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© The Rockefeller University Press, 0021-9525/1999//85 $5.00
The Journal of Cell Biology, Volume 146, Number 1, , 1999 85-98

Original Article

A Cell-Free Assay Allows Reconstitution of Vps33p-Dependent Transport to the Yeast Vacuole/Lysosome



Thomas Vidaa and Brenda Gerhardta

a Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas 77030
Department of Integrative Biology and Pharmacology, The University of Texas Medical School, 6431 Fannin, Houston, TX 77030.(713) 500-7455(713) 500-7445

tvida{at}farmr1.med.uth.tmc.edu

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, ~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.

Key Words: carboxypeptidase Y • lysosome • membrane fusion • Saccharomyces cerevisiae • vacuole

© 1999 The Rockefeller University Press

1.used in this paper: CPY, carboxypeptidase Y; CDCFDA, dichlorocarboxyfluorescein diacetate; PLC, prelysosomal compartment; PrA, proteinase A; PVC, prevacuolar compartment


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