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  • Title: Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae.
    Author: Cheng MY, Pollock RA, Hendrick JP, Horwich AL.
    Journal: Proc Natl Acad Sci U S A; 1987 Jun; 84(12):4063-7. PubMed ID: 3295876.
    Abstract:
    Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter. In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa). When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction. When OTCase enzymatic activity was assayed in fractions similarly derived from an S. cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction. A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined. As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity. Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine. We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S. cerevisiae.
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