J. Biol. Chem., Vol. 259, Issue 9, 5392-5395, May, 1984

Newly processed ornithine transcarbamylase subunits are assembled to trimers in rat liver mitochondria

F Kalousek, MD Orsulak and LE Rosenberg

We have characterized further the biogenesis in vitro of ornithine transcarbamylase, a homotrimeric mitochondrial matrix enzyme synthesized in the cytoplasm as a larger precursor. When cell-free translation mixtures containing the ornithine transcarbamylase precursor (40 kDa) were chromatographed on Bio-Gel P-200 columns, all of the precursor eluted as aggregates or complexes with molecular weights greater than 200 kDa. None of the precursor bound to a ligand affinity column containing delta-N-(phosphonoacetyl)-L-ornithine (delta- PALO), a transition-state analog and competitive inhibitor of carbamyl phosphate binding, which recognizes native ornithine transcarbamylase. In contrast, a significant portion of the labeled mature-sized subunits, formed when intact mitochondria processed the precursor, bound specifically to the delta-PALO column, were eluted by carbamyl phosphate, and chromatographed on a Bio-Gel P-300 column with a mobility identical to that of native, trimeric ornithine transcarbamylase. No such binding to delta-PALO was observed for the mature-sized monomer or dimer, or for the intermediate-sized ornithine transcarbamylase polypeptide. Moreover, processing by a mitochondrial matrix fraction failed to yield trimeric enzyme, despite producing ample amounts of mature-sized monomer. We conclude that delta-PALO recognizes only trimeric ornithine transcarbamylase composed of mature- sized subunits and that such trimers can be assembled in vitro by intact mitochondria following translocation and proteolytic processing.
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				F. Firgaira, J. Hendrick, F Kalousek, J. Kraus, and L. Rosenberg RNA required for import of precursor proteins into mitochondria Science, December 14, 1984; 226(4680): 1319 - 1322. [Abstract] [PDF]