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 pell et is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacu oles, is used as "acceptor" membranes. When these membranes are incubated t ogether with ATP and cytosolic extracts, similar to 50% of the radiolabeled proCPY is processed to mature CPY. Maturation eras inhibited by dilution o f donor and acceptor membranes during incubation, showed a 15-min lag perio d, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (wh ich 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 effi cient at driving transport. Moreover, antibodies against Vps33p (a Sec1 hom ologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhib ited assays. This cell-free system has allowed the demonstration of reconst ituted intercompartmental transport coupled to the function of a VPS gene p roduct.