Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5808-5812, June 1993 Biology Folding and intracellular transport of the yeast plasma-membrane H+-ATPase: Effects of mutations in KAR2 and SEC65 AMY CHANG*t, MARK D. ROSEt, AND CAROLYN W. SLAYMAN* *Department of Genetics, Yale University School of Medicine, New Haven, CT 06510; and tDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544 Communicated by Gerald R. Fink, March 10, 1993

ABSTRACT We have developed two independent assays to homologs of the 19-kDa and 54-kDa subunits of mammalian study the integration, folding, and intracellular transport ofthe SRP (6-11); SEC61, -62, and -63, the products ofwhich form polytopic plasma membrane H+-ATPase in yeast. To follow a membrane-bound multisubunit complex required for the folding, controlled trypsinolysis was used to disdnguish be- translocation of several secretory into the ER lumen tween the El conformation of the ATPase (favored in the (12, 13); SSAI, -2, -3, and 4, which encode cytoplasmic presence of ADP) and the E2 conformation (favored in the members of the heat shock (hsp)70 family (14); and presence of vanadate). By this criterion, wild-type ATPase KAR2, which specifies a different hsp7o immunoglobulin appears to recognize its ligands and assume distinct confor- heavy chain binding protein (BiP)/78-kDa glucose-regulated mations within a short time after its biosynthesis. To follow protein (GRP78) that resides in the ER, interacts with nascent intracellular transport, we have exploited the fact that export polypeptides at the lumenal side of the membrane, and of newly synthesized ATPase from the participates in the translocation event itself (15-18). Al- is accompanied by kinase-mediated phosphorylation, leading though these gene products are clearly required for the to a shift in electrophoretic mobility. Because proper folding is translocation/integration and folding ofparticular proteins, it required for transport from the endoplasmic reticulum, the is far from certain that they define a single pathway for all mobility shift also serves as a convenient bioassay for correct proteins entering the ER. Indeed, several lines of evidence folding. As a first step toward identifying cell components have suggested that there may be a multiplicity of such important in folding of the nascent ATPase, we have used the pathways. Neither SEC65 nor SRP54 is required for cell dual assays to examine the role of KAR2, encoding the yeast viability, even though loss of function of either gene leads to homolog of immunoglobulin heavy chain binding protein/ a defect in the translocation/integration of some nascent 78-kDa glucose-regulated protein, and SEC65, encoding a polypeptides (7, 10, 11). Furthermore, genes have been subunit of the yeast signal recognition particle. Although identified that are uniquely required for the folding and ER mutation of KAR2 caused defective translocation of several export of a specific class of membrane proteins (SHR3; ref. secretory precursors into the endoplasmic reticulum lumen, 19) or that display differential specificity in the integration of ATPase folding and intracellular transport were unperturbed. membrane fusion proteins (SEC70, -71, -72; ref. 20). For this By contrast, in a sec65 mutant, the folding and intracellular reason, it is critical to dissect the requirements for the transport ofnewly synthesized ATPase were delayed. Our data biogenesis ofa variety ofproteins to understand the full array suggest that conformational maturation of the ATPase is a of pathways and mechanisms that may exist. rapid process in wild-type cells and that membrane integration In this study we have focused on the plasma-membrane mediated by signal recognition peptide is important for the H+-ATPase of Saccharomyces cerevisiae. The ATPase, en- proper folding of this polytopic protein. coded by PMAI, is a major constituent of the cell surface, composing 5-10% of the plasma membrane protein (21, 22). Hydropathy analysis of the deduced amino acid sequence Newly synthesized proteins destined for the Golgi complex, predicts a polytopic topology with four transmembrane seg- , or plasma membrane, as well as those destined for ments at the N terminus and four to six additional transmem- , enter the secretory pathway at the endoplasmic brane segments at the C terminus. The large central cyto- reticulum (ER). In mammalian cells, a primary route of plasmic domain contains sites for ATP binding and hydroly- targeting to the ER depends on the signal recognition particle sis, and only -4% of the molecule is predicted to be (SRP), which serves as an adaptor to mediate the interaction extracytoplasmic (22). Because the ATPase has no cleaved of a signal sequence with a receptor at the ER membrane (1). N-terminal signal sequence, its membrane targeting and Although the molecular details of protein translocation and integration are presumed to occur via internal signal and integration are not clear, it has been suggested that proteins stop-transfer sequences. Newly synthesized ATPase is de- traverse the ER membrane through a proteinaceous pore/ livered to the cell surface via the secretory pathway (23), and channel (2). It is thought that integration of transmembrane intracellular transport is accompanied by posttranslational proteins is dictated by stop transfer sequences, and the phosphorylation of the ATPase (24). orientation of polytopic proteins (with multiple membrane In the study to be described, we have developed dual spanning domains) is determined by alternating signal and assays to monitor the folding and intracellular transport of stop transfer sequences (3). Proper integration and folding are newly synthesized ATPase and have examined the role of likely prerequisites for the export of nascent proteins from KAR2 and SEC65 in the biogenesis of this polytopic mem- the ER (4, 5). brane protein. By contrast with BiP/GRP78, which resides in In recent years, a growing number of genes has been the ER lumen and act at a relatively late stage in protein identified in yeast that are required for the translocation/ may integration and folding ofnewly synthesized proteins. Among translocation and folding, SRP appears to interact with these genes are SEC65 and SRP54 (SRHI), which encode Abbreviations: SRP, signal recognition particle; DPAP B, dipeptidyl aminopeptidase B; CPY, carboxypeptidase Y; hsp, heat shock pro- The publication costs ofthis article were defrayed in part by page charge tein; ER, endoplasmic reticulum. payment. This article must therefore be hereby marked "advertisement" tPresent address: Whitehead Institute for Biomedical Research, 9 in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cambridge Center, Cambridge, MA 02142. 5808 Downloaded by guest on September 28, 2021 Cefl Biology: Chang et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5809 nascent polypeptides at the earliest steps in protein biogen- RESULTS esis (1). Our results with temperature-sensitive mutants sug- Folding of Newly Synthesized ATPase Assayed by Limited gest that while loss of KAR2 protein function causes defec- Trypsinolysis. Like other members of the E1E2 class, the tive translocation of several secretory precursors into the ER yeast plasma membrane ATPase undergoes conformational lumen, ATPase folding and intracellular transport are not changes during the catalytic cycle (22, 27). The E1 confor- perturbed. By contrast, conformational maturation of newly mation of the enzyme is favored in the presence of MgADP, made ATPase is delayed in a sec65 mutant, suggesting a whereas the E2 conformation is favored in the presence ofthe facilitative role for SRP in ATPase integration and folding. transition-state analogue vanadate. To analyze folding of newly synthesized ATPase, we used controlled trypsinolysis in the presence and absence of MgADP and vanadate. MATERIALS AND METHODS Wild-type cells were pulse-labeled for 2 min with a mixture of [35S]Cys and [35S]Met; at various times of chase, mem- Strains and Growth Media. S. cerevisiae strain NY 13 branes were isolated and trypsinized; then ATPase digestion (MATa, ura3-52), used as the wild type, and NY 431 (MATa, products were immunoprecipitated and analyzed by SDS/ ura3-52, secl8-1) were provided by Peter Novick (Yale Uni- PAGE. By 2 hr of chase, newly synthesized ATPase has versity). RSY 457 (Mata, ura3-52, ade2, trpl-1, leu2-3,-112, presumably arrived at the plasma membrane (24). Fig. 1A his3, sec65-1) was provided by Randy Schekman (University (anes 5-7) shows that at this time, newly made ATPase is of California, Berkeley). The kar2 mutant used in this study folded in the membrane such that it is fairly resistant to was MS 177 (Mata, ura3-52, ade2-101, kar2-159). The PMA1 trypsin; some intact ATPase survived tryptic cleavage alto- Lys-379-* Gin mutation was expressed in SY4 strain, in which gether. In the presence of vanadate, specific fragments of 92 the chromosomalPMAI gene is under the control ofthe GAL] kDa (arrowhead) and 60 kDa (arrow) were protected from promoter [Mata, ura3-52, leu2-3,-112, his4-619, sec64, GAL, degradation. In the presence of ADP, fragments of 92 kDa pmal::YIpGAL-PMA1 (pRR219)] (25). Cells were grown in and 50 kDa (arrow) were protected. Identical fragments were minimal medium containing 0.7% yeast nitrogen base without protected in the presence ofligands when plasma membranes amino acids (Difco), 2% glucose, and the appropriate nutri- were analyzed by immunoblot (data not shown). To deter- tional supplement(s) (26). mine when the ATPase first becomes competent to assume Membrane All strains distinct conformational states, tryptic sensitivity at 1 min of Metabolic Labeling and Preparation. chase was examined. Interestingly, folding of newly made except SY4 were grown and labeled as described (24). ATPase, expected to reside predominantly within the ER at Briefly, cells were grown to midlogarithmic phase at 25°C in this time, appeared similar to that seen at late times of chase. low-sulfate minimal medium (100 ,uM Na2SO4/2% glucose). The ATPase at 1 min of chase displayed resistance to tryptic The cells were preincubated at 25°C or 37°C for 30 min in digestion (Fig. 1A, lane 2), and vanadate- and ADP-protected minimal medium (25 ,uM Na2SO4) before labeling with fragments were observed (lanes 3 and 4). In a parallel Expre35S35S (10-20 ,uCi per OD600; NEN). SY4 cells harbor- experiment (data not shown), the same tryptic patterns were ing the PMA1 Lys-379 -* Gln mutation were grown in seen when exit from the ER was blocked at 37°C in the secl8 minimal medium/2% galactose and then shifted to medi- mutant (28). Taken together, the data suggest that the ability um/2% glucose for 3 hr at 25°C to inhibit expression of to assume specific conformations is acquired at the ER. wild-type ATPase. The cells were then resuspended in me- dium/25 ,uM Na2SO4/2% glucose, shifted to 37°C for 30 min A lm 2h B im C K379Q to allow mutant ATPase expression, and pulse-labeled as - - - - SDS was terminated 10 described above. Radiolabeling by adding - + + + Trypsin mM cysteine and 10 mM methionine. At various times of V04 ADP VO, ADPJ V04 AI)R VO ADP were with beads. To chase, cells lysed by vortexing glass t.", prepare a total membrane fraction, cell lysate was centrifuged g~~~~~~~~~N at 100,000 x g for 1 hr. To determine 35S-labeled protein content, aliquots of lysate and 250 pg of bovine serum albumin were precipitated with 10o trichloroacetic acid for =...... - 30 min on ice. The samples were washed twice with 5% - trichloroacetic acid, and the pellets were counted in the presence of Optifluor. Controlled Tryptic Digestion and Immunoprecipitation. For controlled trypsinolysis, membranes (1-5 pg ofprotein) were suspended in 250 mM sucrose/5 mM MgCl2/20 mM Hepes, pH 7.5 buffer. Bovine serum albumin (usually =80 pg) was FIG. 1. Tryptic digestion of newly synthesized ATPase. Cells added to give a final protein concentration of 1 mg/ml. The were pulse-labeled for 2 min with Expre35S35S and chased. (A) After samples were allowed to equilibrate briefly with vanadate 1 min (1m) and 2 hr (2h) ofchase, total membranes were prepared and (0.1 mM) or MgADP (5 mM) before trypsin addition. Diges- incubated at 25°C in the absence and presence of trypsin for 30 min. tion at a trypsin/protein ratio of 1:50 was done for 30 min at Vanadate (0.1 mM) and ADP (5 mM) were included, as indicated. Tryptic fragments were immunoprecipitated and resolved by SDS/ 25°C. The reaction was terminated by the addition of 1 mM PAGE and fluorography. The arrowhead indicates the 92-kDa frag- diisopropylfluorophosphate, and the tryptic fragments were ment protected by both vanadate and ADP; arrows indicate the immunoprecipitated. unique vanadate-protected 60-kDa band and the ADP-specific 50- Samples, normalized to trichloroacetic acid-precipitable kDa band. (B) ATPase degradation pattern from membranes from 1 cpm, were immunoprecipitated, as described (24). Rabbit min of chase resuspended in the presence of 1% SDS for 5 min at antiserum to Kar2 protein was prepared, as described (17). 25°C. The sample was then diluted with tryptic digestion buffer to an SDS concentration of 0.05%, and trypsinolysis and immunoprecip- Rabbit antisera to dipeptidyl aminopeptidase B (DPAP B) and itation was as described above. (C) Tryptic degradation pattern ofthe carboxypeptidase Y (CPY) were provided by Tom Stevens ATPase Lys-379 -- Gln (K379Q) mutant. The following molecular (University of Oregon) and Randy Schekman (University of weight markers (at left) were used: M, 205,000, Mr 116,000, Mr California, Berkeley). 97,400, Mr 66,000, Mr 45,000, and Mr 29,000. Downloaded by guest on September 28, 2021 5810 Cell Biology: Chang et al. Proc. Natl. Acad. Sci. USA 90 (1993) Several control experiments were done to confirm the ER membrane. Cleavage of the N-terminal signal sequence accuracy of the trypsinolysis assay. As expected, tryptic and core glycosylation generate a 67-kDa pl form; modifi- sensitivity increased dramatically when the ATPase was first cation in the Golgi produces a 69-kDa p2 form; and proteo- denatured by SDS; no intact ATPase remained, and no lytic cleavage upon arrival in the vacuole produces the degradation fragments were recovered (Fig. 1B). In addition, 61-kDa mature enzyme (30). Fig. 3A (Right) shows pl CPY we examined a PMA1 point mutant (Lys-379 -- Gln), which immunoprecipitated from wild-type cells at 1 min of chase, is known to be defective in intracellular transport (29). The and the pl, p2, and mature forms at 15 min of chase. In the mutant protein showed increased susceptibility to tryptic kar2 mutant, pre-pro-CPY (58 kDa) accumulated without degradation, as well as insensitivity to vanadate and ADP glycosylation or signal cleavage. The translocation of newly (Fig. 1C), consistent with the notion that it is misfolded. By synthesized KAR2 protein, a resident of the ER lumen, was contrast, normal tryptic patterns were observed in a Lys-379 similarly assayed (15, 17). In Fig. 3A (Left) mature KAR2 -- Arg mutant, which is transport-competent (data not protein (79 kDa) was immunoprecipitated from wild-type shown). Thus, the trypsinolysis assay appears to reflect cells at 1 min of chase, whereas the 82-kDa KAR2 precursor faithfully the state of membrane integration and protein was seen in the kar2 mutant. The precursor persisted even at folding. 2 hr of chase in kar2 (data not shown). Intracellular Transport of Newly Synthesized ATPase. We The folding and intracellular transport of the plasma mem- have previously shown that phosphorylation at serine and brane ATPase were then examined in the kar2 mutant. As in threonine residues accompanies intracellular transport of wild-type cells, newly synthesized ATPase in kar2 was newly synthesized ATPase. Phosphorylation can be detected quantitatively associated with the membrane fraction (data as a shift in electrophoretic mobility on SDS/polyacrylamide not shown). By limited trypsinolysis, the ATPase degrada- gels and is maximal by 2 hr of chase (24). This behavior was tion pattern observed in kar2 membranes at 1 min of chase exploited to assay the export of newly synthesized ATPase appeared the same as that previously seen in wild-type from the ER. Fig. 2 shows that, in wild-type cells, there is an membranes (compare Fig. 3B with Fig. 1); fragments of 92 increase in the apparent Mr of newly synthesized ATPase kDa and 60 kDa were protected in the presence of vanadate, from 1 min to 2 hr ofchase; as expected, alkaline phosphatase and the 92-kDa and 50-kDa fragments were seen in the treatment ofthe 2-hr sample converted it to the low-Mr form. presence of ADP. Furthermore, intracellular transport of When exit from the ER was blocked at the restrictive newly synthesized ATPase in the mutant appeared unper- temperature in a secl8 mutant, no increase in apparent Mr turbed, as evidenced by the shift in its electrophoretic was detected at 2 hr of chase. Similarly, no mobility change mobility during chase (Fig. 3C). These data indicate that, was observed for the Lys-379 -> Gin mutant ATPase that although loss of KAR2 function caused defective transloca- showed increased susceptibility to degradation in the trypsin assay. Thus, the mobility shift ofthe ATPase at 2 hr of chase A Controls (Fig. 2, arrowhead) can be taken as a measure ofexport from CPY Kar2p the ER. Because proper integration and folding are likely WT kar2 required for transport from the ER (4), the mobility shift WT kar2 Im 15m Im serves as a bioassay for correct folding. Furthermore, be- cause the entire ATPase band undergoes the shift, it appears .-_P2 that essentially all the molecules undergo proper folding and intracellular transport. ATPase Folding and Intraceflular Transport in a kar2 Mu- tant. We used the trypsinolysis and electrophoretic mobility assays to ask whether KAR2 plays a role in the folding and B ATPase folding ATPase intracellular transport ofnewly synthesized ATPase. For this C intraceltllar transport purpose, the temperature-sensitive kar2-159 mutant was - + + + Try,psin kar2 secl8 lm 2h lm 2h shifted to 37°C and biosynthetically labeled. As a control, V04 ADP newly synthesized CPY and KAR2 protein were analyzed to confirm the translocation defect of the kar2 mutant (17, 18). -w fs In wild-type cells the vacuolar protease CPY enters the ~~~~~~~~~Pi secretory pathway by translocation ofprepro-CPY across the a. A SEC18 K379Q WT Im 2h lm 2h lm 2h- 2h+ FIG. 3. Folding and intracellular transport of newly synthesized AT- Pase in kar2 cells. kar 2-159 cells were shifted to 37°C and then pulse- __ labeled and chased. CPY and KAR2 protein (Kar2p) were immunoprecip- itated from cell lysate; ATPase was immunoprecipitated from a total membrane fraction and analyzed by SDS/PAGE and fluorography. (A) A - - Expression of mutant phenotype: newly synthesized prepro-CPY from kar2 at 1 min of chase and pl, FIG. 2. Intracellular transport of newly synthesized ATPase. p2, and m (mature) CPY forms from wild type at 1 min (1m) and 15 Cells were pulse-labeled and chased as described in Fig. 1 legend. min (1Sm) of chase (Right); newly synthesized KAR2 precursor (82 ATPase was immunoprecipitated from total membranes and ana- kDa) from kar2 cells, and the mature KAR2 protein (79 kDa) from lyzed by SDS/PAGE (8% run-off gel, as described in ref. 24) and wild-type cells (Left). (B) ATPase folding in kar2-159: Total mem- fluorography. ATPase from wild-type cells is compared with the brane isolated after 1 min of chase was trypsinized, and ATPase Lys-379 -. Gln ATPase mutant, and ATPase accumulated in secl8. degradation products were immunoprecipitated. (C) ATPase intra- ATPase immunoprecipitated from wild-type cells at 2 hr ofchase was cellular transport in kar2-159: the increase in ATPase apparent Mr at incubated in the absence (2h-; arrowhead) and presence (2h+) of 1 2 hr (2h) of chase is indicated by the arrowhead. ATPase from secl8 unit of alkaline phosphatase for 1 hr at 37°C. cells are shown for size comparison on an 8% run-off gel. Downloaded by guest on September 28, 2021 Cell Biology: Chang et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5811 tion of some proteins, the integration, folding, and transport conditions but without trypsin. ATPase was then immuno- of the polytopic ATPase were unaffected. precipitated from an equal aliquot of each sample, calculated Involvement of SEC65 in ATPase Folding and Transport. on the basis of total 35S-labeled protein. In wild-type cells, Because SRP appears to play a role in the earliest steps in newly synthesized ATPase was quite stable in vitro during protein biogenesis (1), we wished to examine its role in the the entire chase period (Right). By contrast, the ATPase in integration and folding ofnewly synthesized ATPase. For this sec65 was degraded at early times of chase (1-30 min) and purpose, we used a strain defective in SEC65, encoding the only became stable to incubation by 1-2 hr of chase (Left). yeast homologue ofthe 19-kDa subunit ofmammalian SRP (8, ATPase degradation in the sec65 mutant occurred in vitro 10). The sec65-1 mutant was originally isolated by selection for only since intact ATPase was quantitatively recovered when temperature-sensitive mutants defective in integration of a samples were boiled in SDS immediately after cell lysis (data chimeric membrane protein and the vacuolar protein DPAP B not shown). (9). DPAP B is anchored in the by a single Because SRP appears to function during protein targeting transmembrane segment with the C-terminal bulk of the mol- across the ER (1), the effect of the sec65 mutation on ecule facing the lumen. Integration of newly synthesized membrane integration ofthe ATPase was assayed. Cell lysate DPAP B is accompanied by core glycosylation of a 96-kDa precursor to generate a 110- to 113-kDa species, and transport was incubated with 0.1 M Na2CO3 at pH 11.5 and was then to the Golgi results in further sugar addition (31). Consistent centrifuged to separate membranes from the supernatant. At with a previous report (9), the unglycosylated 96-kDa precur- 1 min of chase, newly synthesized ATPase was recovered in sor was accumulated in sec65 cells at the restrictive temper- the pellet fraction of sec6S (Fig. 5B), indicating that at least ature, indicating defective integration of DPAP B (Fig. 4A, one of its many transmembrane-spanning domains is embed- Right). By contrast, the translocation of KAR2 protein was ded in the membrane (32). Therefore, the susceptibility to partially blocked in sec65 (Fig. 4A, Left), and there was no degradation in vitro seen at early times of chase seems to effect on CPY translocation, since core-glycosylated CPY (P1) reflect the vulnerability ofan incompletely integrated and/or was observed at 1 min of chase (Fig. 4A, Middle). folded molecule. The sec65 mutation proved to have a complex effect on In spite ofthe perturbation occurring at early times ofchase, ATPase integration and folding. At 1 min of chase, newly the ATPase eventually became properly folded and exported synthesized ATPase showed increased sensitivity to tryptic from the ER in sec65 cells. By 2 hrofchase, newly synthesized digestion (data not shown). Surprisingly, even in the absence ATPase from sec65 remained stable during incubation in the of trypsin, the newly synthesized protein was degraded absence of trypsin, while its trypsinolysis pattern resembled during the in vitro incubation. Fig. 5A shows a pulse-chase that ofwild-type cells (Fig. 4B). Furthermore, ATPase at 2 hr experiment in which total membranes isolated at various ofchase was quantitatively recovered and displayed a shift in times of chase were incubated under trypsinolysis assay its electrophoretic mobility (Fig. 4C, compare 1 min and 2 hr). Thus, the sec65 mutation delayed but did not ultimately A Controls prevent ATPase integration and folding. Kar2p CPY DPAP B WT sec65 WT sec65 WT sec65 A 1 5 15 30 60 120 m 1 5 15 30 60 120 m lm 15m lm

p2 sec65 WT -m ._ ___

B ATPase folding C ATPase intracellular transport

+ + + Trypsin sec65 secl8

V04 ADP Im 2h Im 2h

B Im 2h T S P T S P FIG. 4. Folding and intracellular transport of newly synthesized AT- - Pase in sec65 cells. Biosynthetic la- A* _ beling of sec65-1 at 37°C was as de- scribed in Fig. 3. (A) Expression of the mutant phenotype: newly synthe- sized prepro-CPY immunoprecipi- FIG. 5. Time course of ATPase folding in sec6S. Wild-type and tated from sec65 at 1 min of chase sec6S cells were biosynthetically labeled. Immunoprecipitated AT- compared with CPY forms from wild Pase was resolved by SDS/PAGE and fluorography. (A) ATPase type (WT) cells at 1 min (1m) and 15 stability: membranes isolated from wild type (WT; Right) and sec6S min (15 m) of chase (Middle); KAR2 (Left) cells at various times ofchase were incubated under trypsinol- protein from wild-type and mutant cells at 1 min of chase (Left); ysis assay conditions, except trypsin was omitted. Immunoprecipi- newly synthesized DPAP B from mutant and wild-type cells (Right). tation of ATPase from each sample was normalized to acid- (B) ATPase folding at 2 hr of chase, assayed by trypsinolysis. (C) precipitable cpm. (B) ATPase integration: sec6S cell lysate was ATPase intracellular transport, assayed by electrophoretic mobility incubated with 0.2 M Na2CO3, pH 11.5, for 10 min on ice and at 1 min (1m) and 2 hr (2h) (arrowhead) of chase on an 8% run-offgel. centrifuged at 100,000 x g for 1 hr. Total (T) lysate, supernatant (S) ATPase was immunoprecipitated from cell lysate boiled in the fraction, and pellet (P) were boiled in the presence of 1% SDS, and presence of 1% SDS to prevent degradation. ATPase was immunoprecipitated. Downloaded by guest on September 28, 2021 5812 CeU Biology: Chang et al. Proc. Natl. Acad. Sci. USA 90 (1993) DISCUSSION identical to ours (CPY < KAR2 < DPAP B). Therefore, we that the yeast plasma membrane conclude that our results with sec65-1 are not allele-specific Previous work has shown but instead reflect differing requirements of nascent proteins ATPase, like other members of the E1E2 class of enzymes, for SRP. Since requirements for KAR2 (Fig. 3) and other cycles between two major conformational states during ca- genes (19, 20) similarly differ, there appears to be consider- talysis. In the presence of vanadate, the E2 conformation is able plasticity in the mechanism by which proteins enter or favored, whereas the E1 conformation is preferred in the cross the ER membrane. presence of ADP. One established method to study such conformational changes is controlled trypsinolysis (e.g., refs. We are grateful to Randy Schekman, Tom Stevens, and Rajini Rao 27 and 33). In the present study, we have made use of the for generously providing reagents and to Kenneth Allen for excellent trypsinolysis assay in conjunction with an intracellular trans- technical assistance. This work was supported by National Institutes port assay to characterize the conformational state of newly of Health Research Grant GM 17561 to C.W.S. and Postdoctoral synthesized ATPase. Our data indicate that early in its Fellowship GM 11967 to A.C., and by an American Heart Associ- biosynthesis, while presumably still in the ER, the ATPase is ation Grant-in-Aid to A.C. competent to assume distinct conformations in response to vanadate and ADP (Fig. 1). 1. Walter, P. & Lingappa, V. R. (1986) Annu. Rev. Cell Biol. 2, 499-516. We have gone on to examine the effect of kar2 and sec65 2. Simon, S. M. & Blobel, G. (1991) Cell 65, 371-380. mutations on the biogenesis of the ATPase, which serves as 3. Hartmann, E., Rapoport, T. A. & Lodish, H. F. (1989) Proc. an example of an endogenous polytopic membrane protein. Natl. Acad. Sci. USA 86, 5786-5790. Mutation of KAR2 had no detectable effect on the integra- 4. Pelham, H. R. B. (1989) Annu. Rev. Cell Biol. 5, 1-23. tion, folding, and intracellular transport ofnewly synthesized 5. Hurtley, S. M. & Helenius, A. (1989) Annu. Rev. Cell Biol. 5, ATPase, even though it clearly impaired the translocation of 277-307. several other proteins. Perhaps it is not surprising that 6. Hann, B. C., Poritz, M. A. & Walter, P. (1989) J. CellBiol. 109, ATPase integration and folding occur independently of 3223-3230. KAR2 protein, which resides in the ER lumen, since struc- 7. Hann, B. C. & Walter, P. (1991) Cell 67, 131-144. 8. Hann, B. C., Stirling, C. J. & Walter, P. (1992) Nature (Lon- tural predictions indicate that only 4% of the ATPase lies in don) 356, 532-533. the extracytoplasmic space (22). Indeed, a substantial lum- 9. Stirling, C. J., Rothblatt, J., Hosobuchi, M., Deshaies, R. & enal domain may be required for the interaction of a nascent Schekman, R. (1991) Mol. Biol. Cell 3, 129-142. polypeptide with KAR2 protein. Thus, loss ofKAR2 function 10. Stirling, C. J. & Hewitt, E. W. (1992) Nature (London) 356, perturbs specific translocation events and does not result in 534-537. general loss offunction of the secretory pathway. For import 11. Amaya, Y. & Nakano, A. (1991) FEBS Lett. 283, 325-328. into and export from the ER, the plasma membrane ATPase 12. Deshaies, R. J., Kepes, F. & Bohni, P. C. (1989) Trends Genet. may require other cellular components that appear to func- 5, 87-93. tion uninterrupted in kar2. 13. Deshaies, R. J., Sanders, S. L., Feldheim, D. A. & Schekman, R. (1991) Nature (London) 349, 806-808. Interestingly, conformational maturation of the ATPase 14. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, did prove to depend upon SEC65, which encodes a subunit of E. A. & Schekman, R. (1988) Nature (London) 332, 800-805. yeast SRP (8, 10). At early times of chase in a sec65 mutant, 15. Rose, M. D., Misra, L. M. & Vogel, J. P. (1989) Cell 57, newly synthesized ATPase was associated with the mem- 1211-1221. brane but was abnormally susceptible to degradation during 16. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.-J. & incubation in vitro (Fig. 5), suggesting that it was not in a fully Sambrook, J. (1989) Cell 57, 1223-1236. integrated and folded conformation. These results are con- 17. Vogel, J. P., Misra, L. M. & Rose, M. D. (1990) J. Cell Biol. sistent with a role for SRP in membrane integration because 110, 1885-1895. proper folding may well be hindered for a partially integrated 18. Nguyen, T. H., Law, D. T. S. & Williams, D. B. (1991) Proc. Natl. Acad. Sci. USA 88, 1565-1569. polypeptide (in which some membrane-spanning segments 19. Ljungdahl, P. O., Gimeno, C. J., Styles, C. A. & Fink, G. R. have failed to enter the bilayer). Nevertheless, it is intriguing (1992) Cell 71, 463-478. that the sec65 mutation does not appear to cause irreversible 20. Green, N., Fang, H. & Walter, P. (1992) J. Cell Biol. 116, misfolding or mislocalization because the ATPase ultimately 597-604. became stabilized (Fig. SA), assumed a mature conformation 21. Serrano, R., Kielland-Brandt, M. C. & Fink, G. R. (1986) (Fig. 4B), and was transported from the ER (Fig. 4C). Nature (London) 319, 689-693. Thus it appears that SEC65 protein facilitates, but is not 22. Serrano, R. (1989) Annu. Rev. Plant Physiol. 40, 61-94. essential for, the proper integration and folding of nascent 23. Holcomb, C. L., Hansen, W. J., Etchverry, T. & Schekman, ATPase. One interpretation of this result is that there is an R. (1988) J. Cell Biol. 106, 641-648. 24. Chang, A. & Slayman, C. W. (1991) J. Cell Biol. 115, 289-295. SRP-independent mechanism for protein targeting and inte- 25. Nakamoto, R. K., Rao, R. & Slayman, C. W. (1991) J. Biol. gration at the ER. Consistent with this idea is the finding that Chem. 266, 7940-7949. yeast cells remain viable upon deletion of either SRP54 or 26. Sherman, F., Fink, G. R. & Hicks, J. (1986) Methods in Yeast SEC65, encoding different subunits of SRP (7, 10). In addi- Genetics (Cold Spring Harbor Lab. Press, Plainview, NY). tion, it has been observed that translocation/integration of 27. Perlin, D. S. & Brown, C. L. (1987) J. Biol. Chem. 262, some proteins can occur posttranslationally in the absence of 6788-6794. SRP in an in vitro system from yeast (1). 28. Novick, P. & Schekman, R. (1983) J. Cell Biol. 96, 541-547. The consequences of the sec65 mutation clearly depend 29. Rao, R. & Slayman, C. W. (1993) J. Biol. Chem. 268, 6708- upon the nascent polypeptide. Severe defects have been 6713. 30. Stevens, T., Esmon, B. & Schekman, R. (1982) Cell 30, observed in the translocation of invertase and a factor in 439-448. sec65 cells (9). We found that sec65 was not at all restrictive 31. Roberts, C. J., Pohlig, G., Rothman, J. H. & Stevens, T. H. for CPY, partially restrictive for KAR2 protein, and com- (1989) J. Cell Biol. 108, 1363-1373. pletely restrictive for DPAP B (Fig. 4A). Hann and Walter (7) 32. Steck, T. L. & Yu, J. (1973) J. Supramol. Struct. 1, 220-248. have also reported differential effects after depletion of 33. Geering, K., Kraehenbuhl, J.-P. & Rossier, B. C. (1987) J. Cell SRP54 protein, and the order of severity in their study was Biol. 105, 2613-2619. Downloaded by guest on September 28, 2021