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PMID: 8432727 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.

The Journal of cell biology ·Vol. 120 ·No. 4 ·1993-02-00 ·Pages 865-75

Pryer NK, Salama NR, Schekman R, Kaiser CA

Abstract

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.

Related Genes
SEC13
MeSH Terms
Alleles Amino Acid Sequence Base Sequence Cloning, Molecular Consensus Sequence Endoplasmic Reticulum/metabolism,physiology Fungal Proteins/metabolism Genes, Fungal Macromolecular Substances Membrane Proteins/metabolism Molecular Sequence Data Mutagenesis, Insertional Nuclear Pore Complex Proteins Organelles/metabolism Restriction Mapping Saccharomyces cerevisiae/metabolism Saccharomyces cerevisiae Proteins Sequence Alignment
Chemicals
Fungal Proteins Macromolecular Substances Membrane Proteins Nuclear Pore Complex Proteins SEC13 protein, S cerevisiae Saccharomyces cerevisiae Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Pryer N K
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Salama N R
Schekman R
Kaiser C A
References (46)
46 references, click to expand
  1. The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein.
    J Biol Chem. 1992 Jun 15;267(17):12106-15 PMID: 1601878
  2. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  3. Reconstitution of SEC gene product-dependent intercompartmental protein transport.
    Cell. 1988 Jul 29;54(3):335-44 PMID: 3293799
  4. Fusions to staphylococcal protein A.
    Methods Enzymol. 1990;185:144-61 PMID: 2199777
  5. 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):3405-14 PMID: 2192256
  6. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins.
    EMBO J. 1984 Jun;3(6):1429-34 PMID: 6086324
  7. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway.
    Cell. 1990 May 18;61(4):723-33 PMID: 2188733
  8. Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily.
    Mol Cell Biol. 1991 Feb;11(2):872-85 PMID: 1990290
  9. A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast.
    J Cell Biol. 1988 Sep;107(3):851-63 PMID: 3047151
  10. Purification and characterization of a heat-shock element binding protein from yeast.
    EMBO J. 1987 Oct;6(10):3035-41 PMID: 3319580
  11. The structure of transposable yeast mating type loci.
    Cell. 1980 Mar;19(3):753-64 PMID: 6244896
  12. Yeast recombination: the association between double-strand gap repair and crossing-over.
    Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417-21 PMID: 6308623
  13. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
    Genetics. 1989 May;122(1):19-27 PMID: 2659436
  14. Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast.
    Methods Enzymol. 1992;219:124-36 PMID: 1487986
  15. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole.
    Cell. 1982 Sep;30(2):439-48 PMID: 6754086
  16. Comparative analysis of the beta transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1.
    Proteins. 1992 May;13(1):41-56 PMID: 1594577
  17. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs.
    Proc Natl Acad Sci U S A. 1986 Apr;83(7):2162-6 PMID: 3083416
  18. 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):2677-91 PMID: 2512296
  19. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery.
    Cell. 1988 Mar 25;52(6):915-24 PMID: 3127057
  20. A simplified method for the quantitative assay of small amounts of protein in biologic material.
    Anal Biochem. 1973 Feb;51(2):654-5 PMID: 4735559
  21. Vesicle-mediated protein sorting.
    Annu Rev Biochem. 1992;61:471-516 PMID: 1497318
  22. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
  23. Involvement of GTP-binding "G" proteins in transport through the Golgi stack.
    Cell. 1987 Dec 24;51(6):1053-62 PMID: 2826014
  24. Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant.
    J Cell Biol. 1988 Oct;107(4):1465-76 PMID: 3049622
  25. Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum.
    Mol Cell Biol. 1991 Nov;11(11):5727-34 PMID: 1922074
  26. Proteinase mutants of Saccharomyces cerevisiae.
    Genetics. 1977 Jan;85(1):23-33 PMID: 320092
  27. Order of events in the yeast secretory pathway.
    Cell. 1981 Aug;25(2):461-9 PMID: 7026045
  28. The product of the PRP4 gene of S. cerevisiae shows homology to beta subunits of G proteins.
    Cell. 1989 Sep 8;58(5):811-2 PMID: 2528410
  29. A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells.
    J Cell Biol. 1991 Sep;114(6):1113-24 PMID: 1910049
  30. Purification of dihydrofolic reductase from chicken liver by affinity chromatography.
    Biochem Biophys Res Commun. 1971 Aug 6;44(3):608-13 PMID: 5123199
  31. Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway.
    Cell. 1984 Feb;36(2):309-18 PMID: 6420074
  32. A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae.
    J Cell Biol. 1991 Apr;113(2):245-60 PMID: 2010462
  33. Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER.
    J Cell Biol. 1991 Aug;114(4):663-70 PMID: 1907973
  34. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway.
    Cell. 1980 Aug;21(1):205-15 PMID: 6996832
  35. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity.
    Anal Biochem. 1981 Nov 1;117(2):307-10 PMID: 6172996
  36. Unidirectional digestion with exonuclease III in DNA sequence analysis.
    Methods Enzymol. 1987;155:156-65 PMID: 3323819
  37. Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro.
    EMBO J. 1989 Jun;8(6):1677-84 PMID: 2670558
  38. Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.
    J Cell Biol. 1991 Aug;114(4):671-9 PMID: 1907974
  39. Dihydrofolate reductase from a resistant subline of the L1210 lymphoma. Purification by affinity chromatography and ultraviolet difference spectrophotometric and circular dichroic studies.
    Biochemistry. 1977 Jul 12;16(14):3073-9 PMID: 19040
  40. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.
    Mol Cell Biol. 1986 Jul;6(7):2490-9 PMID: 3023936
  41. 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):1587-94 PMID: 3312234
  42. 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):1058-63 PMID: 1985933
  43. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction.
    Proc Natl Acad Sci U S A. 1990 Jan;87(1):355-9 PMID: 2104983
  44. Homologous Recombination between Episomal Plasmids and Chromosomes in Yeast.
    Genetics. 1983 Dec;105(4):843-56 PMID: 17246179
  45. 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):667-76 PMID: 1498369
  46. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles.
    J Cell Biol. 1991 Jul;114(2):219-29 PMID: 1649197
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.120.4.865
Published
1993-02-00
Pages
865-75
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2200066
Subset
IM
Grants
NIGMS NIH HHS · GM 26755 · United States
Databases
GENBANK
L05929
External Links
PubMed source DOI: 10.1083/jcb.120.4.865 PMC Full Text View journal details
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