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PMID: 2269659 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole.

The Journal of cell biology ·Vol. 111 ·No. 6 Pt 2 ·1990-12-00 ·Pages 2871-84

Vida TA, Graham TR, Emr SD

Abstract

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.

Related Genes
vps
MeSH Terms
Adenosine Triphosphate/metabolism Biological Transport Carboxypeptidases/metabolism Cathepsin A Cell Fractionation Cell-Free System Cytoplasm/metabolism Fungal Proteins/metabolism Intracellular Membranes/metabolism Mutation Protein Processing, Post-Translational Protoplasts/metabolism Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins Vacuoles/metabolism
Chemicals
Fungal Proteins Saccharomyces cerevisiae Proteins Adenosine Triphosphate Carboxypeptidases Cathepsin A PRC1 protein, S cerevisiae serine carboxypeptidase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Vida T A
Division of Biology 147-75, California Institute of Technology, Pasadena 91125.
Graham T R
Emr S D
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.111.6.2871
Published
1990-12-00
Pages
2871-84
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2116412
Subset
IM
Grants
NIGMS NIH HHS · GM-32703 · United States
External Links
PubMed source DOI: 10.1083/jcb.111.6.2871 PMC Full Text View journal details
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