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Split ubiquitin as a sensor of protein interactions in vivo

Johnsson, Nils and Varshavsky, Alexander (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America, 91 (22). pp. 10340-10344. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:JOHpnas94

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Abstract

We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C-ub) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (N-ub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered N-ub. However, if C-ub and the altered N-ub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C-ub is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.


Item Type:Article
Additional Information:Copyright © 1994 by the National Academy of Sciences. Communicated by John Abelson, July 11, 1994. We thank W. P. Burmeister for his help in preparing Fig. 1a. We thank current and former members of this laboratory, especially G. Turner, E. Johnson, J. Dohmen, F. Levy, and C. Byrd, for comments on the manuscript. This work was supported by grants to A.V. from the National Institutes ofHealth (GM31530 and DK39520). N.J. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. 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:SACCHAROMYCES-CEREVISIAE, LEUCINE ZIPPER, GENE ENCODES, YEAST, SYSTEM, FAMILY, DNA, IDENTIFICATION, POLYPEPTIDES, PROTEOLYSIS
Record Number:CaltechAUTHORS:JOHpnas94
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:JOHpnas94
Alternative URL:http://www.pnas.org/cgi/content/abstract/91/22/10340
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1396
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Jan 2006
Last Modified:26 Dec 2012 08:44

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