The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins.
SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a SEC13-dihydrofolate reductase (mouse) fusion gene (SEC13:DHFR) was constructed that complements both sec13 temperature sensitive and null mutations. Methotrexate-agarose affinity chromatography facilitated the purification of two forms of the Sec13-dhfrp fusion protein: a monomeric form and a high molecular weight complex. The complex form consists of two subunits: Sec13-dhfrp and a 150 kDa protein (p150). Native immunoprecipitation experiments confirm that Sec13p exists in a complex with p150 in wild type cells. Functional analysis supports a role for both subunits in protein transport. Vesicle budding from the ER in a cell-free reaction is inhibited by Fab antibody fragments directed against either Sec13p or p150. The purified Sec13-dhfrp/p150 complex, but not the Sec13-dhfrp monomer, in combination with two other pure protein fractions (Sar1p and a Sec23/Sec24 protein complex) satisfies the requirement for cytosol in a cell-free vesicle budding reaction. The vesicles formed with the purified protein fractions are competent to fuse with the Golgi and are biochemically distinct from the ER membrane fraction from which they derive.
Salama
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Department of Molecular and Cell Biology, Howard Hughes Research Institute, University of California, Berkeley 94720.
Yeung
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Schekman
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D001692:Biological Transport; D002474:Cell-Free System; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification
D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D011993:Recombinant Fusion Proteins; C079620:SEC13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013762:Tetrahydrofolate Dehydrogenase