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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...

A and alpha cells of the yeast Saccharomyces cerevisiae exhibit an axial budding pattern, whereas a/alpha diploid cells exhibit a bipolar pattern. Mutations in BUD3, BUD4, and AXL1 cause a and alpha cells to exhibit the bipolar pattern, indicating that these genes are necessary to specify the axial budding pattern (Chant, J., and I. Herskowitz. 1991. Cell. 65:1203-1212; Fujita, A., C. Oka, Y. Arikawa, T. Katagi, A. Tonouchi, S. Kuhara, and Y. Misumi. 1994. Nature (Lond.). 372:567-570). We cloned and sequenced BUD4, which codes for a large, novel protein (Bud4p) with a potential GTP-binding motif. Bud4p is expressed and localized to the mother/bud neck in all cell types. Most mitotic cells contain two apparent rings of Bud4 immunoreactive staining, as observed for Bud3p (Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J.R. Pringle. 1995. J. Cell Biol. 129: 767-778). Early G1 cells contain a single ring of Bud4p immunoreactive staining, whereas cells at START and in S phase lack these rings. The level of Bud4p is also regulated in a cell cycle-dependent manner. Bud4p is inefficiently localized in bud3 mutants and after a temperature shift of a temperature-sensitive mutant, cdc12, defective in the neck filaments. These observations suggest that Bud4p and Bud3p cooperate to recognize a spatial landmark (the neck filaments) during mitosis and support the hypothesis that they subsequently become a landmark for establishing the axial budding pattern in G1.

Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by integral membrane proteins. Genetic selections uncovered three Saccharomyces cerevisiae genes (SEC61, SEC62 and SEC63), mutations in which block import of precursor proteins into the ER lumen in vivo and in vitro. The DNA sequences of SEC62 and SEC63 predict multispanning membrane proteins, and biochemical characterization of the SEC62 protein (Sec62) confirms that it is an integral ER membrane protein. Here we show that Sec61, Sec62 and Sec63 are assembled with two additional proteins into a multisubunit membrane-associated complex. These results confirm previous predictions, based upon genetic interactions between the SEC genes, that Sec61, Sec62 and Sec63 act together to facilitate protein translocation into the ER.