A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.
We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.
Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas 77030, USA. tvida@farmr1.med.uth.tmc.edu
T
T
Vida
B
B
Gerhardt
D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism; D002352:Carrier Proteins; D043402:Cathepsin A; D002458:Cell Fractionation; D002498:Centrifugation; D003600:Cytosol / Q000502:physiology; D004792:Enzyme Precursors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D008565:Membrane Proteins; D008851:Micropore Filters; D050765:Qa-SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013104:Spheroplasts / Q000166:cytology / Q000201:enzymology; D013696:Temperature; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D000906:Antibodies; D002352:Carrier Proteins; D004792:Enzyme Precursors; D005656:Fungal Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; C065454:VPS33 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D002268:Carboxypeptidases; D043402:Cathepsin A; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases