Purification and characterization of constitutive secretory vesicles from yeast.
We have developed a purification procedure for the isolation of constitutive post-Golgi secretory vesicles from Saccharomyces cerevisiae. Although the post-Golgi stage of the secretion pathway is normally very rapid, we have used a temperature-sensitive secretory mutant, sec 6-4, to greatly expand the population of secretory vesicles. Following invertase as a marker, intact vesicles are enriched 36-fold from the crude lysate. The final preparation contains few contaminants as assessed by morphologic and biochemical examination. Three proteins (110, 40-45, and 18 kD) co-purify with the vesicle marker enzyme invertase. Metabolic labeling experiments indicate that these vesicle-associated proteins are synthesized during the period of vesicle accumulation. They are not apparent in the corresponding fractions from wild-type cells. Analysis of these proteins indicates that the 110-kD protein is a major glycoprotein residing in the vesicle lumen, while the 40-45- and 18-kD proteins are not glycosylated and are firmly associated with the vesicle membrane, each with at least one domain exposed on the cytoplasmic surface.
Walworth
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Novick
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D002458:Cell Fractionation / Q000379:methods*; D005656:Fungal Proteins / Q000378:metabolism; D007425:Intracellular Membranes / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*
D005656:Fungal Proteins