Abstract
We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.
Full Text
The Full Text of this article is available as a PDF (8.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bacon R. A., Salminen A., Ruohola H., Novick P., Ferro-Novick S. The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants. J Cell Biol. 1989 Sep;109(3):1015–1022. doi: 10.1083/jcb.109.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barlowe C., Orci L., Yeung T., Hosobuchi M., Hamamoto S., Salama N., Rexach M. F., Ravazzola M., Amherdt M., Schekman R. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994 Jun 17;77(6):895–907. doi: 10.1016/0092-8674(94)90138-4. [DOI] [PubMed] [Google Scholar]
- Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
- Deshaies R. J., Schekman R. A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J Cell Biol. 1987 Aug;105(2):633–645. doi: 10.1083/jcb.105.2.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duden R., Hosobuchi M., Hamamoto S., Winey M., Byers B., Schekman R. Yeast beta- and beta'-coat proteins (COP). Two coatomer subunits essential for endoplasmic reticulum-to-Golgi protein traffic. J Biol Chem. 1994 Sep 30;269(39):24486–24495. [PubMed] [Google Scholar]
- Ferro-Novick S., Hansen W., Schauer I., Schekman R. Genes required for completion of import of proteins into the endoplasmic reticulum in yeast. J Cell Biol. 1984 Jan;98(1):44–53. doi: 10.1083/jcb.98.1.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franzusoff A., Redding K., Crosby J., Fuller R. S., Schekman R. Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J Cell Biol. 1991 Jan;112(1):27–37. doi: 10.1083/jcb.112.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franzusoff A., Schekman R. Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation. EMBO J. 1989 Sep;8(9):2695–2702. doi: 10.1002/j.1460-2075.1989.tb08410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gaynor E. C., te Heesen S., Graham T. R., Aebi M., Emr S. D. Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J Cell Biol. 1994 Nov;127(3):653–665. doi: 10.1083/jcb.127.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldstein A., Lampen J. O. Beta-D-fructofuranoside fructohydrolase from yeast. Methods Enzymol. 1975;42:504–511. doi: 10.1016/0076-6879(75)42159-0. [DOI] [PubMed] [Google Scholar]
- Hardwick K. G., Pelham H. R. SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J Cell Biol. 1992 Nov;119(3):513–521. doi: 10.1083/jcb.119.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hasilik A., Tanner W. Carbohydrate moiety of carboxypeptidase Y and perturbation of its biosynthesis. Eur J Biochem. 1978 Nov 15;91(2):567–575. doi: 10.1111/j.1432-1033.1978.tb12710.x. [DOI] [PubMed] [Google Scholar]
- Hicke L., Yoshihisa T., Schekman R. Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum. Mol Biol Cell. 1992 Jun;3(6):667–676. doi: 10.1091/mbc.3.6.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hosobuchi M., Kreis T., Schekman R. SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer. Nature. 1992 Dec 10;360(6404):603–605. doi: 10.1038/360603a0. [DOI] [PubMed] [Google Scholar]
- Julius D., Schekman R., Thorner J. Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell. 1984 Feb;36(2):309–318. doi: 10.1016/0092-8674(84)90224-1. [DOI] [PubMed] [Google Scholar]
- Kaiser C. A., Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
- Klionsky D. J., Herman P. K., Emr S. D. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. doi: 10.1128/mr.54.3.266-292.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Letourneur F., Gaynor E. C., Hennecke S., Démollière C., Duden R., Emr S. D., Riezman H., Cosson P. Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell. 1994 Dec 30;79(7):1199–1207. doi: 10.1016/0092-8674(94)90011-6. [DOI] [PubMed] [Google Scholar]
- Lyons S., Nelson N. An immunological method for detecting gene expression in yeast colonies. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7426–7430. doi: 10.1073/pnas.81.23.7426. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakajima H., Hirata A., Ogawa Y., Yonehara T., Yoda K., Yamasaki M. A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol. 1991 Apr;113(2):245–260. doi: 10.1083/jcb.113.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakańo A., Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Newman A. P., Ferro-Novick S. Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection. J Cell Biol. 1987 Oct;105(4):1587–1594. doi: 10.1083/jcb.105.4.1587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Newman A. P., Shim J., Ferro-Novick S. BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex. Mol Cell Biol. 1990 Jul;10(7):3405–3414. doi: 10.1128/mcb.10.7.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novick P., Ferro S., Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. doi: 10.1016/0092-8674(81)90064-7. [DOI] [PubMed] [Google Scholar]
- Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
- Ossig R., Dascher C., Trepte H. H., Schmitt H. D., Gallwitz D. The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport. Mol Cell Biol. 1991 Jun;11(6):2980–2993. doi: 10.1128/mcb.11.6.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
- Pelham H. R. About turn for the COPs? Cell. 1994 Dec 30;79(7):1125–1127. doi: 10.1016/0092-8674(94)90002-7. [DOI] [PubMed] [Google Scholar]
- Perlman D., Halvorson H. O. Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene. Cell. 1981 Aug;25(2):525–536. doi: 10.1016/0092-8674(81)90071-4. [DOI] [PubMed] [Google Scholar]
- Preuss D., Mulholland J., Franzusoff A., Segev N., Botstein D. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol Biol Cell. 1992 Jul;3(7):789–803. doi: 10.1091/mbc.3.7.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Preuss D., Mulholland J., Kaiser C. A., Orlean P., Albright C., Rose M. D., Robbins P. W., Botstein D. Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy. Yeast. 1991 Dec;7(9):891–911. doi: 10.1002/yea.320070902. [DOI] [PubMed] [Google Scholar]
- Pryer N. K., Salama N. R., Schekman R., Kaiser C. A. Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro. J Cell Biol. 1993 Feb;120(4):865–875. doi: 10.1083/jcb.120.4.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pryer N. K., Wuestehube L. J., Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. doi: 10.1146/annurev.bi.61.070192.002351. [DOI] [PubMed] [Google Scholar]
- Redding K., Holcomb C., Fuller R. S. Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae. J Cell Biol. 1991 May;113(3):527–538. doi: 10.1083/jcb.113.3.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts C. J., Pohlig G., Rothman J. H., Stevens T. H. Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole. J Cell Biol. 1989 Apr;108(4):1363–1373. doi: 10.1083/jcb.108.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts C. J., Raymond C. K., Yamashiro C. T., Stevens T. H. Methods for studying the yeast vacuole. Methods Enzymol. 1991;194:644–661. doi: 10.1016/0076-6879(91)94047-g. [DOI] [PubMed] [Google Scholar]
- Robinson J. S., Graham T. R., Emr S. D. A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation. Mol Cell Biol. 1991 Dec;11(12):5813–5824. doi: 10.1128/mcb.11.12.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothblatt J. A., Deshaies R. J., Sanders S. L., Daum G., Schekman R. Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast. J Cell Biol. 1989 Dec;109(6 Pt 1):2641–2652. doi: 10.1083/jcb.109.6.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothblatt J. A., Meyer D. I. Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986 Feb 28;44(4):619–628. doi: 10.1016/0092-8674(86)90271-0. [DOI] [PubMed] [Google Scholar]
- Rothblatt J., Schekman R. A hitchhiker's guide to analysis of the secretory pathway in yeast. Methods Cell Biol. 1989;32:3–36. doi: 10.1016/s0091-679x(08)61165-6. [DOI] [PubMed] [Google Scholar]
- Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
- Salama N. R., Yeung T., Schekman R. W. The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins. EMBO J. 1993 Nov;12(11):4073–4082. doi: 10.1002/j.1460-2075.1993.tb06091.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmitt H. D., Puzicha M., Gallwitz D. Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium. Cell. 1988 May 20;53(4):635–647. doi: 10.1016/0092-8674(88)90579-x. [DOI] [PubMed] [Google Scholar]
- Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
- Shen S. H., Chrétien P., Bastien L., Slilaty S. N. Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli. J Biol Chem. 1991 Jan 15;266(2):1058–1063. [PubMed] [Google Scholar]
- Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
- Stevens T., Esmon B., Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
- Stirling C. J., Rothblatt J., Hosobuchi M., Deshaies R., Schekman R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. Mol Biol Cell. 1992 Feb;3(2):129–142. doi: 10.1091/mbc.3.2.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waters M. G., Serafini T., Rothman J. E. 'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature. 1991 Jan 17;349(6306):248–251. doi: 10.1038/349248a0. [DOI] [PubMed] [Google Scholar]
- Wilsbach K., Payne G. S. Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. EMBO J. 1993 Aug;12(8):3049–3059. doi: 10.1002/j.1460-2075.1993.tb05974.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wright R., Basson M., D'Ari L., Rine J. Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus. J Cell Biol. 1988 Jul;107(1):101–114. doi: 10.1083/jcb.107.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]