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.     

Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum.

KAR2 encodes the yeast homologue of mammalian BiP, the endoplasmic reticulum (ER) resident member of the HSP70 family. Kar2p has been shown to be required for the translocation of proteins across the ER membrane as well as nuclear fusion. Sec63, an ER integral membrane protein that shares homology with the Escherichia coli DnaJ protein, is also required for translocation. In this paper we describe several specific genetic interactions between these two proteins, Kar2p and Sec63p. First, temperature-sensitive mutations in KAR2 and SEC63 form synthetic lethal combinations. Second, dominant mutations in KAR2 are allele-specific suppressors for the temperature-sensitive growth and translocation defect of sec63-1. Third, the sec63-1, unlike other translocation defective mutations, results in the induction of KAR2 mRNA levels. Taken together, these genetic interactions suggest that Kar2p and Sec63p interact in vivo in a manner similar to that of the E. coli HSP70, DnaK, and DnaJ. We propose that the interaction between these two proteins is critical to their function in protein translocation.     

High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Delta mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of approximately 100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of approximately 480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.     

The yeast SSS1 gene is essential for secretory protein translocation and encodes a conserved protein of the endoplasmic reticulum.

The SEC61, SEC62 and SEC63 yeast gene products are membrane components of the apparatus that catalyses protein translocation into the endoplasmic reticulum (ER). In the hope of uncovering additional components of the translocation apparatus, we sought yeast genes whose overexpression would restore partial thermoresistance in a sec61 translocation-deficient mutant. The first extragenic Sec sixty-one suppressor, SSS1, is an essential single copy gene whose overexpression restores translocation in the sec61 mutant. Another extragenic suppressor was identified as TDH3, which encodes the major isozyme of the most abundant yeast protein, glyceraldehyde-3-phosphate dehydrogenase. TDH3 overexpression could exert an indirect effect by competitively inhibiting protein synthesis, thereby allowing the impaired translocation apparatus to cope with a reduced flow of newly synthesized secretory proteins. Depletion of the Sss1 protein rapidly results in accumulation of multiple secretory or membrane proteins devoid of post-translational modifications; the normally secreted alpha-factor accumulates on the cytosolic side of ER membranes. Thus, the SSS1 gene is required for continued translocation of secretory preproteins beyond their early association to ER membranes. Consistent with its essential role in protein translocation, the Sss1 protein localizes to the ER and homologues were detected in higher eukaryotes.     

Extragenic Suppressors of Mutations in the Cytoplasmic C Terminus of Sec63 Define Five Genes in Saccharomyces Cerevisae

Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.     

Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.     

Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.     

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection. Using temperature-sensitive sec24-1 mutants, we showed previously that a secretory glycoprotein, Hsp150, does not require functional Sec24p for ER exit. Deletion of SFB2, SFB3 or both from wild type or the deletion of SFB2 from sec24-1 cells did not affect Hsp150 transport. SFB3 deletion has been reported to be lethal in sec24-1. However, here we constructed a sec24-1 Deltasfb3 and a sec24-1 Deltasfb2 Deltasfb3 strain and show that Hsp150 was secreted slowly in both. Turning off the SEC24 gene did not inhibit Hsp150 secretion either, and the lack of SEC24 expression in a Deltasfb2 Deltasfb3 deletant still allowed some secretion. The sec24-1 Deltasfb2 Deltasfb3 mutant grew slower than sec24-1. The cells were irregularly shaped, budded from random sites and contained proliferated ER at permissive temperature. At restrictive temperature, the ER formed carmellae-like proliferations. Our data indicate that ER exit may occur in vesicles lacking a full complement of Sec23p/24p and Sec13p/31p, demonstrating diversity in the composition of the COPII coat.     

Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.     

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.     

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.     

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.     

The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.     

Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.     

SED4 encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation

SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage- dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.     

Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.     

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional- lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.     

Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cerevisiae block budding of transport vesicles from the ER. SEC16 was cloned by complementation of the sec16-1 mutation and encodes a 240-kD protein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but not by nonionic detergents or urea. Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorporated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction. Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16p associates with Sec23p as part of the transport vesicle coat structure. Genetic analysis of SEC16 identifies three functionally distinguishable domains. One domain is defined by the five temperature- sensitive mutations clustered in the middle of SEC16. Each of these mutations can be complemented by the central domain of SEC16 expressed alone. The stoichiometry of Sec16p is critical for secretory function since overexpression of Sec16p causes a lethal secretion defect. This lethal function maps to the NH2-terminus of the protein, defining a second functional domain. A separate function for the COOH-terminal domain of Sec16p is shown by its ability to bind Sec23p. Together, these results suggest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.     

Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery.

SEC62 is required for the import of secretory protein precursors into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. The DNA sequence of SEC62 predicts a 32-kDa polypeptide with two potential membrane-spanning segments. Two antisera directed against different portions of the SEC62 coding region specifically detected a 30-kDa polypeptide in cell extracts. A combination of subcellular fractionation, detergent and alkali extraction, and indirect immunofluorescence studies indicated that Sec62p is intimately associated with the ER membrane. Protease digestion of intact microsomes and analysis of the oligosaccharide content of a set of Sec62p-invertase hybrid proteins suggested that Sec62p spans the ER membrane twice, displaying hydrophilic amino- and carboxy-terminal domains towards the cytosol. Sec62p-invertase hybrid proteins that lack the Sec62p C terminus failed to complement the sec62-l mutation and dramatically inhibited the growth of sec62-l cells at a normally permissive temperature. The inhibitory action of toxic Sec62p-invertase hybrids was partially counteracted by the overexpression of Sec63p. Taken together, these data suggest that the C-terminal domain of Sec62p performs an essential function and that the N-terminal domain associates with other components of the translocation machinery, including Sec63p.     

Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.