Synthesis in vitro of precursor-type carnitine acetyltransferase with messenger RNA from Candida tropicalis

Abstract

Carnitine acetyltransferase was synthesized in vitro in the mRNA-dependent reticulocyte system with mRNA from alkane-grown or propionate-grown cells of Candida tropicalis. The protein synthesized in vitro was isolated by immunoprecipitation with antibody against peroxisomal or mitochondrial carnitine acetyltransferase and was compared with peroxisomal carnitine acetyltransferase (Mr of subunits, 64 000 and 57 000) and the mitochondrial enzyme (Mr of subunits, 64 000 and 52 000) of C. tropicalis by electrophoresis in the presence of sodium dodecyl sulfate. Nascent carnitine acetyltransferase prepared in vitro showed a hetero-oligomeric property, like the peroxisomal and mitochondrial enzymes isolated from C. tropicalis. The molecular weights of the subunits of nascent carnitine acetyltransferase were estimated to be 71 000 and 57 000, indicating the existence of the precursor form of the enzyme. By sucrose density gradient centrifugation of total mRNA, these two subunit proteins were shown to be synthesized with respective mRNAs of different sizes. The same precursor-type of carnitine acetyltransferase was obtained with the mRNAs from the alkane-grown cells and the propionate-grown cells. The results obtained suggest that a common precursor will be post-translationally modified to form the peroxisomal and mitochondrial enzymes.