Molecular Biology of the CellVol. 6,15351547, November 1995

Identification of the Sequences in HMG-CoAReductase Required for Karmellae AssemblyMark L. Parrish,* Christian Sengstag,t Jasper D. Rine,* andRobin L. Wright*§

*Department of Zoology, University of Washington, Seattle, Washington 98195; tInstitut furToxikologie, der Eidgenossischen Technischen Hochschule, und der Universitat Zurich, CH-8603Schwerzenbach, Switzerland; and tDivision of Genetics, Department of Molecular and CellularBiology, University of California, Berkeley, California 94720

Submitted June 19, 1995; Accepted August 16, 1995Monitoring Editor: Keith R. Yamamoto

In all eukaryotic cells that have been examined, specific membrane arrays are induced inresponse to increased levels of the ER membrane protein, HMG-CoA reductase. Analysisof these inducible membranes has the potential to reveal basic insights into generalmembrane assembly. Yeast express two HMG-CoA reductase isozymes, and eachisozyme induces a morphologically distinct proliferation of the endoplasmic reticulum.The isozyme encoded by HMG1 induces karmellae, which are long stacks of membranesthat partially enclose the nucleus. In contrast, the isozyme encoded by HMG2 inducesshort stacks of membrane that may be associated with the nucleus, but are frequentlypresent at the cell periphery. To understand the molecular nature of the different cellularresponses to Hmglp and Hmg2p, we mapped the region of Hmglp that is needed forkarmellae assembly. For this analysis, a series of exchange alleles was examined in whicha portion of the Hmg2p membrane domain was replaced with the corresponding Hmglpsequences. Results of this analysis indicated that the ER lumenal loop between predictedtransmembrane domains 6 and 7 was both necessary and sufficient for karmellae assem-bly, when present in the context of an HMG-CoA reductase membrane domain. Immu-noblotting experiments ruled out the simple possibility that differences in the amounts ofthe various chimeric HMG-CoA reductase proteins was responsible for the alteredcellular responses. Our results are consistent with the hypothesis that each yeast isozymeinduces or organizes a qualitatively different organization of ER membrane.

INTRODUCTION

Assembly of specific membranes is an essential pro-cess throughout cell growth and development. Nev-ertheless, the mechanisms by which cells achieve thisspecificity are not understood in even a single case. Auseful approach to unravel these mechanisms focuseson the specific membrane biogenesis induced by asubset of membrane proteins (Chin et al., 1982; VonMeyenburg et al., 1984; Weiner et al., 1984; Elmes et al.,1986; Wright et al., 1988). One of the best characterizedof these proteins is HMG-CoA reductase, an integralER membrane protein that catalyzes the rate-limiting

5 Corresponding author.

step in cholesterol biosynthesis (see Goldstein andBrown, 1990 for recent review). In all organisms thathave been tested, elevations in the level of HMG-CoAreductase lead to the assembly of cell type-specificmembrane arrays (Chin et al., 1982; Anderson et al.,1983; Pathak et al., 1986; Li et al., 1988; Singer et al.,1988; Wright et al., 1988; Andreis et al., 1990; Wright etal., 1990). For example, mammalian cells assemblehexagonal arrays of smooth membrane tubules knownas crystalloid ER (endoplasmic reticulum) (Chin et al.,1982; Anderson et al., 1983; Pathak et al., 1986). Incontrast, yeast cells assemble stacked membrane ar-rays (Wright et al., 1988; Koning and Wright, unpub-lished data).

i 1995 by The American Society for Cell Biology 1535

M.L. Parrish et al.

In animals and fungi, HMG-CoA reductase is boundto membranes via a complex membrane domain thatspans the bilayer at least seven times (Liscum et al.,1985; Olender and Simoni, 1992; Roitelman et al.,1992). Although this domain has no role in the cata-lytic activity of HMG-CoA reductase, it is essential forboth sterol-induced degradation of HMG-CoA reduc-tase (Chin et al., 1985; Gil et al., 1985; Skalnik et al.,1988) and for induction of membrane biogenesis(Jingami et al., 1987; this study). One model to accountfor HMG-CoA reductase-induced membrane biogen-esis is that the membrane-bound domain of the pro-tein delivers a signal for production of specific mem-brane arrays. Cells respond to this signal by producingthe necessary proteins and lipids that are assembledwith HMG-CoA reductase into the membrane array.The nature of the putative signal is unknown, but mayrequire specific sequences in the membrane domain.Consistent with this model, the specific organization

of the membranes formed in response to HMG-CoAreductase is cell-type specific (Wright et al., 1990). Forexample, although the amino acid sequences of themembrane domains of mammalian and yeast HMG-CoA reductase have no sequence homology, each iscapable of inducing membranes in the other cell type.However, the membranes formed are determined bythe cell type in which the membranes are assembled.Consequently, expression of mammalian HMG-CoAreductase in yeast leads to karmellae, rather than crys-talloid ER formation. In contrast, expression of yeastHMG-CoA reductase in mammalian cells leads tocrystalloid ER, rather than karmellae assembly. Theseresults suggest that cell-specific factors are involved inorganization of the amplified membranes.Unlike mammalian cells that have only a single

HMG-CoA reductase gene, yeast carry a pair of genes,HMG1 and HMG2, that each encodes a functionalHMG-CoA reductase isozyme (Basson et al., 1986).HMG1 and HMG2 encode proteins (referred to asHmglp and Hmg2p) with nearly identical catalyticdomains and either gene can support cell viability(Basson et al., 1986). Less homology is present in themembrane domain sequence, although the secondarystructures of the membrane domains are similar, if notidentical (Basson et al., 1986; Sengstag et al., 1990).Interestingly, each isozyme induces a morphologicallydistinct organization of stacked membranes (Wright etal., 1988; Koning and Wright, unpublished observa-tions). Cells that express 10-fold elevated levels ofHmglp assemble karmellae, which are long stacks ofmembranes that partially encircle the nucleus. In con-trast, cells that express 10-fold elevated levels ofHmg2p assemble short stacks of membranes that canbe near the nucleus, but are frequently at the cellperiphery. Thus, although they are functionally inter-changeable and structurally similar, each yeast HMG-

CoA reductase isozyme produces a different andreadily observable cellular response.As a first step in understanding the mechanics and

regulation of specialized membrane biogenesis, thedifferent cellular responses to Hmglp and Hmg2pwere exploited to map the karmellae-inducing se-quences in Hmglp. Specifically, we examined cellsexpressing chimeric proteins that replaced portions ofthe Hmglp membrane domain with the correspond-ing Hmg2p sequences. As expected from similar stud-ies in mammalian cells, the activity of HMG-CoA re-ductase was not required for stimulation of membraneproliferation. In addition, results of this analysis dem-onstrated that a region of the Hmglp predicted to liein the ER lumen was both necessary and sufficient toinduce karmellae assembly. Quantitative differencesin amounts of the chimeric proteins could not accountfor the altered membrane assembly. Thus, these re-sults raise the possibility that the signal for karmellaeassembly may operate through protein-protein inter-actions that occur in the ER lumen.

MATERIALS AND METHODS

Strains and MediaThe yeast strains used in this study are listed in Table 1. Strainscontaining plasmids were grown at 30'C on modified minimalmedium (0.67% yeast nitrogen base without amino acids, 2% glu-cose, 2% casamino acids) supplemented with the appropriate aminoacids or nucleotide bases (Sherman et al., 1986). Strains not contain-ing plasmids were grown on YPD (2% yeast extract, 2% Bacto-peptone, and 2% glucose) (Sherman et al., 1986). Solid mediumcontained 2% agar. Membrane morphology was assayed on culturesat OD6. of 0.5 or less (early logarithmic phase)

Molecular Genetic ManipulationsConstruction of the genes that encode Hmgl:Hmg2 fusion proteinsis described by Sengstag et al. (1990). In brief, the fusion proteinsconsist of the membrane domain of HMG-CoA reductase (amino-terminal 525 amino acids), followed by the first 307 amino acids ofmature invertase, and terminated with the carboxyl-terminal 767amino acids of histidinol dehydrogenase. To produce the differentmembers of this fusion protein series, sequences encoding increas-ing portions of the Hmglp membrane domain were systematicallyreplaced with the corresponding Hmg2p sequences. The junctionsbetween Hmglp and Hmg2p sequences in all of the fusions occur atthe carboxyl terminus of a predicted transmembrane domain. Theresulting series of fusion proteins have progressively increasingproportions of the Hmg2p membrane domain, such that the Hmg2psequences are present at the amino terminus and terminate at apredicted transmembrane domain. In this report, these proteins arereferred to as Hmgl(1_7):Suc2:His4p, Hmg2(1):Hmgl(2-7):Suc2:His4p,and so forth. The numbers in parentheses after "Hmgl" and"Hmg2" refer to the transmembrane domains from the particularisozyme that are present in the fusion protein. Thus, Hmg2(1):Hmgl(2-7):Suc2:His4p contains the first transmembrane domainfrom Hmg2p and the second through seventh transmembrane do-mains from Hmglp (see Table 1).To exchange Hmglp and Hmg2p "Loop G" sequences (the region

between transmembrane domains 6 and 7), site-directed mutagen-esis was used to introduce in-frame SailI and XhoI sites flanking theLoop G-encoding sequences (Altered Sites, Promega, Madison, WI).For Hmglp, an XhoI site was placed at the 5' end and a SaIlI site was

Molecular Biology of the Cell1536

Signal for Karmellae Biogenesis

placed at the 3' end of the Loop G sequence. For Hmg2p, a Sall sitewas placed at the 5' end and an XhoI site was placed at the 3' endof the Loop G sequences. The regions encoding Loop G were ex-changed in two steps, taking advantage of the complementarity ofXhoI and SalI restriction fragments. To fuse the altered Hmglpmembrane domain together with carboxyl terminal Suc2:His4p se-quences, the 2.4-kb EcoRI fragment containing the promoter andmembrane domain coding sequences of Hmgl(Hmg2 Loop G) wasblunted, using the Klenow fragment of DNA polymerase, and li-gated into the SmaI site of pCS3, producing pRW377. This plasmidwas transformed into JRY396, yielding RWY591, which producedHmgl(Hmg2 Loop G):Suc2:His4p. To fuse the altered Hmg2p membranedomain with the Hmg2p carboxyl sequences, the 3-kb SacI-BamHIfragment containing the promoter and membrane domain codingsequences of Hmg2(Hmgl Loop G) was ligated into a modified pJR360in which the BamHI site in the polylinker was destroyed by filling inwith the Klenow fragment of DNA polymerase and religating. Thisplasmid, pMP375, was transformed into JRY396 producingRWY589, which expresses Hmg2(Hmgl Loop G)

Light MicroscopyEarly log-phase cells (OD6. = 0.3-0.5) were stained with 10 Ag/mlDiOC6 (Kodak, Rochester, NY) per 107 cells and the cells wereexamined using fluorescein filter combinations (Excitation 480 ± 20

nm, Barrier 535 ± 40 nm) on a Nikon Microphot-FXA fluorescencemicroscope (Koning et al., 1993). Photographs of DiOC6-stained cellswere taken using Polaroid type 57 film (3000 ASA). Alternately, thestained cells were observed with laser scanning confocal micros-copy using a Bio-Rad MRC6000 laser scanning confocal microscope(488-nm excitation wavelength and BHS emission filter; Richmond,CA).The immunofluorescence procedure was modified from methods

described by Pringle et al. (1989). Specifically, 3-5 ml of an earlylog-phase culture (0.3-0.5 OD600 per ml) was fixed for 2 h in 3.7%formaldehyde. Cells were washed twice by centrifugation and re-suspension in 1.2 M sorbitol, 100 mM potassium phosphate, pH 7.5.For partial removal of the cell wall, the washed cell pellet wasresuspended in 0.5 ml of this buffer to which had been added 1 j,lof ,3-mercaptoethanol, 10 j.l glusulase (Sigma Chemicals, St. Louis,MO), and 50 ,ul of 1 mg/ml zymolyase 20T (ICN, Irvine, CA). Afterincubation at 37°C for 15 min, the cells were washed twice bycentrifugation and resuspension in Tris-buffered saline (TBS). A15-,ul aliquot of the washed cell suspension was applied to each wellof a multiwell slide (Cell-line Associates, Newfield, NJ), which hadpreviously been treated for 15 min with 1% aqueous polyethyleni-mine, then rinsed in tap water, and air dried. The cells were allowedto settle and adhere during a 15-min incubation in a moist chamber;then the nonadherent cells were removed by aspiration and the

Table 1. Yeast strains used in this study

Strain number Description Notes

JRY396- MATa leu2-3 leu2-112 host strain(alias RSY198) prcl::LEU2 suc2D9 ura3-52 invertase and carboxypeptidase Y deficientJRY282a JRY396 + pA produces Hmgl:Suc2:His4 fusion

(multicopy YEp352 vector)JRY283a JRY396 + pX1,6 produces Hmg2(1):Hmgl(2-7): Suc2:His4p fusion

(multicopy YEp352 vector)JRY284a JRY396 + pX2,5 produces Hmg2(1 2):Hmgl(3-7): Suc2:His4p fusion

(multicopy YEp352 vector)JRY285a JRY396 + pX3,4 produces Hmg2(j 3):Hmgl(4-7): Suc2:His4p fusion

(multicopy YEp352 vector)JRY286a JRY396 + pX4,3 produces Hmg2(14):Hmgl(5-7): Suc2:His4p fusion

(multicopy YEp352 vector)JRY287a JRY396 + pX5,2 produces Hmg2(j 5):Hmg1(6-7): Suc2:His4p fusion

(multicopy YEp352 vector)JRY288a JRY396 + pX6,1 produces Hmg2(1 6):Hmgl(7): Suc2:His4p fusion

(multicopy YEp352 vector)JRY289a JRY396 + pX7,0 produces Hmg2:Suc2:His4p fusion

(multicopy YEp352 vector)JRY290- JRY396 + pAD7 produces Hmgl(1 6):Suc2:His4p fusion

(multicopy YEp352 vector)RWY249b JRY396 + YEpHmg2 expresses native Hmg2p

(multicopy YEp352 vector)RWY605 JRY396 + pJR360 expresses native Hmg2pthis work (multicopy pSEY 8 vector)RWY589 JRY396 + pMP375 expresses Hmg2(Hmgl "Loop G") proteinthis work (multicopy pSEY 8 vector)RWY591 JRY396 + pRW377 expresses Hmgl(Hmg2 "Loop G-) Suc2:His4p fusionthis work (multicopy pSEY 8 vector) proteinJRY384a JRY396 + YEp352 control strain

(multicopy vector) contains vector onlyJRY383b JRY396 + pJR59 expresses native Hmglp

(multicopy YEp24 vector)

a Sengstag, et al., 1990.bRine, et al., 1983.c Basson, et al., 1988.

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M.L. Parrish et al.

wells were washed three to five times with 30-,ul droplets of TBS. A10-,u droplet of blocking solution, TBSTO (2% ovalbumin, 0.05%Tween-20 in TBS), was applied and allowed to incubate at roomtemperature for 15 min. An equal volume of antisera diluted inTBSTO was added and incubated for 1-2 h. Following washing (fivetimes by aspiration and application of 30-j,l droplets of TBS), fluo-rescein-labeled goat anti-rabbit antiserum (Cappel/Organon Tech-nica, Durham, NC) diluted 1:2000 in TBSTO was added and allowedto bind for 1 h at room temperature. The wells were washed asbefore and then a 20-,lI droplet of 1 ,ug/ml 4,6,-diamidino-2-phe-nylindole in TBS was added. Following incubation for 5 min, thewells were washed twice with TBS, 5 .l of Citifluor mountingsolution (Ted Pella, Redding, CA) was applied to each well, and acoverslip was added and sealed in place with nail polish. A poly-clonal antiserum against a-tubulin, provided by Frank Solomon(Massachusetts Institute of Technology, Cambridge, MA), was usedas a positive control for the staining procedure. Affinity-purifiedanti-invertase antiserum was provided by Randy Schekman (Uni-versity of California, Berkeley, CA). Antiserum against Kar2p was agift from Dr. Jeff Brodsky (University of Pittsburgh, Pittsburgh, PA).The fluorescence intensity observed with the anti-invertase anti-serum was too low for conventional photography. Consequently,the signal was captured and amplified using a silicon-intensifiedvideo camera and analysis system (DAGE-MTI, Michigan City, IN).Hard-copy images from the video display were made using a videoprinter (Seikosha, Mahwah, NJ).

PAGE and ImmunoblottingCrude membrane fractions were prepared from logarithmic phasecultures using modifications of a method described by Deschenesand Broach (1987). Cultures were harvested and resuspended inlysis buffer (0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl2, and 10 mMTris-HCl, pH 7.4) containing 1 ,ug/ml TPCK, leupeptin, pepstatin A,and 1 mM PefaBloc SC (Boehringer Mannheim Biochemica, India-napolis, IN). Acid-washed glass beads were added to the meniscusand the sample was agitated for 1 min in a Mini-Beadbeater (Bio-spec, Bartlesville, OK). The lysate was removed from the beads andcleared by several 4-s spins in a microcentrifuge. (A small amount ofcross-reactive material was present in this pellet). Membranes werepelleted by centrifugation for 20 min at top speed in a microcentri-fuge, resuspended in lysis buffer, and repelleted. The final pelletwas resuspended in 10 ,ul of lysis buffer per 107 cells and dividedinto aliquots before storing at -75°C. For SDS-PAGE, an equalvolume of 2 x sample buffer (8 M urea, 4% SDS, 10% ,B-mercapto-ethanol, 20% glycerol, 0.125 M Tris-HCl, pH 6.8) was added, and thesample was heated for 15 min at 55-60°C. The proteins were sep-arated on 7.5% or 10% polyacrylamide minigels, using a 3% stackinggel that contained 8 M urea. Following electrophoresis, the proteinswere transferred to nitrocellulose in a semi-dry transfer apparatus(Hoefer, San Francisco, CA) operated at 100 mA for 1 h. Afterblocking overnight in TBSTM (2% nonfat dry milk, 0.05% Tween-20in TBS), the blot was incubated for 2 h in anti-invertase antiserumdiluted 1:10,000 in TBSTM or anti-Hmg2p diluted 1:500 in TBSTM(the antisera was produced against the C-terminal 15 amino acids ofHmg2p: QPSNKGPPCKTSALL). Following three washes in TBSTM,the blot was incubated for 1 h in alkaline phosphatase-conjugatedgoat anti-rabbit antiserum (Promega, Madison, WI) diluted 1:7500in TBSTM. The blot was then washed three times in TBSTM, twicein TBST and twice in TBS, and then developed using 10 ,ug/mlnitroblue tetrazolium and 5 ,ug/ml 5-bromo-4-choro-3-indolyl phos-phate in 0.1 M Tris-HCl, pH 9.5, 0.1 mM NaCl, 5 mM MgCl2 assubstrates. Dried gels and immunoblots were digitized using aScan-X Color Scanner (HSD Microcomputer, Mountain View, CA).Relative amounts of total protein loaded per lane as well as relativeamounts of Hmglp or Hmg2p on the immunoblots were deter-mined using the NIH Image 1.54 Analysis software. Serial dilutionsof samples were loaded and analyzed to evaluate whether theimmunoblots were in the linear range of the alkaline phosphatasereaction.

RESULTS

Experimental StrategyTo map the karmellae-inducing region of Hmglp, weexamined cells expressing a series of recombinantforms of HMG-CoA reductase in which increasingamounts of the Hmglp membrane domain were re-placed with the corresponding Hmg2p sequences. Allfusion genes were present on multi-copy plasmidsthat were present in approximately 10 copies per cell.Thus, the increased gene dosage led to concomitantincreases in the amount of the HMG-CoA reductasemembrane domain encoded by the particular plasmidcarried by the cell. The fusion gene containing theentire Hmglp membrane domain was expressed fromthe HMG1 promoter. However, the other fusion genesin this series were expressed from the HMG2 pro-moter. With one exception, the HMG-CoA reductasecarboxyl terminus, which contains the catalytic do-main, was replaced with sequences from Suc2p andHis4p (Sengstag et al., 1990). The Suc2p sequencesserved as an immunological tag and the His4p se-quences encoded histidinol dehydrogenase, an activ-ity that is functional only when in the cytoplasm (De-Shaies and Schekman, 1987). This Suc2p-His4pcarboxyl terminus was useful for in vivo and in vitrotests of yeast HMG-CoA reductase topology (Sengstaget al., 1990).The ability of the fusion proteins to induce karmel-

lae was determined by staining living cells with thelipophilic fluorescent dye DiOC6. Under the condi-tions used in this study, DiOC6 specifically stained thenuclear envelope and endoplasmic reticulum, allow-ing the presence of membrane proliferations to berapidly assessed and quantitated (Koning et al., 1993).Cells that lacked karmellae were distinguished bydim, uniform nuclear envelope staining (Figure 1A).In contrast, cells with karmellae membrane prolifera-tions were characterized by bright, asymmetric nu-clear envelope staining (Figure 1, B-D).

Identification of the Sequences in theHmglp Membrane Domain Required forKarmellae AssemblyCells expressing either intact Hmglp (JRY383) or thefusion protein containing the entire Hmglp mem-brane domain fused to Suc2:His4p (JRY282) possessedkarmellae (summarized in Figure 2). This result con-firmed that, as observed in mammalian cells, the se-quences required for membrane induction werepresent in the membrane domain. Thus, elevations ofHMG-CoA reductase activity were not required forinduction of membrane biogenesis. Rather, the mem-brane-associated region was sufficient for karmellaeinduction. Karmellae were observed in 20% of the cellsexpressing the intact Hmglp protein (Figure 3A) and

Molecular Biology of the Cell1538

Signal for Karmellae Biogenesis

DNV6~-

K

Figure 1. DiOC6-staining rapidly revealed the presence and dis-tribution of karmellae membranes in yeast cell populations. (A)DiOC6 staining patterns of a vector-containing control strain thatdid not assemble karmellae (JRY384). Nuclei that lacked karmellaewere readily distinguished by the uniform intensity of nuclearenvelope staining. Cells with this staining pattern were scored askarmellae minus. However, it is possible that the staining was notsufficiently sensitive to visualize nuclei with only one or two kar-mellae layers. Consequently, DiOC6 staining may underestimate thenumber of cells in a population that possesses karmellae. In addi-tion, karmellae were scored as present in approximately 1% of thecells observed from staining, but not overproducing HMG-CoAreductase. These observations presumably reflect the intrinsic errorrate in scoring. All experiments were conducted in a manner inwhich the person scoring the karmellae frequency was unaware ofstrain identity. (B and C) DiOC6 staining patterns of a strain thatoverproduced Hmglp revealed that karmellae were present in 32%of the population (RY282). Karmellae-containing nuclei had dis-tinctly asymmetric staining of the nuclear envelope. (D) A reverseimage of JRY282 stained with DiOC6. A prominent karmellae-con-taining nucleus is present in the lower right of the image (markedwith the arrowhead and the letter K). The other arrow (with theletter N) points to a nucleus that contained much less prominentkarmellae. Karmellae are not visible in the other four cells. Fluores-cence microscopy, 1200X.

in 32% of cells expressing the Hmgl(1_7):Suc2:His4pfusion protein (Figure 3B). Because karmellae are re-tained within the mother cell at mitosis, karmellae areexpected to be in at most 50% of cells in a log phaseculture. A key issue is how well the DiOC6 stainingreported the presence of karmellae. Based on elec-tron microscopy, karmellae membranes are presentin 35% of log-phase cells expressing intact Hmglp(Wright and Rine, 1989). The frequency of karmellaeobserved by DiOC6 staining in strains expressing

the Hmgl(1_7):Suc2:His4p fusion protein was nearthis expected value of 35%; however, as judged byDiOC6 staining, the frequency of membrane prolif-erations was lower than expected in strains express-ing the intact Hmglp protein. This lower frequencymay reflect limitations of the DiOC6 staining proce-dure. If so, the frequencies reported in Figure 2under-represented the presence of karmellae. Nev-ertheless, karmellae were both more frequently ob-served and more prominent in cells expressing theHmgl(1_7):Suc2:His4p fusion protein than in thoseexpressing the intact Hmglp protein.We examined the complete series of fusion proteins

in which increasing portions of Hmglp were replacedwith the corresponding Hmg2p sequences. All pro-teins that contained at least the last loop and trans-membrane domain of Hmglp (i.e., Loop G and TMD7)were able to induce karmellae formation (Figure 2 andFigure 3, C-H). Thus, karmellae membranes were as-sembled in cells that expressed fusion proteins con-taining this portion of Hmglp, even when the remain-der of the membrane domain consisted of Hmg2psequences (see Figure 4). In contrast, only 3% of thecells expressing the fusion protein that contained theentire Hmg2p membrane domain assembled karmel-lae. Although short stacks of membranes are presentat the cell periphery and near the nucleus of cellsexpressing Hmg2p (Koning and Wright, unpublishedobservations), karmellae are not observed by electronmicroscopy or DiOC6 staining in cells expressing wild-type Hmg2p. Thus, even the small number of karmel-lae observed in the strain expressing Hmg2(1_7):Suc2:His4p strain was unexpected.A straightforward interpretation of these results was

that information needed to generate karmellae wasnot present in the first six transmembrane domains ofHmglp nor in the loops that link these transmem-brane domains. In addition, these results suggestedthat a karmellae-inducing signal was present in thecarboxyl-terminal portion of the Hmglp membranedomain, perhaps extending from Loop G throughtransmembrane domain 7 (Figure 4). Consistent withthis possibility, a fusion that placed the Suc2:His4pdomain in the center of Loop G, thus deleting trans-membrane domain 7 and a large portion of Loop G,did not induce karmellae assembly (Hmgl(1_6):Suc2:His4p, Figure 2).

Differences in the Steady-state Amounts of FusionProteins Did Not Account for Differences inKarnellae-inducing AbilityIn principle, quantitative differences in the amount offusion proteins accumulated by the cells might under-lie at least some of the differences in karmellae bio-genesis. If so, the chimeric proteins that were unable toinduce karmellae might simply have been present at

Vol. 6, November 1995 1539

M.L. Parrish et al.

Karme llae

Hmgl

Hmg2

Hmgl (1 .7):Suc2:His4

Hmg2(1 ):Hmgl (2-7):Suc2:His4

Hmg2(1 2):Hmgl (37):SUC2:His4Hmg2(1 3):Hmgl (4-7)Suc2:His4

Hmg2(1 .4):Hmgl (5-7):Suc2:His4

Hmg2( 15):Hmgl (6-7):Suc2:His4Hmg2(1 6):Hmgl (7):Suc2:His4

Hmg2(1 7):Suc2:His4

Hmgl (1 6):Suc2:His4

Hmgl(Hmg2 "Loop G):Suc2:HiS4Hmg2(Hmgl "Loop G")

1 2 3 4 5 6 7HMG1 calaiytodomrn3

VAM MiA-VI-X,------

VKA VA 18HJV4 ff

Su.c2;His4 .

Suc2:Hls4H s[ M1iVJIVA' i li5{ l>lrsAl suc2 HiS i.

Q Hmg1p Sequences

0 Hmgl p Trans-W membrane Domains

* Hmg2p Sequences

Hmg2p Trans-_ membrane domains

20%

4%

32%

20%

7%

18%

15%

20%

11%

3% Figure 2. Summary of kar-mellae assembly in popula-tions of cells expressing the

0% chimeric Hmglp:Hmg2p pro-teins. The fusion proteinsused in this study are dia-

4% grammed together with thepercentage of karmellae ob-

26% served in yeast strains ex-pressing each protein. Thepercentages reflected a cu-mulative score of at least 800DiOC6-stained cells, scoredfrom at least three indepen-dent experiments.

lower amounts than chimeric proteins that inducedkarmellae. To examine this possibility, the relativelevels of fusion proteins expressed in each strain wereevaluated by immunoblots using antiserum againstthe Suc2 gene product, invertase (Figure 5). Althoughvariations in protein amount were present, these vari-ations did not correlate with the ability of a fusionprotein to induce karmellae. For example, cells ex-pressing Hmg2(1_7):Suc2:His4p contained higher lev-els of fusion protein than Hmg2(1 3):Hmgl(4-7):Suc2:His4p, Hmg2(1_4):Hmgl(-7):Suc2:His4p, or Hmg2(1_5):Hmgl(6-7):Suc2:His4p. However, the cells expressingHmg2(1_7):Suc2:His4p assembled karmellae in only 3%of the cells, whereas the other strains assembled 15-20%. This observation indicated that the ability toinduce karmellae was due to qualitative rather thanquantitative differences.

Perinuclear Localization Was Insufficient toInduce KarmellaeThe ability of a protein to induce karmellae mightdepend upon its specific localization within the cell.If so, proteins that induced karmellae would bepresent in a particular subcellular compartment,whereas proteins that cannot induce karmellae

would be present in a different compartment. Toexamine this possibility, the subcellular localizationof the fusion proteins was determined by immuno-fluorescence (Figure 6). Intact Hmglp protein waspresent in the nuclear envelope, and enriched inkarmellae membranes (Figure 6A). As expected, theHmgl(1_7):Suc2:His4p fusion protein had a similarlocalization pattern as the intact Hmglp protein,indicating that the Hmglp membrane domain wassufficient for localization (Figure 6B). All of theother fusion proteins with chimeric Hmg2:Hmglpmembrane domains were also present in the nuclearenvelope, in many cases displaying an asymmetriclocalization pattern like that of the intact Hmglpprotein (data not shown). For example, the Hmg2(1-7):Suc2:His4p fusion protein was present within thenuclear envelope (Figure 6C). In addition, ratherthan being uniformly present throughout the nu-clear envelope, Hmg2(1_7):Suc2:His4p was asym-metrically localized in the nuclear envelope, pro-ducing a pattern that resembled the localization ofHmglp. Surprisingly, Hmg2p had a different local-ization pattern. Instead of being localized in thenuclear envelope, Hmg2p was present in discretepatches just beneath the plasma membrane (Figure

Molecular Biology of the Cell1540

Signal for Karmellae Biogenesis

Figure 3. Analysis of exchange alleles demonstrated that transmembrane domain 7 and Loop G from Hmglp were necessary for karmellaeassembly. Confocal micrographs of DiOC6-stained cells expressing the following proteins: (A) Hmgl:Suc2:His4p (JRY282), a strain expressinga fusion protein containing the entire HMG1 membrane domain fused to SUC2:HIS4 (karmellae present in 32% of cells); (B) Hmgl(¶):Hmg2(2-7):Suc2:His4p (JRY283), a strain expressing a fusion protein containing the first transmembrane domain of HMG2, and the remainingmembrane domain of HMG1 fused to SUC2:HIS4 (karmellae scored in 20% of cells); (C) Hmgl(1_2):Hmg2(3-7):Suc2:His4p (JRY284), a strainexpressing a fusion protein containing the first two transmembrane domains of HMG2, and the remaining membrane domain ofHMG1 fusedto SUC2:HIS4 (karmellae scored in 7% of cells); (D) Hmgl(1_3):Hmg2(4-7):Suc2:His4p (JRY285), a strain expressing a fusion protein containingthe first three transmembrane domains of HMG2, and the remaining membrane domain of HMG1 fused to SUC2:HIS4 (karmellae scored in18% of cells); (E) Hmgl(1_4):Hmg2(>7):Suc2:His4p (JRY286), a strain expressing a fusion protein containing the first four transmembranedomains of HMG2, and the remaining membrane domain of HMG1 fused to SUC2:HIS4 (karmellae scored in 15% of cells); (F) Hmgl(l]5):Hmg2(6-7):Suc2:His4p (JRY287), a strain expressing a fusion protein containing the first five transmembrane domains of HMG2, and theremaining membrane domain of HMG1 fused to SUC2:HIS4 (karmellae scored in 20% of cells); (G) Hmgl(1_6):Hmg2(7):Suc2:His4p (JRY288),a strain expressing a fusion protein containing the first six transmembrane domains of HMG2, and the remaining membrane domain of HMG1fused to SUC2:HIS4 (karmellae scored in 11% of cells). The karmellae in this strain frequently looped away from the nucleus, as shown inthis cell; (H) Hmg2:Suc2:His4p (JRY289), a strain expressing a fusion protein containing the HMG2 membrane domain fused to SUC2:HIS4(karmellae scored in 3% of cells). Confocal microscopy; Bar, 2 ,um.

6D). Thus, it appeared that sequences in the car-boxyl terminus of Hmg2p were important for local-ization. Regardless of this unexpected result, theHmg2(1_7):Suc2:His4p protein was present in the nu-clear envelope but did not induce karmellae forma-tion. Therefore, localization of the Hmg2p mem-brane domain in the same compartment as Hmglpwas not sufficient to induce karmellae.

The Loop between Transmembrane Domains 6 and 7of Hmglp Was Necessary and Sufficient to InduceKarmellae AssemblyThe results thus far suggested that a karmellae-induc-ing signal was present in the Hmglp membrane do-main region that included Loop G and transmem-brane domain 7. To test whether the signal might belimited to Loop G sequences, we constructed a newfusion in which the Hmglp Loop G was replaced withthe Hmg2p Loop G sequences. As expected, theHmgl(H.g2 Loop G):Suc2:His4p fusion was unable to effi-

ciently induce karmellae (Figures 2 and 7B), indicatingthat the Hmglp Loop G sequences were necessary forkarmellae assembly.To test whether or not the sequences in the Hmglp

Loop G were sufficient for karmellae formation, weconstructed a complementary fusion in which theHmglp Loop G sequences were placed into theHmg2p context. This fusion differed from the othersdescribed in this report in that it contained the nativeHmg2p catalytic domain rather than a Suc2:His4p car-boxyl terminus. The Hmg2p protein with the HmglpLoop G efficiently assembled karmellae, indicatingthat Hmglp Loop G contained sequences that weresufficient for karmellae induction, at least in the gen-eral context of an HMG-CoA reductase membranedomain (Figures 2 and 7D).For both Loop G exchange alleles, immunoblotting

experiments were used to examine the relative steady-state levels of the altered HMG-CoA reductase pro-tein. For this analysis, Hmgl (17):Suc2:His4p levels

Vol. 6, November 1995 1541

M.L. Parrish et al.

Predicted Topology of Yeast HMG-CoA Reductase A

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Figure 4. Diagram of the predicted topology of the karmellae-inducing region of HMG1. Current models for yeast HMG-CoAreductase topology predict seven transmembrane domains. How-ever, experiments that test topology of mammalian HMG-CoA re-ductase are consistent with the presence of eight transmembranedomains (Roitelman et al., 1992). The region of HMG1 that wassufficient for karmellae induction is depicted by the thicker lines.

were compared with Hmgl(Hmg2 Loop G):Suc2:His4pusing anti-invertase antiserum and Hmg2p' levelswere compared with Hmg2(Hmgl Loop G) using anti-serum against the carboxyl terminal 15 amino acids ofHmg2p. In both cases, similar amounts of altered pro-tein were produced relative to controls, ruling out thepossibility that Loop G simply affected protein. quan-tity (Figure 8).

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A Membrane-inducing Signal Was Present in theHmglp Membrane DomainAs a step toward understanding the processes in-volved in membrane biogenesis, we have focused onkarmellae, the specialized membrane array assembledby yeast cells in response to increases in HMG-CoAreductase levels. To identify the sequences of theHmglp protein that were required for karmellae bio-genesis, a series of recombinant proteins was ana-lyzed. These proteins contained chimeric membranedomains in which different amounts of the Hmglpmembrane domain, which induces karmellae, werereplaced with the corresponding region of the Hmg2pmembrane domain, which induces different mem-brane arrays.Our analysis demonstrated that the information re-

quired for karmellae biogenesis was present withinthe Hmglp Loop G sequence (Figure 4). The ability ofthis region to induce karmellae was assayed in thecontext of the normal topology of an HMG-CoA re-ductase membrane domain that provided the best con-trolled environment to test its function. In addition toLoop G, ultrastructural analysis indicated that theloop between the 5th and 6th transmembrane domains

Figure 5. The abilities of different proteins to induce karmellaewas unlikely to be due to quantitative differences in protein amount.(A) Immunoblot of total crude membrane preparations probed withantiserum that recognized the invertase (Suc2p) portion of the fu-sion protein. Note that Hmgl(1.7):Suc2:His4p (lane A) accumulatedthe greatest amount of the fusion protein and also generated kar-mellae in the highest proportion of cells in the population. Incontrast, Hmg2(j.7):Suc2:His4p (lane H), which does not assemblekarmellae, accumulated higher amounts of fusion protein than

Hm2(.3):Hg(4-7) (lane D) or Hmg2(1)4):Hm1p(:Suc2:His4p(lane E), which assembled karmellae in 15--20% of the population.Thus, the inability of Hmg2:Suc2:His4p to induce karmellae did notreflect a decreased accumulation of the protein relative to the kar-mellae-inducing proteins. As a control for gel loading, the blot wasalso probed with antisera against the ER protein Kar2p. (B) Thegraph compares the fusion protein levels, normalized to total pro-tein, and the amount of karmellae induced by each chimeric protein.There was no simple correlation between the amount of fusionprotein and the capacity to induce karmellae, indicating that qual-itative differences in the proteins were important determinants ofkarmellae-inducing ability.

("Loop E") appeared to be important for close associ-ation of the resulting membranes with the nucleus(Wright, unpublished observations). Interestingly,Loop E is predicted to be located in the cytoplasm,whereas Loop G is predicted to lie within the ERlumen (Sengstag et al., 1990).

1542~~~~~~~~~~~~~~~~~~~~~~~~~MolecularBiology of the Cell1542

Signal for Karmellae Biogenesis

Figure 6. The Hmg2:Suc2:His4p fusion protein had an unexpected subcellular localization. (A-D) Immunofluorescence localization ofHMG-CoA reductase or fusion proteins. (A) Hmglp (JRY383), localization of intact HMG1 using affinity-purified antiserum against theHMG1 catalytic domain. HMG1 was present in the nuclear envelope, enriched in the karmellae membrane layers. (B) Hmgl:Suc2:His4p(JRY282), localization of fusion protein using affinity-purified antiserum against invertase (recognizes the Suc2p portion of the fusion protein).This fusion protein had a similar localization pattem as HMG1, asymmetrically localized within the nuclear envelope. (C) Hmg2:Suc2:His4p(JRY289), localization of fusion protein using affinity-purified antiserum against invertase (recognizes the Suc2p portion of the fusion protein).Unexpectedly, this fusion protein was not present in the same location as the intact Hmg2 protein (see panel D). Instead, in many cells, it waspresent in the nuclear envelope. The localization was not uniform, but also asymmetric as observed for Hmglp and the Hmgl:Suc2:His4pfusion proteins. (D) Hmg2p (JRY249), localization of Hmg2p protein using affinity-purified antiserum against the carboxyl-terminal 15 aminoacids of the catalytic domain. Hmg2p was not localized in the nuclear envelope. Instead, it was present in patches near the cell periphery.Although not obvious in this photograph, the brightly stained region was present just beneath the plasma membrane. Cells contained oneor more of these Hmg2p-containing structures. (see also Figure 7C). (E-F) DiOC6 staining of representative cells from the same cultures asthose processed for immunofluorescence. Note that these micrographs do not correspond to the same cells shown in panels A-D. (Currentimmunofluorescence protocols are not compatible with retention of DiOC6 staining pattems, making co-localization impossible.) (E) Hmglp(JRY383). (F) Hmgl:Suc2:His4p (RY282). (G) Hmg2:Suc2:His4p (JRY289). (H) Hmg2p (RWY249). Fluorescence microscopy, 1200X.

Several conclusions concerning HMG-CoA reduc-tase-induced membrane assembly came from thesedata. First, fusion proteins in which the entire catalyticdomain was replaced with heterologous sequencesassembled karmellae. Thus, the membrane domainwas both necessary and sufficient for induction ofmembrane biogenesis and for determining the organi-zation of the resulting membranes. This result ruledout the possibility that membrane biogenesis in re-sponse to HMG-CoA reductase was merely due toincreased sterol biosynthesis. Second, the region re-quired for karmellae biogenesis was present in a spe-cific, contiguous region of the membrane domain.Third, the ability to induce karmellae did not correlatewith either quantitative differences between the fusionproteins nor with differences in their subcellular local-ization. Thus, the inability of Hmg2p to induce kar-mellae was not due to lower relative amounts of theprotein nor to its presence in a membrane that wasincapable of assembling karmellae. Taken together,

our results supported the notion that induction ofkarmellae biogenesis involved interactions betweenthe Hmglp membrane domain Loop G and other cel-lular components.The HMG-CoA reductase membrane domain has

three known functions in mammalian cells. It isrequired for subcellular localization of the enzyme(Skalnik et al., 1988), for control of HMG-CoA re-ductase half-life in response to sterols (Chin et al.,1985; Gil et al., 1985; Jingami et al., 1987), and forinduction of membrane biogenesis in response toincreases in HMG-CoA reductase levels (Chin et al.,1982; Skalnik et al., 1985, 1988; Chun and Simoni,1992). Specific regions of the membrane domainhave been identified that are necessary for regula-tion of half-life in response to sterols (Jingami et al.,1987; Skalnik et al., 1988; Chun and Simoni, 1992). Inaddition, the role of certain membrane regions ininduction of membrane biogenesis has been exam-ined (Jingami et al., 1987). Interestingly, an HMG-

Vol. 6, November 1995 1543

M.L. Parrish et al.

Figure 7. The ER-lumenal Loop G from Hmglp was necessary andsufficient to induce karmellae. (A) Karmellae were present in ap-proximately 30% of DiOC6-stained cells expressing Hmgl:Suc2:His4p. (B) Replacement of the Hmglp Loop G sequences with thecorresponding Hmg2p sequences (in Hmgl:Suc2:His4p) attenuatedkarmellae-inducing ability to 4%. (C) Karmellae were not observedin cells expressing Hmg2p. Instead, a morphologically differentproliferation of membranes was present. Frequently, one or moreshort strips of membrane were located near the plasma membraneof these cells, as shown in this figure. (D) Replacement of theHmg2p Loop G sequences with the corresponding Hmglp se-quences enabled Hmg2p to induce karmellae membranes efficiently.Confocal microscopy, Bar, 2 gm.

CoA reductase allele in which the sequences encod-ing transmembrane domains 4 through 5 weredeleted was capable of inducing membrane prolif-

eration, but the membranes were not organized intothe highly structured crystalloid ER structures in-duced by wild-type mammalian HMG-CoA reduc-tase (Jingami et al., 1987). The picture that is begin-ning to emerge indicates that the membrane domainof HMG-CoA reductase comprises a mosiac of dif-ferent functions that map to discrete regions(Skalnik et al., 1988; Chun et al., 1990; Chun andSimoni, 1992).Recent work has shown the membrane domain

also mediates half-life control of the yeast Hmg2p(Hampton and Rine, 1994). In these studies, thesequences in Hmg2p needed for regulated degrada-tion are present in "Loop B" (Hampton and Rine,1994). In contrast, results presented in this paperdemonstrate that the sequences of Hmglp neededfor karmellae assembly were present in Loop G.Thus, different functions of the membrane domainappear to be mediated by different regions of themembrane domain.The amino acid sequences of the Hmglp and

Hmg2p Loop G are compared in Figure 9. Loop G,which was necessary and sufficient for induction ofkarmellae biogenesis, is one of the membrane do-main regions with the least homology betweenHmglp and Hmg2p (Basson et al., 1988). Of the 76amino acids in this sequence, 33 are identical (43%).However, the homology is not uniformly distrib-uted throughout the sequence. Instead, the differ-ences cluster into certain areas (boxed in Figure 9).Based on Chou and Fasman algorithms performedby the program MacDNASIS (Hitachi Software En-gineering, San Bruno, CA), the predicted secondarystructures of Hmglp and Hmg2p Loop G are alsodifferent (Figure 9). Note the helix and turn struc-tures in the first boxed region of Hmglp versus thepredicted sheet structure for the corresponding re-gion of Hmg2p. In addition, the predicted structure

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Figure 8. Exchanges of Loop G sequences did notalter relative steady-state levels of protein. (A) Im-munoblot of total crude membrane preparationsprobed with antiserum that recognized either theinvertase (Suc2p) portion of the fusion protein (bloton left) or the Hmg2p carboxyl terminus (blot onright). Similar amounts of protein were present inHmgl(1_7):Suc2:His4p and Hmgl(Hmg2 Loop G):Suc2:His4p. However, the level of Hmg2(Hmgl Loop G) wasapproximately 25% less than that of Hmg2p. Never-theless, even though cells expressing Hmg2(HmglLoop G) contained less of the protein, they still assem-bled karmellae efficiently, whereas cells expressinghigher levels of intact Hmg2p did not. The blotswere also probed with antiserum against the ERprotein Kar2p, as a control for gel loading.

Molecular Biology of the Cell1544

Signal for Karmellae Biogenesis

Comparison of Amino Acid Sequences in Loop G ofHMG1 and HMG2 Membrane Domains

Figure 9. Comparison of theamino acid sequences and pre-dicted secondary structures ofLoop G sequences from Hmglpand Hmg2p. Amino acid differ-ences are marked in bold print.Nonconservative changes aremarked by a star. The boxed re-gions are areas of high diver-gence. Schematic representationsare based on the Chou and Fas-man method of secondary proteinstructure prediction, performedby the MacDNAsis program.Black areas represent a-helicalstructure. Diagonal lines repre-sent areas of ,3 sheets. Gray areasrepresent ,B turn regions. Verticalline patterns represent areas ofcoiled structure.

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of the second block of divergent amino acids ofHmglp is a coil, whereas Hmg2p is a sheet. Thesestructural and sequence differences are consistentwith the abilities of Hmglp and Hmg2p to inducedifferent cellular responses.

Coupling of Membrane Assembly to Synthesis ofSpecific Membrane Proteins Is Not CommonConceivably, alterations in the amount of any mem-brane protein could affect membrane assembly. How-ever, surprisingly few membrane proteins have beenidentified that can produce readily observable alter-ations in membrane assembly or organization. In pro-karyotes, three cases have been reported. Whenexpressed at 25-50 times greater levels than in wild-type Escherichia coli, fumarate reductase (Weiner et al.,1984; Elmes, 1986), ATP synthase (Von Meyenburg etal., 1984), or sn-glycerol-3-phosphate acyltransferase(Wilkenson et al., 1986, 1992) cause formation ofplasma membrane invaginations that appear as tu-bules intruding into the cytoplasm. These membraneproliferations consist of protein/lipid crystals inwhich as much as 90% of the protein present withinthe membranes is the overproduced protein or pro-teins. Perhaps not surprisingly, cells containing thesetubules grow very slowly, or not at all, indicating thatthe presence of these membranes disrupts essentialcellular functions.

Alteration of membrane organization occurs inmany situations in eukaryotic cells (for examples:Ghadially, 1976; Karnaky et al., 1984; York and Dick-son, 1985; Braunbeck et al., 1987; Ho, 1987; Nott andMoore, 1987; Lopez-Iglesias and Puvion-Dutilleul,1988; Thiaw et al., 1988; Kandasamy and Kristen, 1989;Kerr and Weiss, 1991). However, as in prokaryotic

cells, few specific proteins have been identified thatare capable of mediating these alterations. The twobest characterized examples involve the smooth ERproteins, cytochrome P450 and HMG-CoA reductase(Orrenius et al., 1965; Orrenius and Ericsson, 1966;Black, 1972; Black et al., 1979; Chin et al., 1982; Wrightand Rine, 1989). In these cases, the membranes containthe inducing protein, as well as additional proteins,while the presence of these membranes produces few,if any observable growth defects (Orrenius et al., 1965;Chin et al., 1982; Kochevar and Anderson, 1987). Con-sequently, the membrane proliferations produced ineukaryotes are more complex than the simple lipid/protein crystals observed in prokaryotic cells. In fact,secretory proteins can pass through the crystalloid ER,indicating that it can function as an intermediate of thesecretory pathway (Bergman and Fusco, 1990). Takentogether, these observations raise the possibility thatthe coupling of membrane biogenesis to a select groupof proteins may reflect basic control circuits for thesynthesis of specific cellular membranes. Regardless,the ability to control membrane biogenesis by selec-tively altering the level of a single protein provides auseful vantage point for gaining a molecular perspec-tive on how cells regulate the synthesis and organiza-tion of specific cellular membranes.

ACKNOWLEDGMENTSWe thank Ann J. Koning for suggesting the successful strategy forobtaining the Loop G exchanges. We are also indebted to Pek Lumand Steve Castillo for their help in the preparation of the figures.This research was supported by grants from the American CancerSociety, the American Heart Association (California Division), andthe National Institutes of Health (GM-45726 to R.W. and GM-35827to J.R.), as well as a grant from the Swiss National Science Founda-tion (C.S.).

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Signal for Karmellae Biogenesis

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