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.
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-betagal (beta-galactosidase) and Cys-betagal. Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) 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 approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.
Kwon
Y T
YT
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Kashina
A S
AS
Varshavsky
A
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D001224:Aspartic Acid; D001483:Base Sequence; D002461:Cell Line, Transformed; D002467:Cell Nucleus; D003545:Cysteine; D003600:Cytosol; D018076:DNA, Complementary; D004331:Drosophila melanogaster / Q000235:genetics; D005091:Exons; D005786:Gene Expression Regulation; D018698:Glutamic Acid; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D001616:beta-Galactosidase
D018076:DNA, Complementary; D001224:Aspartic Acid; D018698:Glutamic Acid; D019881:Aminoacyltransferases; C019727:arginyltransferase; D001616:beta-Galactosidase; D003545:Cysteine