Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Aug 1;105(2):633–645. doi: 10.1083/jcb.105.2.633

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum

PMCID: PMC2114772  PMID: 3305520

Abstract

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bernstein M., Hoffmann W., Ammerer G., Schekman R. Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum. J Cell Biol. 1985 Dec;101(6):2374–2382. doi: 10.1083/jcb.101.6.2374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Briggs M. S., Gierasch L. M., Zlotnick A., Lear J. D., DeGrado W. F. In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides. Science. 1985 May 31;228(4703):1096–1099. doi: 10.1126/science.3158076. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Connolly T., Gilmore R. Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein. J Cell Biol. 1986 Dec;103(6 Pt 1):2253–2261. doi: 10.1083/jcb.103.6.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Emr S. D., Schauer I., Hansen W., Esmon P., Schekman R. Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2347–2355. doi: 10.1128/mcb.4.11.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Emr S. D., Schekman R., Flessel M. C., Thorner J. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7080–7084. doi: 10.1073/pnas.80.23.7080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Esmon B., Novick P., Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell. 1981 Aug;25(2):451–460. doi: 10.1016/0092-8674(81)90063-5. [DOI] [PubMed] [Google Scholar]
  10. Evans E. A., Gilmore R., Blobel G. Purification of microsomal signal peptidase as a complex. Proc Natl Acad Sci U S A. 1986 Feb;83(3):581–585. doi: 10.1073/pnas.83.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feldman R. I., Bernstein M., Schekman R. Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum. J Biol Chem. 1987 Jul 5;262(19):9332–9339. [PubMed] [Google Scholar]
  12. 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]
  13. Ferro-Novick S., Novick P., Field C., Schekman R. Yeast secretory mutants that block the formation of active cell surface enzymes. J Cell Biol. 1984 Jan;98(1):35–43. doi: 10.1083/jcb.98.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilmore R., Blobel G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell. 1985 Sep;42(2):497–505. doi: 10.1016/0092-8674(85)90107-2. [DOI] [PubMed] [Google Scholar]
  15. Gilmore R., Walter P., Blobel G. Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J Cell Biol. 1982 Nov;95(2 Pt 1):470–477. doi: 10.1083/jcb.95.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
  17. Hemmings B. A., Zubenko G. S., Hasilik A., Jones E. W. Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Jan;78(1):435–439. doi: 10.1073/pnas.78.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoffmann W. Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae. A transmembrane protein without N-terminal hydrophobic signal sequence. J Biol Chem. 1985 Sep 25;260(21):11831–11837. [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ito K., Bassford P. J., Jr, Beckwith J. Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell. 1981 Jun;24(3):707–717. doi: 10.1016/0092-8674(81)90097-0. [DOI] [PubMed] [Google Scholar]
  22. Ito K., Wittekind M., Nomura M., Shiba K., Yura T., Miura A., Nashimoto H. A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins. Cell. 1983 Mar;32(3):789–797. doi: 10.1016/0092-8674(83)90065-x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kaiser C. A., Botstein D. Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase. Mol Cell Biol. 1986 Jul;6(7):2382–2391. doi: 10.1128/mcb.6.7.2382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Keesey J. K., Jr, Bigelis R., Fink G. R. The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide. J Biol Chem. 1979 Aug 10;254(15):7427–7433. [PubMed] [Google Scholar]
  26. Kubota S., Yoshida Y., Kumaoka H., Furumichi A. Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. J Biochem. 1977 Jan;81(1):197–205. doi: 10.1093/oxfordjournals.jbchem.a131436. [DOI] [PubMed] [Google Scholar]
  27. Kumamoto C. A., Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol. 1983 Apr;154(1):253–260. doi: 10.1128/jb.154.1.253-260.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kurjan J., Herskowitz I. Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell. 1982 Oct;30(3):933–943. doi: 10.1016/0092-8674(82)90298-7. [DOI] [PubMed] [Google Scholar]
  29. Kurzchalia T. V., Wiedmann M., Girshovich A. S., Bochkareva E. S., Bielka H., Rapoport T. A. The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nature. 1986 Apr 17;320(6063):634–636. doi: 10.1038/320634a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  32. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  34. Nakayama N., Miyajima A., Arai K. Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae. EMBO J. 1985 Oct;4(10):2643–2648. doi: 10.1002/j.1460-2075.1985.tb03982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Novick P., Schekman R. Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1858–1862. doi: 10.1073/pnas.76.4.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ohno-Iwashita Y., Wolfe P., Ito K., Wickner W. Processing of preproteins by liposomes bearing leader peptidase. Biochemistry. 1984 Dec 4;23(25):6178–6184. doi: 10.1021/bi00320a044. [DOI] [PubMed] [Google Scholar]
  39. Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
  40. Parker R., Guthrie C. A point mutation in the conserved hexanucleotide at a yeast 5' splice junction uncouples recognition, cleavage, and ligation. Cell. 1985 May;41(1):107–118. doi: 10.1016/0092-8674(85)90065-0. [DOI] [PubMed] [Google Scholar]
  41. Perlman D., Halvorson H. O., Cannon L. E. Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence. Proc Natl Acad Sci U S A. 1982 Feb;79(3):781–785. doi: 10.1073/pnas.79.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Rothblatt J. A., Meyer D. I. Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism. EMBO J. 1986 May;5(5):1031–1036. doi: 10.1002/j.1460-2075.1986.tb04318.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  45. Schauer I., Emr S., Gross C., Schekman R. Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985 May;100(5):1664–1675. doi: 10.1083/jcb.100.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Scott J. H., Schekman R. Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol. 1980 May;142(2):414–423. doi: 10.1128/jb.142.2.414-423.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sharma S., Rodgers L., Brandsma J., Gething M. J., Sambrook J. SV40 T antigen and the exocytotic pathway. EMBO J. 1985 Jun;4(6):1479–1489. doi: 10.1002/j.1460-2075.1985.tb03806.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Simon K., Perara E., Lingappa V. R. Translocation of globin fusion proteins across the endoplasmic reticulum membrane in Xenopus laevis oocytes. J Cell Biol. 1987 May;104(5):1165–1172. doi: 10.1083/jcb.104.5.1165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  51. Tanaka J., Fink G. R. The histidine permease gene (HIP1) of Saccharomyces cerevisiae. Gene. 1985;38(1-3):205–214. doi: 10.1016/0378-1119(85)90219-7. [DOI] [PubMed] [Google Scholar]
  52. Valls L. A., Hunter C. P., Rothman J. H., Stevens T. H. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. doi: 10.1016/0092-8674(87)90085-7. [DOI] [PubMed] [Google Scholar]
  53. Walter P., Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. doi: 10.1073/pnas.77.12.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Walter P., Blobel G. Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature. 1982 Oct 21;299(5885):691–698. doi: 10.1038/299691a0. [DOI] [PubMed] [Google Scholar]
  55. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Waters M. G., Blobel G. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol. 1986 May;102(5):1543–1550. doi: 10.1083/jcb.102.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Waters M. G., Chirico W. J., Blobel G. Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor. J Cell Biol. 1986 Dec;103(6 Pt 2):2629–2636. doi: 10.1083/jcb.103.6.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wickerham L. J. A Critical Evaluation of the Nitrogen Assimilation Tests Commonly Used in the Classification of Yeasts. J Bacteriol. 1946 Sep;52(3):293–301. [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES