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Molecular and Cellular Biology, January 1999, p. 182-193, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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
Yong Tae
Kwon,
Anna S.
Kashina, and
Alexander
Varshavsky*
Division of Biology, California Institute of
Technology, Pasadena, California 91125
Received 13 August 1998/Returned for modification 21 September
1998/Accepted 6 October 1998
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
ate1
Saccharomyces cerevisiae in the presence of test
substrates that included Asp-
gal (
-galactosidase) and Cys-
gal.
Both forms of the mouse R-transferase conferred instability on
Asp-
gal (but not on Cys-
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.
*
Corresponding author. Mailing address: Division of
Biology, 147-75, Caltech, 1200 East California Blvd., Pasadena, CA
91125. Phone: (626) 395-3785. Fax: (626) 440-9821. E-mail:
avarsh{at}cco.caltech.edu.
Molecular and Cellular Biology, January 1999, p. 182-193, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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