Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.     

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.     

Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae

To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1. Sanders et al. (Sanders, S. L., K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman. 1992. Cell. 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore. We now show that mutation of a conserved residue (A181-->T) (Nelson, M. K., T. Kurihara, and P. Silver. 1993. Genetics. 134:159- 173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203. We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired. Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect. Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane.     

ER membrane protein complex required for nuclear fusion

Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.     

A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70s

Eukaryotic cells contain multiple Hsp70 proteins and DnaJ homologues. The partnership between a given Hsp70 and its interacting DnaJ could, in principle, be determined by their cellular colocalization or by specific protein-protein interactions. The yeast SCJ1 gene encodes one of several homologues of the bacterial chaperone DnaJ. We show that Scj1p is located in the lumen of the endoplasmic reticulum (ER), where it can function with Kar2p (the ER-lumenal BiP/Hsp70 of yeast). The region common to all DnaJ homologues (termed the J domain) from Scj1p can be swapped for a similar region in Sec63p, which is known to interact with Kar2p in the ER lumen, to form a functional transmembrane protein component of the secretory machinery. Thus, Kar2p can interact with two different DnaJ proteins. On the other hand, J domains from two other non-ER DnaJs, Sis1p and Mdj1p, do not function when swapped into Sec63p. However, only three amino acid changes in the Sis1p J domain render the Sec63 fusion protein fully functional in the ER lumen. These results indicate that the choice of an Hsp70 partner by a given DnaJ homologue is specified by the J domain.     

A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome

Reconstituted proteoliposomes derived from solubilized yeast microsomes are able to translocate a secreted yeast mating pheromone precursor (Brodsky, J. L., S. Hamamoto, D. Feldheim, and R. Schekman. 1993. J. Cell Biol. 120:95-107). Reconstituted proteoliposomes prepared from strains with mutations in the SEC63 or KAR2 genes are defective for translocation; the kar2 defect can be overcome by the addition of purified BiP (encoded by the KAR2 gene). We now show that addition of BiP to wild-type reconstituted vesicles increases their translocation efficiency three-fold. To identify other ER components that are required for translocation, we purified a microsomal membrane protein complex that contains Sec63p. We found that the complex also includes BiP, Sec66p (gp31.5), and Sec67p (p23). The Sec63p complex restores translocation activity to reconstituted vesicles that are prepared from a sec63-1 strain, or from cells in which the SEC66 or SEC67 genes are disrupted. BiP dissociates from the complex when the purification is performed in the presence of ATP gamma S or when the starting membranes are from yeast containing the sec63-1 mutation. We conclude that the purified Sec63p complex is active and required for protein translocation, and that the association of BiP with the complex may be regulated in vivo.     

Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.     

SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.     

Sec61p and BiP directly facilitate polypeptide translocation into the ER.

Secretory proteins are segregated from cytosolic proteins by their translocation into the endoplasmic reticulum (ER). A modified secretory protein trapped during translocation across the ER membrane can be crosslinked to two previously identified proteins, Sec61p and BiP (Kar2p). The dependence of this cross-linking upon proteins and small molecules was examined. Mutations in SEC62 and SEC63 decrease the ability of Sec61p to be cross-linked to the secretory polypeptide trapped in translocation. ATP is also required for interaction of Sec61p with the secretory protein. Three kar2 alleles display defective translocation in vitro. Two of these alleles also decrease the ability of Sec61p to be cross-linked to the secretory protein. The third allele, while exhibiting a severe translocation defect, does not affect the interaction of Sec61p with the secretory protein. These results suggest that Sec61p is directly involved in translocation and that BiP acts at two stages of the translocation cycle.     

BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane.

We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.     

The Brl domain in Sec63p is required for assembly of functional endoplasmic reticulum translocons

Protein translocation into the endoplasmic reticulum occurs at pore-forming structures known as translocons. In yeast, two different targeting pathways converge at a translocation pore formed by the Sec61 complex. The signal recognition particle-dependent pathway targets nascent precursors co-translationally, whereas the Sec62p-dependent pathway targets polypeptides post-translationally. In addition to the Sec61 complex, both pathways also require Sec63p, an integral membrane protein of the Hsp40 family, and Kar2p, a soluble Hsp70 located in the ER lumen. Using a series of mutant alleles, we demonstrate that a conserved Brl (Brr2-like) domain in the COOH-terminal cytosolic region of Sec63p is essential for function both in vivo and in vitro. We further demonstrate that this domain is required for assembly of two oligomeric complexes of 350 and 380 kDa, respectively. The larger of these corresponds to the heptameric "SEC complex" required for post-translational translocation. However, the 350-kDa complex represents a newly defined hexameric SEC' complex comprising Sec61p, Sss1p, Sbh1p, Sec63p, Sec71p, and Sec72p. Our data indicate that the SEC' complex is required for co-translational protein translocation across the yeast ER membrane.     

Suppression of a sec63 mutation identifies a novel component of the yeast endoplasmic reticulum translocation apparatus.

Mutations in the SEC63 gene are associated with defects in protein translocation into the endoplasmic reticulum (ER) as well as in nuclear protein localization in Saccharomyces cerevisiae. To identify proteins that might interact and/or function with SEC63p, we cloned a high copy suppressor (HSS1) of the temperature-sensitive lethal phenotype of the sec63-101 mutant. HSS1 is an allele-specific sec63 suppressor that encodes an integral ER membrane glycoprotein of 206 amino acids with the N-terminus in the ER lumen and C-terminal region in the cytoplasm. Haploid strains disrupted for HSS1 are temperature-sensitive for growth and accumulate precursor forms of Kar2p and invertase. The HSS1 null allele is synthetically lethal in combination with mutations affecting ER translocation. We propose that HSS1p is important for ER translocation and interacts with previously identified components of the yeast translocation apparatus. HSS1 is identical to SEC66, which encodes a glycoprotein complexed with SEC62p and SEC63p.     

The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S–transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.     

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.     

Saccharomyces cerevisiae Rot1p is an ER-localized membrane protein that may function with BiP/Kar2p in protein folding

The 70-kDa heat shock protein (Hsp70) family of molecular chaperones cooperates with cofactors to promote protein folding, assembly of protein complexes and translocation of proteins across membranes. Although many cofactors of cytosolic Hsp70s have been identified, knowledge about cofactors of BiP/Kar2p, an endoplasmic reticulum (ER)-resident Hsp70, is still poor. Here we propose the Saccharomyces cerevisiae protein Rot1p as a possible cofactor of BiP/Kar2p involved in protein folding. Rot1p was found to be an essential, ER-localized membrane protein facing the lumen. ROT1 genetically interacted with several ER chaperone genes including KAR2, and the rot1-2 mutation triggered the unfolded protein response. Rot1p associated with Kar2p, especially under conditions of ER stress, and maturation of a model protein, a reduced form of carboxypeptidaseY, was impaired in a kar2-1 rot1-2 double mutant. These findings suggest that Rot1p participates in protein folding with Kar2p. Morphological analysis of rot1-2 cells revealed cell wall defects and accumulation of autophagic bodies in the vacuole. This implies that the protein folding machinery in which Rot1p is involved chaperones proteins acting in various physiological processes including cell wall synthesis and lysis of autophagic bodies.     

J Domain Co-chaperone Specificity Defines the Role of BiP during Protein Translocation

Hsp70 chaperones can potentially interact with one of several J domain-containing Hsp40 co-chaperones to regulate distinct cellular processes. However, features within Hsp70s that determine Hsp40 specificity are undefined. To investigate this question, we introduced mutations into the ER-lumenal Hsp70, BiP/Kar2p, and found that an R217A substitution in the J domain-interacting surface of BiP compromised the physical and functional interaction with Sec63p, an Hsp40 required for ER translocation. In contrast, interaction with Jem1p, an Hsp40 required for ER-associated degradation, was unaffected. Moreover, yeast expressing R217A BiP exhibited defects in translocation but not in ER-associated degradation. Finally, the genetic interactions of the R217A BiP mutant were found to correlate with those of known translocation mutants. Together, our results indicate that residues within the Hsp70 J domain-interacting surface help confer Hsp40 specificity, in turn influencing distinct chaperone-mediated cellular activities.     

Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast

BiP/GRP78 is an essential member of the HSP70 family that resides in the lumen of the endoplasmic reticulum. In yeast, BiP/GRP78 is encoded by the KAR2 gene. A temperature sensitive mutation was isolated in KAR2 and found to cause a rapid block in protein secretion. Secretory precursors of a number of proteins (invertase, carboxypeptidase Y, alpha-factor, and BiP) accumulated that were characteristic of a block in translocation into the lumen of the ER. Protease protection experiments confirmed that the precursors accumulated on the cytoplasmic side of the ER membrane. Moreover, depletion of wild-type KAR2 protein also resulted in a block in translocation of secretory proteins. These results implicate BiP/GRP78 function in the continued translocation of proteins into the lumen of the ER.     

A Role for the DnaJ Homologue Scj1p in Protein Folding in the Yeast Endoplasmic Reticulum

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Δscj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Δscj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.     

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway.     

Mammalian Sec61 is associated with Sec62 and Sec63

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.