RGS4 is arginylated and degraded by the N-end rule pathway in vitro.
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.
Davydov
I V
IV
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Varshavsky
A
A
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D006868:Hydrolysis; D051379:Mice; D014176:Protein Biosynthesis; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D020710:RGS Proteins; D012333:RNA, Messenger; C098799:RGS4 protein; D001120:Arginine; D003545:Cysteine