Vida, Thomas A. and Graham, Todd R. and Emr, Scott D. (1990) In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. Journal of Cell Biology, 111 (6, Pt.). pp. 2871-2884. ISSN 0021-9525 http://resolver.caltech.edu/CaltechAUTHORS:VIDjcb90
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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.
|Additional Information:||Copyright © 1990 by The Rockefeller University Press Received for publication 20 June 1990 and in revised form 16 August 1990. We thank David Baker and Randy Scbekman (University of California, Berkley) for communicating results before publication. We are also grateful to Jeremy Thorner for antiserum against phosphoglycerol kinase and to the members of the Emr laboratory for useful discussions and comments on the manuscript. This work was supported by Public Health Service grant GM-32703 from the National Institutes of Health. T.A. Vida and T.R. Graham were both supported by Postdoctoral Research Fellowships from the American Cancer Society.|
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|Deposited On:||30 Nov 2006|
|Last Modified:||26 Dec 2012 09:19|
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