Molecular mechanism of protein import into mitochondria

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Ornithine, transcarbamylase (OTC) is initially synthesized as a precursor with a transient NH_2-terminal presequence of 32 amino acid residues, then is imported posttranslationally into the mitochondrial matrix. We expressed precursor and mature form of rat OTC in Escherichia coli, purified it in a denatured form, and showed that the purified. OTC precursor could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate. When guanidine-HCl-denatured mature OTC was diluted and incubated at OﾟC, it was reactivated to a specific activity of 18% of that of the purified mature enzyme. Unexpectedly, guanidine-denatured OTC precursor was activated to a similar value under similar conditions. The native and reactivated OTC sedimented on sucrose gradients as trimmers. These observations indicate that the presequence does not prevent the OTC precursor from folding into an enzymatically active trimetic form. Sucrose gradient centrifugation analysis showed that the OTC precursor, but not mature OTC, formed a complex with a protein factor(s) present in reticulocyte lysate.The cytosolic protein factor was highly purified from reticulocyte lysate by affinity chromatography using OTC precursor-bound Sepharose. This factor was named PBF (presequence binding factor). Urea-denatured OTC precursor formed an insoluble aggregate in the absence of PBF, but formed a soluble complex in its presence.Formation of the OTC precursor-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of the OTC precursor. The purified PBF markedly stimulated the import of purified OTC precursor into the isolated mitochondria. Thus, binding of PBF to precursor proteins may well be the first step of mitochondrial protein import. PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form.