Lévy, Frédéric and Johnsson, Nils and Rümenapf, Tillmann and Varshavsky, Alexander (1996) Using ubiquitin to follow the metabolic fate of a protein. Proceedings of the National Academy of Sciences of the United States of America, 93 (10). pp. 4907-4912. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:LEVpnas96
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We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.
|Additional Information:||Copyright © 1996 by the National Academy of Sciences. Contributed by Alexander Varshavsky, December 15, 1995. We thank L. Larson for her participation at the beginning of this work, M. Ghislain and M. Gonzalez for discussions, and A. Webster for bringing ref. 19 to our attention. We also thank J. Johnston, M. Gonzalez, G. Turner, and A. Webster for their comments on the manuscript. This work was supported by National Institutes of Health Grants DK39520 and GM31530 (to A.V.). F.L. was supported initially by an EMBO postdoctoral fellowship and later by the Swiss National Fund for Research. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||proteolysis, pulse-chase, half-life, N-end rule, cell culture, degradation, pathway, cells|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||06 Jan 2006|
|Last Modified:||26 Dec 2012 08:43|
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