Davydov, Ilia V. and Varshavsky, Alexander (2000) RGS4 is arginylated and degraded by the N-end rule pathway in vitro. Journal of Biological Chemistry, 275 (30). pp. 22931-22941. ISSN 0021-9258. doi:10.1074/jbc.M001605200. https://resolver.caltech.edu/CaltechAUTHORS:DAVjbc00
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Abstract
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.
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Additional Information: | © 2000 by the American Society for Biochemistry and Molecular Biology. Received for publication, February 28, 2000, and in revised form, April 22, 2000. Published, JBC Papers in Press, April 26, 2000. We thank T. Rümenapf for providing Ub-X-nsP4-encoding plasmids, which were used as PCR templates in the present work, and for sharing unpublished data on the N-end rule-dependent degradation of truncated Tyr-nsP4 in reticulocyte lysate. We are grateful to G. Hathaway for expert assistance with the radiochemical sequencing of RGS4M19A-GST and to Y.T. Kwon and N. Barteneva for the mouse brains used to prepare the cDNA library. We thank C.K. Chen and M.I. Simon for the gifts of pcDNA3-RGS16 and a Gβ5L-coding DNA fragment; H. Sorimachi and K. Suzuki for the gifts of p94- and p94C129A-encoding plasmids; and J. S. Elce for the gift of pET-80k. We gratefully acknowledge discussions of the paper and comments on the manuscript by E. Smirnova and members of the Varshavsky laboratory. | ||||||
Subject Keywords: | RNA-PROTEIN TRANSFERASE; YEAST METHIONINE AMINOPEPTIDASE; MUSCLE-SPECIFIC CALPAIN; SHORT-LIVED PROTEIN; SACCHAROMYCES-CEREVISIAE; UBIQUITIN SYSTEM; ALPHA-SUBUNIT; DEGRADATION; PROTEASOME; ACTIVATION | ||||||
Issue or Number: | 30 | ||||||
DOI: | 10.1074/jbc.M001605200 | ||||||
Record Number: | CaltechAUTHORS:DAVjbc00 | ||||||
Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:DAVjbc00 | ||||||
Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||
ID Code: | 5075 | ||||||
Collection: | CaltechAUTHORS | ||||||
Deposited By: | Lindsay Cleary | ||||||
Deposited On: | 28 Sep 2006 | ||||||
Last Modified: | 08 Nov 2021 20:22 |
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