca Morelos 62508,Mexico ¶ Institute of Biochemistry, University of Stuttgart, Stuttgart, D-70569, Germany || Department ofMolecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine,Cincinnati, Ohio 45267 ** Department of Dermatology, Groningen University Hospital, 9700 RB Groningen,The Netherlands ‡‡ Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto,606-8304, Japan §§ Department of Dermatology, Juntendo University School of Medicine, Tokyo 113-8421,Japan

AbstractRelatively few clues have been uncovered to elucidate the cell biological role(s) of mammalianATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitouslyexpressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarilyaffects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting indefects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) aswell as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereasdegradation of a nonglycosylated misfolded secretory protein substrate appears unaffected.Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins,nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ERstress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. Thesedata point to the important contributions of the Golgi-localized ATP2C1 protein in homeostaticmaintenance throughout the secretory pathway.

The secretory pathway calcium pool is multifunctional, regulating activities within theendoplasmic reticulum, Golgi complex, and secretory vesicles, including secretory proteinfolding and molecular chaperone function, post-translational modifications, and intracellulartransport and secretion (1) as well as intracellular signaling and cell viability during ER1 stress(2–8). Two P-type ATPases have been identified in yeast that contribute to secretory pathway

*This work was supported in part by National Institutes of Health Grants DK40344 and DK48280 (to P. A.) and a research grant fromthe AlphaOne Foundation (to P. A.), as well as a mentor-based postdoctoral award from the American Diabetes Association (to P. A.).¶¶ To whom correspondence should be addressed: Division of Metabolism, Endocrinology, and Diabetes, 5560 MSRB2, University ofMichigan, 1500 E. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-936-5505; Fax: 718-936-6684; E-mail: [email protected] abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; siRNA, small interfering RNA; NRK,normal rat kidney; PBS, phosphate-buffered saline; mAb, monoclonal antibody; HA, hemagglutinin; PDI, protein disulfide isomerase;endo H, endoglycosidase H; UPR, unfolded protein response; DAPI, 4′,6-diamidino-2-phenylindole; BiP, binding protein; SERCA, sarco/endoplasmic reticulum calcium ATPase.

NIH Public AccessAuthor ManuscriptJ Biol Chem. Author manuscript; available in PMC 2008 September 1.

Published in final edited form as:J Biol Chem. 2005 March 11; 280(10): 9467–9473. doi:10.1074/jbc.M413243200.

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divalent cation homeostasis (9,10), one of them being Pmr1p, which pumps Ca2+ and Mn2+

from the cytosol into the lumen of the secretory pathway (11–15). In addition to members ofthe ER-localized, thapsigargin-sensitive SERCA pumps encoded by the ATP2A family ofgenes, mammalian cells express the thapsigargin-insensitive ATP2C1 gene product (16–18).The latter protein shares a high level of identity with Pmr1p pumps of yeast and Caenorhabditiselegans that operate at the Golgi complex (12,17,19,20).

In yeast, absence of PMR1 results in a strain that grows well in standard media but exhibitsdefective growth in the presence of calcium chelators (14) or during various imposed stressconditions (9,10,17). Carbohydrate processing of N-linked glycoproteins is also abnormal inpmr1 mutants (10–12,14). In addition, although Pmr1p is localized to the Golgi, pmr1 wasidentified as der5 in a genetic screen for mutants defective in ER-associated degradation(ERAD) (21), thereby stabilizing the misfolded CPY* glycoprotein (14).

In humans, loss of one functional ATP2C1 allele results in Hailey-Hailey disease (22,23).Affected individuals achieve normal development, fertility, and a normal life span but developred blistering skin erosions attributable to defective cell adhesion of keratinocytes in theepidermis. Because the ATP2C1 gene product is thought to be ubiquitously expressed, it isunknown why the disease phenotype should be limited to keratinocytes. Using small interferingRNA (siRNA), knockdown of ATP2C1 in mammalian cells has been reported to inhibitcalcium accumulation in the Golgi complex (24) and also, significantly, in the ER (25).However, ATP2C1-related secretory pathway phenotypes have remained largely unexploredin mammalian cells.

In this report, we examine endogenous ATP2C1 Ca2+ pump expression byimmunofluorescence, the detection of which is markedly increased in cells incubated in lowCa2+ media. Using siRNA-mediated knockdown of ATP2C1, we show that such cells exhibitdefects in glycan processing of wild-type thyroglobulin (a secretory glycoprotein), ERAD ofmutant thyroglobulin, and hypersensitivity to ER stress. These findings underscore animportant role of ATP2C1 in secretory pathway homeostasis.

EXPERIMENTAL PROCEDURESMaterials

MG115 was from Calbiochem; brefeldin A and cycloheximide were from Sigma. Anti-thyroglobulin antibodies have been described elsewhere (26).

Cells293, COS-7, NRK, FRT, and HaCat cells (kindly provided by Dr. G. Bokoch, Scripps Institute,La Jolla, CA) and primary human fibroblasts were grown in Dulbecco’s modified Eagle’smedium (25 mM glucose) supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 units/ml-100 μg/ml) except in Fig. 2 in which NRK cells and primaryfibroblasts were cultured under serum-free conditions. Primary human keratinocytes werecultured in EpiLife supplemented with “human keratinocyte growth supplement” (cells andmedia from Cascade Biologics) at a calcium concentration of 0.09 mM. In preparation forexperiments Ca2+ was supplemented to 1.25 mM in selected samples for 48 h before fixationand preparation for immunofluorescence.

Transfection of 293 and FRT cells at ~80% confluence was performed in individual wells ofa 6-well plate using 2 μg of 3×HA (hemagglutinin)-rATP2C1 plasmid (below) and 4 μl ofLipofectamine (Invitrogen) in Opti-MEM for 4 h, and then the cells were supplemented with10% fetal bovine serum for 12 h, after which it was replaced with fresh growth medium.Individual clones were selected with 800 μg/ml G418 and maintained with 400 μg/ml G418.

Ramos-Castañeda et al.

J Biol Chem. Author manuscript; available in PMC 2008 September 1.

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Preparation of Anti-ATP2C1 AntibodyA cDNA (derived from Gen-Bank™ accession number NM131907) encoding residues 369 to475 of the rat ATP2C1 protein was subcloned into pGEX-3X, encoding a glu-tathione S-transferase-fusion protein. (More than 40% of the residues in this sequence differ from that inthe distinct rATP2C2 gene product (GenBank™ accession number NP604457)). Afterisopropyl 1-thio-β-D-galactopyranoside induction, bacterial lysates were purified onglutathione-Sepharose. Purified fusion protein was used as antigen, and three rabbit antisera(160, 161, and 304) were obtained. Of these, antiserum 161 yielded a better signal in identifyingan ATP2C1-specific band in transfected COS-7 cells. Immunoglobulins from 161 werepurified on protein A-agarose. Despite purification, antibody 161 could not reliably detect astrong signal of endogenous ATP2C1 by Western blotting, although it yielded a strong signalby immunofluorescence.

ImmunofluorescenceIn 24-well plates, cells seeded at 104 cells/well on 0.1% poly-D-lysine-coated coverslips weregrown for ~2 days. All subsequent steps were performed at room temperature. The cells werefixed with 3.7% formaldehyde in PBS, permeabilized in 0.1% Nonidet P-40, and blocked with10% goat serum containing 0.1% Nonidet P-40 in PBS. Coverslips were then incubated withrabbit anti-ATP2C1 antibody or mAb anti-HA (Covance), mAb anti-PDI (ABR), mAb anti-EEA1 (ABR), mAb anti-GM130 (BD Biosciences), or mAb anti-Golgi mannosidase II (fromDr. B. Burke, University of Florida, Gainesville, FL). After washing, coverslips were incubatedwith Cy3- and Cy2-conjugated secondary antibodies (Jackson ImmunoResearch) and mounted(in ProLong, Molecular Probes). Epifluorescence images were captured with a CCD camera.

3×HA Tagging the rATP2C1 cDNATo create a start codon and N-terminal 3×HA tag, a synthetic oligonucleotide primer wasdesigned to include a HindIII site immediately preceding codon 8 of rATP2C1 (GenBank™

accession number NM131907), and a reverse primer including an EcoRI site was designed tobegin at nucleotide 894 of the rATP2C1 sequence. The PCR product includes within it a uniqueClaI site of rATP2C1. A 2449-bp ClaI fragment of the pMT SPCA vector was subcloned viathe ClaI site to generate 3×HA-rATP2C1 in pCDNA3.1, encoding a 953-residue gene productin which 41 residues including the 3×HA tag replace the first 7 residues of rATP2C1. The finalproduct was confirmed by DNA sequencing.

ATP2C1 Complementation in pmr1 YeastA pmr1 yeast strain bearing disruption of PMR1 by eliminating a central XbaI-MstI fragmentwas employed (strain YR1234, W303 strain background, ade2–1; can1–100; his3–11,15;leu2–3,112; trp1–1; ura3–1; pmr1::LEU2). This strain was then transformed either withPMR1 on a URA3-marked centromeric plasmid or the URA3-marked empty pYES2 vector(bearing the GAL1 promoter) or pYES2 bearing the 3×HA-rATP2C1 insert. Transformantswere tested for growth after 3 days at 30 °C on plates containing complete medium minus uracilwith 2% galactose plus 2% ethanol and 10 mM EGTA. All strains in the study grew well onthe same plates in the absence of calcium chelator.

ATP2C1 KnockdownA double-stranded RNA oligonucleotide target sequence for rATP2C1 was designed followingthe AA(N)19 rule (27) and chosen from rATP2C1 positions 384–406: AAC CAT TAT GGAAGA AGT ACA TT. This sequence differs at four positions (bold letters) from that found inhuman ATP2C1: AGC CAC TGT GGA AGA AGT ATA TT. The double-strandedoligonucleotide (siRNA) for rATP2C1 was synthesized with and without a fluoresceinisothiocyanate tag on the coding strand (Dharmacon). As a control, we also synthesized the



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scrambled double-stranded oligonucleotide CAC TAT GTG AGA AGA TAA ACA TT.Transfection of the siRNA (0.6 mM oligonucleotide) employed Oligofectamine (Invitrogen)diluted in Opti-MEM. Cells at 50–80% confluence were incubated with this mixture for 6 h at37 °C and then supplemented with 10% fetal bovine serum for 15 h. Cells were then washedand returned to the complete growth medium. By direct fluorescence of fluoresceinisothiocyanate-tagged oligoribonucleotides, a very high fraction of cells (>70%) incorporatedthe siRNA in these cell types (not shown). Thus, we did not routinely use fluorescence-activated cell sorting to separate cells bearing or lacking the siRNA.

Pulse-Chase and Western BlottingFor pulse-chase experiments, control and siRNA-treated cells were preincubated for 30 minin Met/Cys-deficient Dulbecco’s modified Eagle’s medium at 37 °C. Labeling for 30 or 60min employed Expre35S35S (PerkinElmer Life Sciences). At various chase times, cells werelysed in 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 2 mM EDTA, and 10 mM Tris-HCl, pH7.4, plus complete protease inhibitor mixture (Roche Applied Science). Lysates were pre-cleared with 30 μl of zysorbin (Zymed Laboratories Inc.), and then primary antibody was addedto the supernatant for overnight incubation at 4 °C and immunoprecipitation. When necessary,immunoprecipitates were diluted into endoglycosidase H denaturing buffer (New EnglandBiolabs) and heated to 100 °C for 1 min. Zysorbin was then pelleted and the supernatant splitin two equal portions to be either digested with 1,000 units of recombinant endo H (NewEngland Biolabs) or mock-digested with buffer for 1 h at 37 °C. Finally, with or without endoH digestion, immunoprecipitates were solubilized in gel sample buffer and boiled before SDS-PAGE and fluorography or phosphorimaging. For mutant insulin, cells were lysed,immunoprecipitated, and analyzed as described previously (28).

For Western blotting, cells lysates included complete protease inhibitor mixture (RocheApplied Science) and phosphatase inhibitor mixture (Sigma). For detection of 3×HA-rATP2C1, gel samples were not boiled before SDS-PAGE (for all other purposes, sampleswere boiled). After transfer, nitrocellulose was blocked in PBS-0.1% Tween 20 (PBS-T) plus5% nonfat dried milk followed by incubation with primary antibody, including either mAb ratanti-HA (horseradish peroxidase-conjugated, Roche Applied Science), goat anti-glycerol-3-phosphate dehydrogenase (horseradish peroxidase-conjugated, Research Diagnostics, Inc.),chicken anti-calreticulin (Affinity BioReagents), rabbit polyclonal anti-BiP (26), mAb anti-PDI (Stressgen), mAb anti-eIF2α (BIOSOURCE International), rabbit anti-eIF2α-(Ser51)-phosphate (BIOSOURCE International), or rabbit anti-ATF6α (29). The nitrocellulose waswashed and when necessary incubated with secondary antibody (anti-mouse IgG, anti-rabbitIgG, or anti-chicken IgY; all horse-radish peroxidase conjugates from JacksonImmunoResearch) with bands detected by chemiluminescence.

ER Stress Induction and Crystal Violet StainingIn 6-well plates, FRT cells without or with ATP2C1 knockdown were exposed to differentconcentrations of tunicamycin (Calbiochem) for 48 h, washed three times in PBS, and stainedwith 0.2% (w/v) crystal violet (Sigma) in 2% ethanol.

Reverse Transcription-PCR for XBP-1Briefly, after incubation with tunicamycin, 293s22 cells were washed twice with PBS, and totalRNA was isolated with RNeasy (Qiagen). Forward 5′-CCT TGT AGT TGA GAA CCA GG-3′and reverse 5′-GGG GCT TGG TAT ATA TGT GG-3′ (GenBank™ accession numbersAB076384 and AB076383) primers were used with 5 μl of cellular RNA suspension for reversetranscription-PCR using the one-step reverse transcription-PCR (Invitrogen) kit. Reversetranscription was carried out at 50 °C for 30 min, and PCR was performed in 35 cycles, using



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57 °C in the annealing step. After PCR, the synthesized products were resolved by 4% agarosegel electrophoresis and stained with ethidium bromide.

RESULTSLocalization and Expression of the Endogenous ATP2C1 Protein

A rabbit polyclonal antiserum was prepared against a glutathione S-transferase fusion proteinencoding residues 369–475 in a large cytoplasmic loop of the polytopic membrane proteinencoded by ATP2C1 (Fig. 1, inset) (16). Upon characterization of the antibody we found itmost suitable for immunofluorescence studies, revealing a Golgi localization (i.e. selectivelyco-localizing with Golgi mannosidase II and redistributing to a pattern similar to that of ER-localized protein disulfide isomerase in brefeldin A-treated cells) as has been reportedpreviously (17,20,30,31). It was therefore of interest to examine endogenous expression ofATP2C1 in keratinocytes relative to other cells, as patients with ATP2C1 haploinsufficiencyexhibit a keratinocyte cell adhesion defect (32). Cells maintained in serum-free medium (as isstandard for keratinocyte cultures) were examined after a 2-day exposure to calcium added toa level of 1.25 or 0.09 mM (the latter also standard for primary keratinocyte cultures). Primaryhuman keratinocytes had very bright immunofluorescent expression of the ATP2C1 geneproduct in comparison with other cells, and addition of 1.25 mM Ca2+ to the culture mediummarkedly decreased this (Fig. 2). A low-calcium, serum-free culture environment is morechallenging for growth of other cell types; nevertheless, similar behavior was also observed inprimary human fibroblast cultures and NRK cells under these conditions. HaCat keratinocytecell cultures grow in the presence of 10% fetal bovine serum (free Ca2+ ~0.09 mM), and inthese too, the addition of 1.25 mM calcium provoked loss of immunofluorescent ATP2C1expression (whereas Golgi GM130 expression persisted). Together these results indicate anespecially robust expression of the ATP2C1 gene product in nonmutant keratinocytes as wellas a clear relationship between calcium availability and immunofluorescent ATP2C1expression.

Expression of Epitope-tagged Rat ATP2C1To follow more sensitively the ATP2C1 gene product by Western blotting orimmunoprecipitation, we constructed a rat ATP2C1 cDNA that encoded an N-terminal 3×HAepitope tag (i.e. 3×HA-rATP2C1). To establish that the tagged construct is functional, a yeastGAL1 promoter was subcloned behind the 3×HA-rATP2C1 cDNA, which was transformedinto pmr1 yeast. When transformed only with the empty high copy vector (pYES2), pmr1 cellsare unable to grow on an EGTA plate; this phenotype is complemented by expressing PMR1on a plasmid as demonstrated previously (14). Moreover, in multiple independenttransformants in which 3×HA-rATP2C1 was expressed from the pYES2 vector, pmr1 cellgrowth on an EGTA plate was rescued (Fig. 3); similar rescue was observed by rATP2C1without the HA epitope tag (not shown). Although not quantitatively assessing enzymefunction, these crude complementation experiments clearly established that 3×HA-rATP2C1encodes a bioactive protein. When the 3×HA-rATP2C1 construct was subcloned into amammalian expression vector, the anti-HA immunofluorescence was confined to ajuxtanuclear compartment, which co-localized with the endogenous ATP2C1 gene product intransiently transfected 293 cells (Fig. 4, upper panels) as well as in stably transfected 293s22cells in which anti-HA immunofluorescence co-localized with Golgi mannosidase II andGM130 (Fig. 4, lower panels). Thus, the epitope-tagged rATP2C1 construct both functionsand is localized normally in mammalian cells.

Knockdown of Rat ATP2C1 and Human ATP2C1 ExpressionFor knockdown of ATP2C1 protein expression, we selected a synthetic 21-meroligoribonucleotide duplex (corresponding to nucleotides 385–408 of the rat ATP2C1 coding



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sequence). We proceeded to test the siRNA duplex in either rat-derived FRT cells, human-derived 293 cells, or the 293s22 clone that stably expresses 3×HA-rATP2C1. The siRNA wasnot expected to have an impact in the 293 cells (27) because the human mRNA in this regiondiffers at four positions from that found in rodents (see “Experimental Procedures”).Nevertheless, because of the 100% identity that exists along many stretches shared betweenthe rat and human mRNA sequences (overall 97% identity), we were curious as to thepossibility that 293s22 cells made to express rATP2C1 would be rendered susceptible toknockdown of total ATP2C1 (endogenous hATP2C1 as well as rATP2C1 after siRNAtreatment (33)).

Five days after siRNA treatment of FRT cells, we observed a dramatic loss ofimmunofluorescence expression of the rATP2C1 gene product (Fig. 5, upper left panels). Byimmunoblotting with anti-HA in FRT or 293s22 cells, we also observed profound knockdownof rATP2C1 at 2 days, 5 days, or 8 days post-siRNA treatment (Fig. 5, upper right). Control293 cells that had not been transfected to express rATP2C1 were not susceptible to knockdownof endogenous hATP2C1, which showed a normal distribution in the Golgi complex (Fig. 5,lower left panel). However in 293s22 cells, although GM130 remained properly localized (Fig.5, lower middle) as did Golgi mannosidase II (not shown), anti-HA immunofluorescence wasrendered virtually undetectable (lower right), and the endogenous hATP2C1 gene productcould no longer be detected as well (lower middle and right). Although the extent to whichhATP2C1-specific mRNA fragments might be generated by dicer activity upon treatment of293s22 cells with rATP2C1-specific siRNA is unknown, the utility of the above finding is thatthe persistence of hATP2C1 expression in control 293 cells provides an outstanding controlfor specificity of siRNA effects in 293 cells. (Independent controls comprised of nooligoribonucleotide, single-stranded oligoribonucleotide, or scrambled duplexoligoribonucleotide sequence all worked successfully as controls as well.)

Effects of ATP2C1 on Post-translational Modifications Including Acquisition ofEndoglycosidase H Resistance and Sorting in the Golgi Complex

In addition to pumping Ca2+, ATP2C1 pumps Mn2+ (17,19), which is thought to allow foroptimal activity of a variety of enzymes involved in the post-translational processing of glycanson glycoproteins (34–36). To check for phenotypes related to these activities, FRT-thyroglobulin cells (37) treated or untreated with ATP2C1 siRNA were pulse-labeledwith 35S-amino acids and chased for either 15 min or 5 h. Acquisition of endoglycosidase Hresistance for ~80% of the many N-linked glycans on each thyroglobulin molecule occurs uponthe arrival of thyroglobulin at the medial Golgi complex (38); normally, this is accompaniedby a slowing of thyroglobulin band mobility upon SDS-PAGE. However, upon knockdown ofATP2C1, acquisition of endo H resistance for newly synthesized thyroglobulin was notaccompanied by the same degree of slowing of thyroglobulin band mobility, indicatingalterations in the fidelity of secretory pathway post-translational glycan processing (Fig. 6A).(As an aside, thyroglobulin acquisition of endo H resistance occurred a little more quickly insiRNA-treated cells; however, these effects were subtle, and the ultimate extent ofthyroglobulin export did not differ.)

Effects of ATP2C1 on ERAD, ER Stress Response, and ER Stress-induced Cell ToxicityWe have previously shown that the cog thyroglobulin mutation encodes a mutant glycoproteinthat serves as a substrate for ERAD via the ubiquitin-proteasome system (26). To examine therole of ATP2C1 in this process, FRT or 293s22 cells expressing the cog thyroglobulin cDNAwere transfected with the ATP2C1 siRNA. In mock-transfected FRT cells, labeled cogthyroglobulin present in the cells 1 h after synthesis (Fig. 6B, lane 1) had completelydisappeared at 24 h (Fig. 6B, lane 3), whereas in siRNA-treated FRT cells, labeled cogthyroglobulin persisted even after 1 day of chase (Fig. 6B, lane 4). In control 293 cells bearing



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only endogenous hATP2C1, treatment with the rat-specific siRNA had no effect on thecomplete degradation of cog thyroglobulin (Fig. 6B, lanes 9–12). However, in 293s22 cells asin the rat-derived FRT cells, knockdown of ATP2C1 dramatically stabilized the cogthyroglobulin mutant, persisting even after 1 day of chase (Fig. 6B, lane 8). Quantitativerecovery data from this representative experiment (Fig. 6B, bar graph at right) were replicatedin three independent experiments, and no mutant thyroglobulin was detectably secreted fromany of the samples (not shown).

For comparison, we examined intracellular degradation of a mutant single-chain insulin, whichlacks glycans and has been shown to be completely defective for secretion because of theabsence of one of three evolutionarily conserved disulfide bonds (28). Although modestslowing of ERAD could be observed by the addition of the proteasome inhibitor MG115,ATP2C1 knockdown afforded no protection from ERAD for the nonglycosylated substrate(Fig. 6C). These data indicate that ATP2C1 knockdown does not inhibit the general ERADmachinery but has specificity to distinct ERAD substrates.

In some instances, ERAD defects have been found to be associated with the induction of ERchaperone synthesis as part of the unfolded protein response (UPR) (39–42). We comparedBiP levels in 293 cells treated with the rat-specific siRNA (a negative control) to 293s22 cellswith or without knockdown of ATP2C1. As shown in Fig. 7A, knockdown of ATP2C1expression did not induce an increase in BiP and also had no effect on intracellular levels ofcalreticulin or PDI. We considered the possibility that biosynthesis of BiP might indeed beinduced but might fail to be properly retained in the ER and become secreted, leaving steady-state levels unaffected (43). However as shown in Fig. 7B, BiP synthesis was unaltered byknockdown of ATP2C1, and BiP was efficiently retained within cells over a 5-h period. Thesefindings exclude the possibility that knockdown of ATP2C1 itself significantly activates theER stress pathways leading to ER chaperone induction.

Deletion of the PMR1 gene renders yeast cells hypersensitive to ER stress. In FRT cells exposedfor 16 h to various low doses of tunicamycin (Fig. 7C), cells transfected with the controloligoribonucleotide remained in culture and could be stained with crystal violet (wells at left).By contrast, as the tunicamycin dose was increased up to 1 μg/ml, almost no cells withknockdown of ATP2C1 remained (Fig. 7C, wells at right). After challenge with 100 nMthapsigargin, hypersensitivity was again observed selectively in cells with insufficiency ofATP2C1 (not shown). Thus, expression of ATP2C1 appears to facilitate survival in the faceof ER stress.

Because failure to activate ER stress pathways (most notably PERK (44)) can result in cellulartoxicity (45), we considered the possibility that ATP2C1 deficiency might impair UPRactivation. We examined that question using either low dose or high dose tunicamycintreatment protocols (Fig. 8). We could dismiss the notion that ATP2C1 knockdown preventseither proteolytic cleavage and disappearance of full-length ATF6 or PERK-mediatedphosphorylation of the eIF2α subunit of the eIF2B translational initiation factor because theseactivation events were clearly detected after tunicamycin treatment (Fig. 8A). We did find itmore difficult to detect the active cleaved ATF6 transcription factor in cells treated with theATP2C1 siRNA, but this was not a consistent finding (Fig. 8B, upper panel). Additionally,ATP2C1 knockdown did not prevent Ire1-mediated splicing of the XBP-1 mRNA that wasseen after high-dose tunicamycin treatment (Fig. 8B, lower panel). Thus, increased toxicityfrom ER stress in cells with insufficiency of ATP2C1 cannot be explained by an inability toactivate the ER stress response pathways.



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DISCUSSIONATP2C1 encodes a polytopic ion pump that resides in the Golgi complex of all eukaryoticcells. Despite the fact that the Hailey-Hailey disease phenotype is restricted to the skin ofheterozygous individuals, no higher eukaryotic models have been identified to date in whichboth functional alleles of ATP2C1 are lacking, suggesting the possibility that homozygotesmay suffer from a more general phenotype, which may be lethal. With this in mind, it isinteresting to note that most of the cell biological role(s) of the ATP2C1 gene product in themammalian secretory pathway have yet to emerge.

In this report, immunofluorescent expression of the ATP2C1 gene product is most apparentwhen extracellular calcium availability is limiting (Fig. 2). Teleologically, given that theATP2C1 encoded protein becomes less detectable when extra-cellular calcium is increased, itseems unlikely that ATP2C1 subserves a primary purpose in the sequestration/lowering ofCa2+ from cytosolic pools (which would instead predict more ATP2C1 protein when moreCa2+ enters the cytosol from the extracellular environment) but rather in uphill concentration/accumulation of Ca2+ (and potentially other divalent ions (17,19)) within the lumen of thesecretory pathway. Thus, the present data strongly suggest that as keratinocytes migrate tomore peripheral skin, farther from the vascular and interstitial supply of extracellular calcium,their differentiation program is likely to include strong ATP2C1 expression that facilitatesproper delivery of cellular adhesive proteins via the secretory pathway (32). This may makekeratinocytes especially vulnerable to secretory pathway dysfunction in states of ATP2C1haploinsufficiency. Additionally, our data (Fig. 2) suggest that similar ion-dependentregulation of the abundance of the ATP2C1 gene product occurs in many cell types, suggestinga general homeostatic role for ATP2C1 in the secretory pathway of all cells.

To explore this role, we have engineered an siRNA that is effective in ATP2C1 knockdown inrat cells but not human cells (despite the fact that the gene products are 97% identical),providing a valuable specificity control, and we have engineered a cDNA encoding epitope-tagged rat ATP2C1 for expression in human-derived (293) cells. The construct clearly encodesa functional rATP2C1 protein (Fig. 3), which co-localizes with endogenous ATP2C1 and Golgimarkers (Fig. 4). The siRNA treatment results in knockdown of ATP2C1 in rat-derived (FRT)cells but not 293 cells. Interestingly, however, expression of rat ATP2C1 in human-derived293s22 cells renders these cells susceptible to knockdown of the endogenous gene product intrans upon treatment with the rat-specific siRNA (Fig. 5). This produces secretory pathwaydefects, including differences in N-linked glycosylation of newly synthesized thyroglobulin(Fig. 6A). Furthermore, despite the fact that the ATP2C1 gene product is localized to the Golgicomplex, this pump has impact on regulation within the ER. Specifically, after ATP2C1knockdown, we note a major defect in the ERAD of misfolded thyroglobulin encoded by thecog mutant (Fig. 6B) that occurs without secretion of cog thyroglobulin. Indeed, as mentionedin the Introduction, a mutant of the yeast homolog, pmr1, is synonymous with der5, beingdefective in the degradation of the ER misfolded CPY* glycoprotein. The severity of the ERADdefect after ATP2C1 knockdown may be related to the presence of N-glycans, as it is not at alldetectable for a mutant form of insulin, which is nonglycosylated (Fig. 6C).

The effect of the Golgi-localized pump encoded by ATP2C1 on glycoprotein ERAD may entailseveral possibilities that are not mutually exclusive. First, ER-Golgi traffic in both anterogradeand retrograde directions may facilitate the ERAD of misfolded secretory glycoproteins (46–49), possibly giving substrates at least transient exposure to the Golgi ionic environment.Second, the ATP2C1 gene product might pump ions directly into the ER either when it is newlysynthesized or if the pump should cycle between the Golgi and ER compartments. Third,divalent cations pumped into the Golgi lumen might be delivered by retrograde trafficking tothe lumen of the ER. Certainly, yeast cells lack the SERCA class of pumps and nevertheless



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have ER luminal calcium that is likely to be maintained by the Golgi-localized PMR1 geneproduct. This supports the recent conclusion that ATP2C1, the mammalian homolog of PMR1,is at least a contributor to ER luminal calcium levels (25). Nevertheless, ATP2C1 knockdownis distinct from thapsigargin treatment (which disables SERCA pumps). Our characterizationof ATP2C1 knockdown shows that it leads neither to marked UPR activation (Fig. 7A), nordoes it block the ability of cells to activate UPR pathways in response to other ER stresses (Fig.8). At the same time, the ATP2C1 gene product is important for cell survival after ER stress.This was demonstrated both in FRT cells (Fig. 7C) and in 293s22 cells (by increased annexinV staining, not shown) indicating a general finding that is also reported in yeast cells lackingPMR1 (14). Although proximal steps in ER stress signaling occur in ATP2C1-deficient cells,more work is needed to identify which if any downstream targets of UPR activation may beeither hyporesponsive or hyperresponsive in ATP2C1 deficiency, thereby predisposing themto cellular toxicity.

One function of secretory pathway calcium accumulation is to provide an adequate ionicenvironment for ER luminal chaperones that are known calcium-binding proteins (5). ERchaperones such as BiP require calcium binding for their full function (50,51) and are necessaryfor their anti-apoptotic activity (2,52). Thus, based on the studies presented here, we proposethat the ion pump activity of the ATP2C1 gene product in mammalian cells, like that of thePMR1 gene product in yeast cells (14), can account for its role in homeostatic maintenance ofsecretory pathway function, including cellular resistance to ER stress.

AcknowledgementsWe thank M. Scheinfeld for preliminary studies in this project and Dr. A. Chang (University of Michigan, Ann Arbor,MI) for helpful discussions.

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Fig. 1. Immunofluorescence characterization of Ab-161 directed against a large peptide encodedby ATP2C1 in mammalian cellsThe immunofluorescence in NRK cells exhibits no overlap with the distribution of EEA1 (toprow), superficial similarity but limited pixel overlap with fluorescein isothiocyanate-taggedtransferrin (FITC-Trnf, second row), and extensive co-localization with Golgi mannosidase II(Mann-II, third row). Below the line, before a 1-h exposure to 5 μg/ml brefeldin A (+BFA),ATP2C1 is concentrated in a juxtanuclear region that does not overlap with the endoplasmicreticulum marker PDI. After brefeldin A (bottom row), localization of ATP2C1 begins todisperse in the cytoplasm, taking on an endoplasmic reticulum-like pattern (bottom row). DAPIwas used to stain nuclei. Inset at bottom right, a topological map of the ATP2C1 gene productshowing pictorially the sites of antibody recognition of rATP2C1 as well as epitope taggingand siRNA-mediated silencing.



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Fig. 2. Calcium-dependent immunofluorescence of the ATP2C1 gene product in primary cells andcell linesAbove the line, human keratinocytes, NRK cells, and primary human fibroblasts were culturedunder serum-free conditions. Below the line, the human-derived HaCat keratinocyte cell linewas cultured under serum-containing conditions. Cells were cultured at low calcium asdescribed in the text. 1.25 mM Ca2+ was added to the media for 48 h before processing thesamples for immunofluorescence of ATP2C1. In images above the line, DAPI was used tostain nuclei.



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Fig. 3. Functional expression of 3×HA-rATP2C1 in pmr1 null yeastYeast bearing deletion of the PMR1 gene were transformed either with the empty vectorpYES2, the same vector in which the GAL1 promoter drives expression of the mammalian3×HA-rATP2C1, or a centromeric plasmid bearing endogenous yeast PMR1. On a galactose/EGTA-containing plate, pmr1 cells cannot grow, and this growth is rescued either by re-expression of PMR1 or in five independent transformants (22-3,22-6,22-7,22-10,22-14)expressing 3×HA-rATP2C1.



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Fig. 4. Expression of 3×HA-rATP2C1 in mammalian cellsAbove the line, 293 cells were transiently transfected and then processed forimmunofluorescence with anti-HA. DAPI was used to stain nuclei. All cells are positive forendogenous expression of ATP2C1, but only transfected cells are positive for anti-HA, whichyields an identical intracellular distribution to that of the endogenous ATP2C1 gene product.Below the line, a stable G418-resistant clone of 293 cells (293s22) was generated. Theimmunofluorescent distribution with anti-HA matches that of the Golgi markers mannosidaseII (Mann-II) and GM130. The merged panels include DAPI stains of nuclei.



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Fig. 5. Knockdown of ATP2C1Upper left panels, 2 days after rATP2C1 siRNA treatment, rat-derived FRT cells exhibited aprofound loss of immunofluorescent expression of the endogenous gene product. N denotesnuclei. Upper right panels, the FRT(A11) or 293s22 clones, both of which stably express3×HA-rATP2C1, were transfected with rATP2C1 siRNA. At the times indicated, the cellswithout prior fluorescence-activated cell sorting were lysed, and equal aliquots of cellularprotein were analyzed by SDS-PAGE and Western blotting with anti-HA. Below the line, eithercontrol 293 cells or 293s22 cells were transfected with ATP2C1 siRNA. In control 293 cells,the ATP2C1 siRNA was without effect, resulting in continued expression of endogenoushuman ATP2C1 that co-localized with the Golgi marker GM130 (panel at left). In identicallytreated 293s22 cells, GM130 was unchanged, but detection of ATP2C1 with either anti-ATP2C1 or anti-HA was lost. DAPI was used to stain nuclei.



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Fig. 6. Phenotypic effects of ATP2C1 knockdownA, FRT-thyroglobulin (Tg) cells ± ATP2C1 knockdown were pulse-labeled with 35S-aminoacids for 60 min and chased as indicated. After 5 h in control cells without ATP2C1 knockdown,the newly synthesized thyroglobulin shifted to a position of slower mobility by SDS-PAGE,which includes the appearance of an endo H-resistant form. In cells with ATP2C1 knockdown,thyroglobulin did not develop the same degree of slowing of band mobility, indicatingdifferences in thyroglobulin post-translational processing. B, FRT cells, 293 cells, and 293s22cells were transfected or not transfected with ATP2C1 siRNA. After 3 days, 293 and 293s22cells were transfected again to express cog thyroglobulin whereas the FRT cells were alreadya stable clone expressing cog thyroglobulin). 2 days after this transfection, the cells were pulse-labeled with 35S-amino acids and chased either for 1 or 24 h. No cog thyroglobulin was secreted(not shown). In control 293 cells, the newly synthesized cog thyroglobulin was completelydegraded by 24 h (quantified in bar graph at right). After ATP2C1 knockdown, cogthyroglobulin persisted in either FRT cells (open bars at right) or 293s22 cells (closed bars atright). C, 293s22 cells were transfected either with control oligoribonucleotide or siRNA andwere subsequently transfected with a cDNA encoding a single-chain insulin with a 7-aminoacid peptide sequence (-MGGGGGM-) that links the insulin B- and A-chains and additionallycarries the mutations C7S, C44S that abrogate the B7-A7 disulfide bond (28). 2 days after thistransfection, the cells were pulse-labeled with 35S-amino acids and chased in the presence orabsence of 108 μM MG115. No mutant insulin was secreted at any chase time (not shown).MG115 inhibited ERAD, but there was no effect of ATP2C1 knockdown on the rate or extentof ERAD of this nonglycosylated protein substrate.



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Fig. 7. ER chaperone levels, secretion, and sensitivity to ER stress in cells with knockdown ofATP2C1A, 293s22 cells, either expressing ATP2C1 or at 5 days after siRNA treatment, were lysed,normalized for total protein, and immunoblotted with anti-HA (upper row), anti-BiP, anti-calreticulin, anti-PDI, or anti-glycerol-3 phosphate dehydrogenase (G3PDH, used as a loadingcontrol). B, FRT cells ± ATP2C1 knockdown were pulse-labeled with 35S-amino acids andchased for 5 h as in Fig. 8. Both the cell lysates (C) and media (M) were analyzed byimmunoprecipitation with anti-BiP. ATP2C1 knockdown did not increase BiP secretion fromthe cells. C, 293s22 cells were transfected either with single-stranded sense oligoribonucleotideor double-stranded siRNA. Prior to the 5th day post-transfection, cells were exposed overnightto the doses of tunicamycin (Tun) indicated. Cells surviving this treatment were fixed andstained with crystal violet, and the entire plate was scanned to generate the image shown. Cellspretreated with control oligo survived the ER stress, whereas after knockdown of ATP2C1,almost no cells remained after exposure to 1 μg/ml tunicamycin.



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Fig. 8. ER stress response pathways in cells with knockdown of ATP2C1A, 293s22 cells ± ATP2C1 knockdown were treated with 0.5 μg/ml tunicamycin for the timesindicated. Samples were lysed and immunoblotted for ATF6α, eIF2α-Ser51-phosphate, andtotal eIF2α. In cells with ATP2C1 knockdown, loss of preexisting full-length ATF6α wascomparable in kinetics and efficiency. Increased phosphorylation of eIF2α- occurred in bothsets of cells with the same timing, initially appearing in the 5-h sample. unglycos.,unglycosylated. B, 293s22 cells ± ATP2C1 knockdown were treated with 10 μg/ml tunicamycinfor the times indicated. Samples were lysed and analyzed both for loss of preexisting full-lengthATF6α by immunoblotting and for splicing of the XBP-1 mRNA as measured by reversetranscription-PCR.



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Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum...

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