Analysis of a conditional degradation signal in yeast and mammalian cells

Details

Serval ID
serval:BIB_04D9E4E741E1
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Analysis of a conditional degradation signal in yeast and mammalian cells
Journal
European Journal of Biochemistry
Author(s)
Levy  F., Johnston  J. A., Varshavsky  A.
ISSN
0014-2956 (Print)
Publication state
Published
Issued date
01/1999
Volume
259
Number
1-2
Pages
244-52
Notes
Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: Jan
Abstract
The N-end rule pathway is a ubiquitin-dependent proteolytic system, the targets of which include proteins that bear destabilizing N-terminal residues. The latter are a part of the degradation signal called the N-degron. Arg-DHFRts, an engineered N-end rule substrate, bears N-terminal arginine (a destabilizing residue) and DHFRts [a temperature-sensitive mouse dihydrofolate reductase (DHFR) moiety]. Previous work has shown that Arg-DHFRts is long-lived at 23 degreesC but short-lived at 37 degreesC in the yeast Saccharomyces cerevisiae. In the present work, we extended this analysis, and found that the degradation of Arg-DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low-Mr ligand of DHFR. In S. cerevisiae, Arg-DHFRts is degraded at 37 degreesC exclusively by the N-end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety. In mouse cells, MTX completely inhibits the degradation of Arg-DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N-degron. When the N-terminus of Arg-DHFRts was extended with a 42-residue lysine-lacking extension, termed eDeltaK, the resulting Arg-eDeltaK-DHFRts was rapidly degraded at both 23 degreesC and 37 degreesC. Moreover, the degradation of Arg-eDeltaK-DHFRts, in contrast with that of Arg-DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg-DHFRts at 23 degreesC results, at least in part, from steric inaccessibility of its N-terminal arginine. The N-degron of Arg-DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell-penetrating compound. We discuss implications of this advance and the mechanics of targeting by the ubiquitin system.
Keywords
Animals *Arginine Endopeptidases/*metabolism Folic Acid Antagonists/pharmacology Methotrexate/pharmacology Mice Models, Biological Multienzyme Complexes/genetics/*metabolism Mutation Recombinant Proteins/metabolism Saccharomyces cerevisiae/metabolism Species Specificity Substrate Specificity Tetrahydrofolate Dehydrogenase/genetics/*metabolism Thymidylate Synthase/genetics/*metabolism Ubiquitins/*metabolism
Pubmed
Web of science
Open Access
Yes
Create date
28/01/2008 11:17
Last modification date
20/08/2019 12:26
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