Kwon, Yong Tae and Kashina, Anna S. and Varshavsky, Alexander (1999) Alternative Splicing Results in Differential Expression, Activity, and Localization of the Two Forms of Arginyl-tRNA-Protein Transferase, a Component of the N-End Rule Pathway. Molecular and Cellular Biology, 19 (1). pp. 182-193. ISSN 0270-7306 http://resolver.caltech.edu/CaltechAUTHORS:KWOmcb99
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The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-beta gal (beta -galactosidase) and Cys-beta gal. Both forms of the mouse R-transferase conferred instability on Asp-beta gal (but not on Cys-beta gal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is ~0.1 in the skeletal muscle, ~0.25 in the spleen, ~3.3 in the liver and brain, and ~10 in the testis, suggesting that the two R-transferases are functionally distinct.
|Additional Information:||Copyright © 1999, American Society for Microbiology. Received 13 August 1998/Returned for modification 21 September 1998/Accepted 6 October 1998 The first two authors contributed equally to this work. We thank Gary Hathaway of the Caltech Microchemistry Facility for the sequencing of X-beta gal proteins. We are grateful to Hai Rao, Glenn Turner, Fangyong Du, and Lawrence Peck for helpful suggestions and to Fangyong Du, Federico Navarro-Garcia, Hai Rao, and Youming Xie for comments on the manuscript. This work was supported by grants DK39520 and GM31530 to A.V. from the National Institutes of Health.|
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|Deposited On:||21 Dec 2006|
|Last Modified:||26 Dec 2012 09:25|
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