title: A cell-free system for reconstitution of transport between prevacuolar compartments and vacuoles in Saccharomyces cerevisiae.
abstract: Genetic approaches have revealed more than 50 genes involved in the delivery of soluble zymogens like carboxypeptidase Y (CPY) to the lysosome-like vacuole in Saccharomyces cerevisiae. At least 20 of these genes function in transport between the prevacuolar endosome-like compartment (PVC) and the vacuole. To gain biochemical access to these functions, the authors developed a cell-free assay that measures transport-coupled proteolytic maturation of soluble zymogens in vitro. A polycarbonate filter with a defined pore size is used to lyse yeast spheroplasts after pulse-chase radiolabeling. Differential centrifugation enriches for PVCs containing proCPY (p2CPY) in a 125,000 g membrane pellet and is used as donor membranes. Nonradiolabeled spheroplasts are also lysed with a polycarbonate filter but a 15,000 g membrane pellet enriched for vacuoles is collected and used as acceptor membranes. When these two crude membrane pellets are incubated together with adenosine triphosphate and cytosolic protein extracts, nearly 50% of the radiolabeled p2CPY can be processed to the mature vacuolar form, mCPY. This cell-free system allows reconstitution of intercompartmental transport coupled to the function of VPS gene products.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Vida
mesh_terms: D001681:Biological Assay / Q000379:methods*; D043402:Cathepsin A / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D047468:Immunoprecipitation; D011076:Polycarboxylate Cement; D011498:Protein Precursors / Q000378:metabolism; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D013104:Spheroplasts / Q000201:enzymology; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D011076:Polycarboxylate Cement; D011498:Protein Precursors; C005506:polycarbonate; D043402:Cathepsin A