Selective Inhibitors of the Proteasome-dependent and Vacuolar Pathways of Protein Degradation in Saccharomyces cerevisiae*
- ‡To whom correspondence should be addressed. Tel.: 617-432-1855; Fax: 617-232-0173; E-mail: agoldber{at}bcmp.med.harvard.edu.
Abstract
We have studied whether various agents that inhibit purified yeast and mammalian 26 S proteasome can suppress the breakdown of different classes of proteins in Saccharomyces cerevisiae. The degradation of short-lived proteins was inhibited reversibly by peptide aldehyde inhibitors of proteasomes, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and carbobenzoxyl-leucinyl-leucinyl-norvalinal (MG115), in a yeast mutant with enhanced permeability, but not in wild-type strains. Lactacystin, an irreversible proteasome inhibitor, had no effect, but the β-lactone derivative of lactacystin, which directly reacts with proteasomes, inhibited the degradation of short-lived proteins. These inhibitors also blocked the rapid ubiquitin-dependent breakdown of a β-galactosidase fusion protein and caused accumulation of enzymatically active molecules in cells. The degradation of the bulk of cell proteins, which are long-lived molecules, was not blocked by proteasome inhibitors, but could be blocked by phenylmethylsulfonyl fluoride. This agent, which inhibits multiple vacuolar proteases, did not affect the proteasome or breakdown of short-lived proteins. These two classes of inhibitors can thus be used to distinguish the cytosolic and vacuolar proteolytic pathways and to increase the cellular content of short-lived proteins.
Footnotes
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↵* This work was supported by research grants from the Human Frontier Science Program and the National Institutes of Health (NIGMS). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- MG101 (NAc-LLnLal)
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N-acetyl-leucinyl-leucinyl-norleucinal (also known as calpain inhibitor-I)
- MG115 (Cbz-LLnVal)
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carbobenzoxyl-leucinyl-leucinyl-norvalinal
- MG132 (Cbz-LLLal)
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carbobenzoxyl-leucinyl-leucinyl-leucinal
- Ub-P-β-gal
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ubiquitin-proline-β-galactosidase
- Suc-LLVY-AMC
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succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin
- PMSF
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phenylmethylsulfonyl fluoride.
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↵2D. H. Lee and A. L. Goldberg, unpublished results.
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↵3L. R. Dick, A. A. Cruikshank, A. T. Destree, L. Grenier, T. A. McCormack, F. D. Melandri, S. L. Nunes, V. J. Palombella, L. A. Parent, L. Plamondon, and R. L. Stein, submitted for publication.
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- Received August 13, 1996.
- Revision received September 5, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
