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PMID: 10402462 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

The Journal of cell biology ·Vol. 146 ·No. 1 ·1999-07-12 ·Pages 85-98

Vida T, Gerhardt B

Abstract

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.

MeSH Terms
Adenosine Triphosphate/metabolism Antibodies Aspartic Acid Endopeptidases/genetics,metabolism Biological Transport Carboxypeptidases/metabolism Carrier Proteins Cathepsin A Cell Fractionation Centrifugation Cytosol/physiology Enzyme Precursors/metabolism Fungal Proteins/genetics,physiology Gene Deletion Gene Expression Genetic Complementation Test Intracellular Membranes/enzymology,metabolism Lysosomes/enzymology,metabolism Membrane Proteins Micropore Filters Qa-SNARE Proteins Saccharomyces cerevisiae/cytology,enzymology,genetics,metabolism Saccharomyces cerevisiae Proteins Spheroplasts/cytology,enzymology Temperature Vacuoles/enzymology,metabolism Vesicular Transport Proteins
Chemicals
Antibodies Carrier Proteins Enzyme Precursors Fungal Proteins Membrane Proteins Qa-SNARE Proteins Saccharomyces cerevisiae Proteins VAM3 protein, S cerevisiae VPS33 protein, S cerevisiae Vesicular Transport Proteins Adenosine Triphosphate Carboxypeptidases Cathepsin A aspartic proteinase A PEP4 protein, S cerevisiae Aspartic Acid Endopeptidases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Vida T
Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas 77030, USA. [email protected]
Gerhardt B
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.146.1.85
Email
[email protected]
Published
1999-07-12
Pages
85-98
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2199724
Subset
IM
Grants
NCI NIH HHS · CA16672 · United States
NIGMS NIH HHS · GM52092 · United States
External Links
PubMed source DOI: 10.1083/jcb.146.1.85 PMC Full Text View journal details
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