Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec6l, 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 sec6l 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 a-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 Sec6l protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec6lp antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec6lp appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

Abstract. 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°C and show a marked enhancement of the translocation defect. The transloeation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-a-factor (ppctF) 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 ppc~F into the ER can proceed posttranslationally.I s spite of significant advances that have clarified the structure and function of molecules that mediate targeting of secretory proteins to the endoplasmic reticulum (for review, see Walter and Lingappa, 1986;, the actual process of polypeptide transfer across the ER membrane is poorly understood. A common view is that a proteinaceous translocation pore complex within the ER membrane facilitates transfer of the hydrophilic nascent polypeptide across the hydrophobic core of the ER membrane (Blobel and Dobberstein, 1975;Blobel, 1980). The participation of membrane proteins in translocation, as well as in targeting, is indicated by the fact that translocation activity of microsomes is sensitive to proteolysis and chemical alkylation (Walter et al., 1979; Dobberstein, 1980a, 1980b;Gilmore et al., 1982;. Translocating proteins appear to lie within a polar environment in the bilayer because intermediates interrupted in penetration may be solubilized by agents that disrupt protein structure without solubilizing membrane lipids (Gilmore and Blobel, 1985). A lack of specific probes or inhibitors has frustrated biochemical approaches to identifying functional translocator components. Recently, an ER membrane pro-J. A. Rothblatt's present address is the Department of Biological Sciences, Dartmouth College, Hanover, NH 03755. G. Daum was on sabbatical leave from the Institut fiir Biochemie and Lebensmittelchemie, Technische Universitat Graz, A-8010 Graz, Austria. tein, termed signal sequence receptor (SSR), t that interacts directly with the signal peptide of nascent proteins has been identified by chemical cross-linking (Wiedmann e...

). 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 Hsp7O (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 Sec6lp, 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/Sec6lp/gp3l.5/p23 complex.We have previously described a set of temperature-sensitive lethal mutants of Saccharomyces cerevisiae that fail to localize a signal peptide-bearing cytosolic enzyme chimera to the lumen of the endoplasmic reticulum (ER) (14, 38). Characterization of these mutants showed that the products of four genes, SEC61, SEC62, SEC63, and SEC65, are required for translocation of secretory precursor proteins across the ER membrane (14, 38, 45a). Additional alleles of SEC63 that inhibit protein import into the nucleus have also been described (39). In addition to the SEC genes described above, several other genes are required for the proper targeting and insertion of presecretory proteins into the ER. Hsp7O homologs appear to be required in the cytosol and ER lumen for translocation to occur. Cytosolic Hsp7O, encoded by the SSA genes, is required for efficient translocation of prepro-a-factor into the ER both in vivo and in vitro (8,12). An ER luminal Hsp7O homolog, BiP (the KAR2 gene product), has been identified, and some alleles of this gene block translocation of presecretory proteins (33,37,50 pletely purified replication system, the DnaJ and DnaK proteins are required to mediate the disassembly of the A 0-some complex, which activates the helicase activity of DnaB protein to initiate X DNA replication (2, 27, 52). The homology of Sec63p to DnaJ and the requirement for Hsp7O homologs for efficient translocation suggest that one role of Sec63p may be to interact with an Hsp7O homolog to promote protein translocation.To gain a better understanding of Sec63p and its function, we have analyzed the int...

Abstract. When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome ca, 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 c~, but expressing the hybrid NLScytochrome c, 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 c, to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPLI) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein, npU-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 npU 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 np11/sec63 cells at the nonpermissive temperature. Thus, NPLI/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, npll may indirectly alter assembly of proteins into the nucleus.ACH organelle in a eukaryotic cell has a distinct set of proteins that are necessary for its specific function. Certain peptides can act as signals to localize proteins to particular organelles such as the ER, the mitochondria (Verner and Schatz, 1988) and the nucleus (Silver and Hall, 1988). Several proteins have been identified that mediate the recognition of ER-destined proteins and their subsequent translocation across or assembly into the ER membrane (Walter and Blobel, 1980;Meyer et al., 1982;Tajima et al., 1986; Wiedrnan et al., 1987). Receptors have been proposed for mitochondrial signal peptides (Pfaller and Neupert, 1987;Pfanner et al., 1987) and recently a receptor for protein import into chloroplasts has been identified (Pain et al., 1988). By analogy, similar components may exist for localization of proteins to the nucleus.Nuclear localization sequences (NLS)' are stretches of amino acids that are capable of redirecting nonnuclear pro-1. Abbreviations used in this paper: DAPI, diamidinophenylindole; EMS, ethyl methanesulfonate; NLS, nuclear localization sequences; preproCPY, preprocarboxypeptidase Y.teins such as/$-galactosidase to the nucleus. When the first 74 amino acids of the yeast DNA binding protein GAL4 are joined to/~-galactosidase, the result is a fusion protein that is found exclusively in the yeast nucleus as...