The precursor of the biotin-binding subunit of mammalian propionyl-CoA carboxylase can be translocated into mitochondria as apo- or holoprotein.
We have investigated the biogenesis of the biotin-binding alpha-subunit of propionyl-CoA carboxylase (alpha PCC) in cultured Buffalo rat liver cells. Cells were pulse-labeled with [35S]methionine, and the newly synthesized alpha PCC was immunoprecipitated with anti-alpha PCC antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biotinylation of the alpha-subunit was detected by a mobility-shift assay following incubation with avidin. In the presence of biotin and the uncoupler, 2,4-dinitrophenol (DNP), alpha PCC precursor accumulated in the cytosol and was quantitatively biotinylated. Subsequent removal of the uncoupler in a "chase" protocol allowed the accumulated precursor to be translocated into mitochondria and cleaved to its mature form. When cells were grown in biotin-depleted medium and labeled in the presence of DNP, no biotinylation of the cytosolic precursor was observed. Nonetheless, the accumulated precursor was efficiently imported into mitochondria and cleaved to mature alpha PCC upon removal of the uncoupler. In parallel experiments in the absence of DNP, non-biotinylated mature alpha PCC accumulated in mitochondria; following addition of biotin, the apo-alpha PCC was converted rapidly to its holo-form. We conclude that both the alpha PCC precursor and its mature counterpart are competent for biotinylation and that biotinylated and nonbiotinylated alpha PCC precursor are competent for import by mitochondria.
Taroni
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Yale University School of Medicine, Department of Human Genetics, New Haven, Connecticut 06510.
Rosenberg
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LE
D000818:Animals; D001051:Apoenzymes / Q000235:genetics* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D043586:Methylmalonyl-CoA Decarboxylase; D008930:Mitochondria, Liver / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011915:Rats, Inbred BUF
D001051:Apoenzymes; D004792:Enzyme Precursors; D046911:Macromolecular Substances; D001710:Biotin; D008715:Methionine; D002262:Carboxy-Lyases; D043586:Methylmalonyl-CoA Decarboxylase