Abstract
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.
Highlights
Ornithine is an important intermediate in the arginine biosynthetic pathway
We show that processing (i) precedes targeting to the mitochondria, (ii) occurs in a heterologous E. coli background, (iii) occurs in vitro, when Arg7p is produced in a coupled transcription/translation system, and (iv) depends on threonine 215
To monitor the ARG7 gene product(s) by Western immunoblotting, we constructed a series of gene fusions expected to produce HA epitope-tagged derivatives of the enzyme. pAA2 yields Arg7p bearing the epitope at its C terminus, pHP1 yields Arg7p tagged at the intact N terminus; pAA3 and pHP3 encode N-tagged Arg7p, but with partial or total truncation of the mitochondrial leader peptide (Fig. 1)
Summary
Yeast Strains—BJ5459 (Mat␣, ura, trp1Ϫ, lys 801, leu2-⌬1, his3-⌬200, pep (alias pra1)::HIS3, prb1-⌬1,6R, can1) is a strain from. Construction of Plasmids pAA31 and pAA32—Wild-type ARG7 and mutant arg encoding T215A Arg7p were PCR-amplified on plasmids pYeA7–1 and pHP12, respectively, using oligonucleotide AA69 as forward primer and AA70 as reverse primer They contain, respectively, an NdeI site followed by an ATG and a BamHI site followed by a stop codon. The rest of the supernatant was immediately mixed with an equal volume of 2ϫ loading buffer (100 mM Tris-HCl, pH 6.8, 10% -mercaptoethanol (freshly added), 5% SDS, 0.2% bromophenol blue, 20% glycerol), incubated for 5 min in a boiling water bath, and either used directly for SDS-PAGE or stored at Ϫ70 °C (in which case, it was reheated in a boiling bath before loading) Such extracts usually contained about 25 mg of protein/ml.
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