The yeast ARG7 gene product is autoproteolyzed to two subunit peptides, yielding active ornithine acetyltransferase.
Yeast ornithine acetyltransferase has been purified from total yeast extracts as a heterodimer of two subpeptides (Liu, Y., Van Heeswijck, R., Hoj, P., and Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296), confirmed to derive from a single ARG7-encoded precursor (Crabeel, M., Abadjieva, A., Hilven, P., Desimpelaere, J., and Soetens, O. (1997) Eur. J. Biochem. 250, 232-241). By Western immunoblotting, we show that Arg7p is also present as two subpeptides in isolated mitochondria, but that processing occurs before targeting to the mitochondria: deletion of the N-terminal leader peptide results in cytosolic accumulation of N-Arg7p, whereas C-Arg7p partially reaches the organelle by itself. When artificially co-expressed from separate genes, the two subpeptides can complement an arg7 mutation; ornithine acetyltransferase activity is measurable. Maturation of Arg7p occurs at threonine 215 (N-side), in the region most conserved among the 17 ornithine acetyltransferases characterized. Changing this conserved residue to alanine completely abolishes maturation. Furthermore, Arg7p is both processed and active in Escherichia coli, a heterologous background, and is also cleaved in vitro when produced by coupled transcription/translation in a reticulocyte lysate. Together, these data suggest classic autoproteolysis initiated by threonine 215. Most importantly, maturation is required for the enzyme to be functional, since the T215A substitution mutant is catalytically inactive and incapable of genetic complementation, despite its correct targeting to the mitochondria.
Abadjieva
A
A
Department of Microbiology of the Vrije Universiteit Brussel and Vlaams Interuniversitair Instituut voor Biotechnologie, CERIA-COOVI, E. Grysonlane 1, B-1070 Brussels, Belgium.
Hilven
P
P
Pauwels
K
K
Crabeel
M
M
D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015003:Yeasts / Q000201:enzymology*
D004792:Enzyme Precursors; D000123:Acetyltransferases; C020646:glutamate acetyltransferase