Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Jul 1;105(1):163–174. doi: 10.1083/jcb.105.1.163

Purification and characterization of constitutive secretory vesicles from yeast

PMCID: PMC2114885  PMID: 3301865

Abstract

We have developed a purification procedure for the isolation of constitutive post-Golgi secretory vesicles from Saccharomyces cerevisiae. Although the post-Golgi stage of the secretion pathway is normally very rapid, we have used a temperature-sensitive secretory mutant, sec 6-4, to greatly expand the population of secretory vesicles. Following invertase as a marker, intact vesicles are enriched 36-fold from the crude lysate. The final preparation contains few contaminants as assessed by morphologic and biochemical examination. Three proteins (110, 40-45, and 18 kD) co-purify with the vesicle marker enzyme invertase. Metabolic labeling experiments indicate that these vesicle-associated proteins are synthesized during the period of vesicle accumulation. They are not apparent in the corresponding fractions from wild-type cells. Analysis of these proteins indicates that the 110-kD protein is a major glycoprotein residing in the vesicle lumen, while the 40-45- and 18-kD proteins are not glycosylated and are firmly associated with the vesicle membrane, each with at least one domain exposed on the cytoplasmic surface.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

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

  1. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  2. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  3. Bowman B. J., Slayman C. W. The effects of vanadate on the plasma membrane ATPase of Neurospora crassa. J Biol Chem. 1979 Apr 25;254(8):2928–2934. [PubMed] [Google Scholar]
  4. Cameron R. S., Cameron P. L., Castle J. D. A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands. J Cell Biol. 1986 Oct;103(4):1299–1313. doi: 10.1083/jcb.103.4.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlson S. S., Wagner J. A., Kelly R. B. Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity. Biochemistry. 1978 Apr 4;17(7):1188–1199. doi: 10.1021/bi00600a009. [DOI] [PubMed] [Google Scholar]
  6. Dunphy W. G., Rothman J. E. Compartmental organization of the Golgi stack. Cell. 1985 Aug;42(1):13–21. doi: 10.1016/s0092-8674(85)80097-0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Gumbiner B., Kelly R. B. Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):318–322. doi: 10.1073/pnas.78.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Howell K. E., Palade G. E. Hepatic Golgi fractions resolved into membrane and content subfractions. J Cell Biol. 1982 Mar;92(3):822–832. doi: 10.1083/jcb.92.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huttner W. B., Schiebler W., Greengard P., De Camilli P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol. 1983 May;96(5):1374–1388. doi: 10.1083/jcb.96.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jahn R., Schiebler W., Ouimet C., Greengard P. A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4137–4141. doi: 10.1073/pnas.82.12.4137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. 3. Dissociation of intracellular transport from protein synthesis. J Cell Biol. 1968 Dec;39(3):580–588. doi: 10.1083/jcb.39.3.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J Cell Biol. 1967 Aug;34(2):577–596. doi: 10.1083/jcb.34.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J Cell Biol. 1967 Aug;34(2):597–615. doi: 10.1083/jcb.34.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J Cell Biol. 1968 Dec;39(3):589–603. doi: 10.1083/jcb.39.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kelly R. B. Pathways of protein secretion in eukaryotes. Science. 1985 Oct 4;230(4721):25–32. doi: 10.1126/science.2994224. [DOI] [PubMed] [Google Scholar]
  18. Kreibich G., Debey P., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations. J Cell Biol. 1973 Aug;58(2):436–462. doi: 10.1083/jcb.58.2.436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Makarow M. Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts. EMBO J. 1985 Jul;4(7):1855–1860. doi: 10.1002/j.1460-2075.1985.tb03860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mason T. L., Poyton R. O., Wharton D. C., Schatz G. Cytochrome c oxidase from bakers' yeast. I. Isolation and properties. J Biol Chem. 1973 Feb 25;248(4):1346–1354. [PubMed] [Google Scholar]
  23. Mueller S. C., Branton D. Identification of coated vesicles in Saccharomyces cerevisiae. J Cell Biol. 1984 Jan;98(1):341–346. doi: 10.1083/jcb.98.1.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nagy A., Baker R. R., Morris S. J., Whittaker V. P. The preparation and characterization of synaptic vesicles of high purity. Brain Res. 1976 Jun 11;109(2):285–309. doi: 10.1016/0006-8993(76)90531-x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Novick P., Schekman R. Export of major cell surface proteins is blocked in yeast secretory mutants. J Cell Biol. 1983 Feb;96(2):541–547. doi: 10.1083/jcb.96.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Opheim D. J. alpha-D-Mannosidase of Saccharomyces cerevisiae. Characterization and modulation of activity. Biochim Biophys Acta. 1978 May 11;524(1):121–130. doi: 10.1016/0005-2744(78)90110-9. [DOI] [PubMed] [Google Scholar]
  30. Orci L., Glick B. S., Rothman J. E. A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell. 1986 Jul 18;46(2):171–184. doi: 10.1016/0092-8674(86)90734-8. [DOI] [PubMed] [Google Scholar]
  31. Orci L., Ravazzola M., Amherdt M., Madsen O., Vassalli J. D., Perrelet A. Direct identification of prohormone conversion site in insulin-secreting cells. Cell. 1985 Sep;42(2):671–681. doi: 10.1016/0092-8674(85)90124-2. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Payne G. S., Schekman R. A test of clathrin function in protein secretion and cell growth. Science. 1985 Nov 29;230(4729):1009–1014. doi: 10.1126/science.2865811. [DOI] [PubMed] [Google Scholar]
  34. Rodriguez Boulan E., Pendergast M. Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells. Cell. 1980 May;20(1):45–54. doi: 10.1016/0092-8674(80)90233-0. [DOI] [PubMed] [Google Scholar]
  35. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Tulsiani D. R., Opheim D. J., Touster O. Purification and characterization of alpha-D-mannosidase from rat liver golgi membranes. J Biol Chem. 1977 May 25;252(10):3227–3233. [PubMed] [Google Scholar]
  38. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  39. Wagner J. A., Kelly R. B. Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4126–4130. doi: 10.1073/pnas.76.8.4126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  42. Willsky G. R. Characterization of the plasma membrane Mg2+-ATPase from the yeast, Saccharomyces cerevisiae. J Biol Chem. 1979 May 10;254(9):3326–3332. [PubMed] [Google Scholar]

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

RESOURCES