Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5808-5812, June 1993 Cell 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 proteins 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 protein (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 endoplasmic reticulum 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- lysosome, or plasma membrane, as well as those destined for ments at the N terminus and four to six additional transmem- secretion, 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.