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A Mouse Amidase Specific for N-terminal Asparagine: the gene, the enzyme, and their function in the N-end rule pathway

Grigoryev, Sergei and Stewart, Albert E. and Kwon, Yong Tae and Arfin, Stuart M. and Bradshaw, Ralph A. and Jenkins, Nancy A. and Copeland, Neal G. and Varshavsky, Alexander (1996) A Mouse Amidase Specific for N-terminal Asparagine: the gene, the enzyme, and their function in the N-end rule pathway. Journal of Biological Chemistry, 271 (45). pp. 28521-28532. ISSN 0021-9258. doi:10.1074/jbc.271.45.28521.

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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. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The ~17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The ~1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located ~500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown.

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Varshavsky, Alexander0000-0002-4011-258X
Additional Information:© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, July 9, 1996) We thank the colleagues whose names are cited in the text for gifts of cell lines, strains, and plasmids. S.G. is grateful to R.J. Dohmen and K. Madura for the introduction to methods of yeast genetics and to L. Larson and F. Lévy for advice on cell culture techniques. N.G.C. and N.A.J. thank D.J. Gilbert for excellent technical assistance. This work was supported by National Institutes of Health Grant DK39520 (to A.V.) and Grant DK32461 (to R.A.B. and S.M.A.) and by NCI under contract with ABL. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U57690[GenBank] (mouse E214K cDNA), U57691[GenBank] (mouse Ntan1 genomic DNA), and U57692[GenBank] (mouse Ntan1 cDNA).
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National Cancer InstituteUNSPECIFIED
Issue or Number:45
Record Number:CaltechAUTHORS:GRIjbc96
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ID Code:5981
Deposited By: Archive Administrator
Deposited On:10 Nov 2006
Last Modified:08 Nov 2021 20:30

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