Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway

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Abstract
THE transport of proteins destined for post-endoplasmic reticulum locations in the secretory pathway is mediated by small vesicular carriers1–3. Transport vesicles have been generated in cell-free assays from the yeast Saccharomyces cerevisiae, and mammalian systems4–14. Yeast genes encoding cytosolic components that par-ticipate in vesicular traffic were first identified from the collection of conditional-lethal sec (secretory) mutants15–17. Mutations in the yeast SEC7 gene disrupt protein transport in the secretory pathway at the nonpermissive temperature18. The SEC7 gene product is a phosphoprotein of relative molecular mass 230,000 that functions from the cytoplasmic aspect of intracellular mem-branes19–20. We report that in a yeast cell-free transport assay, the introduction of antibodies to Sec? protein (Sec7p) results in the accumulation of transport vesicles. These vesicles are retrieved with SecTp-specific antibodies by immuno-isolation for bio-chemical and electron microscopic characterization. SecTp on the surface of the accumulated transport vesicles, in combination with previous genetic and biochemical studies18–20, implicate SecTp as part of a (non-clathrin) vesicle coat. This SecTp-containing coat structure is proposed to be essential for vesicle budding at multiple stages in the yeast secretory pathway.