Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway.
The transport of proteins destined for post-endoplasmic reticulum locations in the secretory pathway is mediated by small vesicular carriers. Transport vesicles have been generated in cell-free assays from the yeast Saccharomyces cerevisiae, and mammalian systems. Yeast genes encoding cytosolic components that participate in vesicular traffic were first identified from the collection of conditional-lethal sec-(secretory) mutants. Mutations in the yeast SEC7 gene disrupt protein transport in the secretory pathway at the nonpermissive temperature. The SEC7 gene product is a phosphoprotein of relative molecular mass 230,000 that functions from the cytoplasmic aspect of intracellular membranes. We report that in a yeast cell-free transport assay, the introduction of antibodies to Sec7 protein (Sec7p) results in the accumulation of transport vesicles. These vesicles are retrieved with Sec7p-specific antibodies by immuno-isolation for biochemical and electron microscopic characterization. Sec7p on the surface of the accumulated transport vesicles, in combination with previous genetic and biochemical studies, implicate Sec7p as part of a (non-clathrin) vesicle coat. This Sec7p-containing coat structure is proposed to be essential for vesicle budding at multiple stages in the yeast secretory pathway.
Franzusoff
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A
Department of Cellular and Structural Biology, University of Colorado Medical School, Denver 80262.
Lauzé
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Howell
K E
KE
D001692:Biological Transport; D002458:Cell Fractionation; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005656:Fungal Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors; D007163:Immunosorbent Techniques; D008280:Magnetics; D000072235:Mating Factor; D015388:Organelles / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D010455:Peptides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*
D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; C066755:Sec7 guanine nucleotide exchange factors; D000072235:Mating Factor