title: Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences.
abstract: Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease-mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed.
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics; D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D046911:Macromolecular Substances; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics; D009491:Neurospora / Q000378:metabolism*; D009492:Neurospora crassa / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D010618:Phenanthrolines; D011498:Protein Precursors; D000226:Mitochondrial ADP, ATP Translocases; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; C025205:1,10-phenanthroline