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J Cell Biol. 1988 October 1; 107(4): 1465–1476.
PMCID: PMC2115264

Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant

Abstract

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Balch WE, Dunphy WG, Braell WA, Rothman JE. 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. [PubMed]
  • Balch WE, Elliott MM, Keller DS. ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi. J Biol Chem. 1986 Nov 5;261(31):14681–14689. [PubMed]
  • Ballou L, Cohen RE, Ballou CE. Saccharomyces cerevisiae mutants that make mannoproteins with a truncated carbohydrate outer chain. J Biol Chem. 1980 Jun 25;255(12):5986–5991. [PubMed]
  • Bangs JD, Andrews NW, Hart GW, Englund PT. Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein. J Cell Biol. 1986 Jul;103(1):255–263. [PMC free article] [PubMed]
  • Beckers CJ, Keller DS, Balch WE. Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell. 1987 Aug 14;50(4):523–534. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Chamberlain JP. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. [PubMed]
  • Chu FK, Trimble RB, Maley F. The effect of carbohydrate depletion on the properties of yeast external invertase. J Biol Chem. 1978 Dec 25;253(24):8691–8693. [PubMed]
  • Esmon B, Novick P, Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell. 1981 Aug;25(2):451–460. [PubMed]
  • Farquhar MG, Palade GE. The Golgi apparatus (complex)-(1954-1981)-from artifact to center stage. J Cell Biol. 1981 Dec;91(3 Pt 2):77s–103s. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Glick BS, Rothman JE. Possible role for fatty acyl-coenzyme A in intracellular protein transport. Nature. 1987 Mar 19;326(6110):309–312. [PubMed]
  • Goud B, Salminen A, Walworth NC, Novick PJ. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. [PubMed]
  • Hansen W, Garcia PD, 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. [PubMed]
  • Haselbeck A, Schekman R. Interorganelle transfer and glycosylation of yeast invertase in vitro. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2017–2021. [PubMed]
  • Jamieson JD, Palade GE. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J Cell Biol. 1968 Dec;39(3):589–603. [PMC free article] [PubMed]
  • Julius D, Brake A, Blair L, Kunisawa R, Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell. 1984 Jul;37(3):1075–1089. [PubMed]
  • 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. [PubMed]
  • Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. [PubMed]
  • 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. [PubMed]
  • Kukuruzinska MA, Bergh ML, Jackson BJ. Protein glycosylation in yeast. Annu Rev Biochem. 1987;56:915–944. [PubMed]
  • Lodish HF, Kong N, Hirani S, Rasmussen J. A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex. J Cell Biol. 1987 Feb;104(2):221–230. [PMC free article] [PubMed]
  • Melançon P, Glick BS, Malhotra V, Weidman PJ, Serafini T, Gleason ML, Orci L, Rothman JE. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. [PubMed]
  • Meyer DI, Krause E, Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. [PubMed]
  • Nakajima T, Ballou CE. Yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3912–3916. [PubMed]
  • Newman AP, 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. [PMC free article] [PubMed]
  • Novick P, Ferro S, Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. [PubMed]
  • 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. [PubMed]
  • Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. [PubMed]
  • Reid GA, Schatz G. Import of proteins into mitochondria. Extramitochondrial pools and post-translational import of mitochondrial protein precursors in vivo. J Biol Chem. 1982 Nov 10;257(21):13062–13067. [PubMed]
  • Rothblatt JA, Meyer DI. 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. [PubMed]
  • Ruohola H, Ferro-Novick S. Sec53, a protein required for an early step in secretory protein processing and transport in yeast, interacts with the cytoplasmic surface of the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8468–8472. [PubMed]
  • Salminen A, Novick PJ. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. [PubMed]
  • Saraste J, Hedman K. Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface. EMBO J. 1983;2(11):2001–2006. [PubMed]
  • Saraste J, Kuismanen E. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell. 1984 Sep;38(2):535–549. [PubMed]
  • Saraste J, Palade GE, Farquhar MG. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6425–6429. [PubMed]
  • Schmitt HD, 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. [PubMed]
  • 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. [PubMed]
  • Simons K, Virta H. Perforated MDCK cells support intracellular transport. EMBO J. 1987 Aug;6(8):2241–2247. [PubMed]
  • Trimble RB, Atkinson PH. Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. J Biol Chem. 1986 Jul 25;261(21):9815–9824. [PubMed]
  • Tsai PK, Frevert J, Ballou CE. Carbohydrate structure of Saccharomyces cerevisiae mnn9 mannoprotein. J Biol Chem. 1984 Mar 25;259(6):3805–3811. [PubMed]
  • 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. [PubMed]
  • Walter P, Blobel G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 1983;96:84–93. [PubMed]
  • Walter P, Gilmore R, Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. [PubMed]
  • Waters MG, 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. [PMC free article] [PubMed]
  • Waters MG, Chirico WJ, 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. [PMC free article] [PubMed]
  • Wickerham LJ. 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]
  • Wiedmann M, Kurzchalia TV, Hartmann E, Rapoport TA. A signal sequence receptor in the endoplasmic reticulum membrane. Nature. 328(6133):830–833. [PubMed]

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