title: Import and processing of hybrid proteins by mammalian mitochondria in vitro.
abstract: Normal and hybrid proteins were synthesized following transcription of pSP64 recombinant plasmids with SP6 polymerase and translation of resultant mRNAs in a rabbit reticulocyte lysate. The precursor to the rat liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = approximately 40,000), was efficiently imported by rat heart mitochondria in vitro and processed to mature protein. Import of the precursor to a second matrix enzyme, carbamyl-phosphate synthetase I (Mr = approximately 165,000), could not be demonstrated. However, a 33-kDa hybrid protein, bearing the precarbamyl-phosphate synthetase I signal sequence (38 amino acids) and 55 amino acids from the amino terminus of the mature enzyme, followed by the carboxyl-terminal 209 amino acids of ornithine carbamyltransferase, was imported and processed under identical conditions. The topogenic function of the preornithine carbamyltransferase signal sequence (32 amino acids) was confirmed by constructing a hybrid protein bearing the signal sequence and five amino acids of mature enzyme, followed by 250 amino acids of the cytosolic enzyme of Escherichia coli, asparagine synthetase; the hybrid was transported to the matrix compartment of heart mitochondria where processing, albeit incorrect, took place. A hybrid asparagine synthetase bearing the pre-ornithine carbamyltransferase signal sequence plus 28 amino acids of mature ornithine carbamyltransferase, however, was imported and processed with apparent fidelity.
authors.forename: M
authors.initials: M
authors.lastname: Nguyen
authors.forename: C
authors.initials: C
authors.lastname: Argan
authors.forename: C J
authors.initials: CJ
authors.lastname: Lusty
authors.forename: G C
authors.initials: GC
authors.lastname: Shore
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D005470:Fluorometry; D008025:Ligases / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D010957:Plasmids; D021382:Protein Sorting Signals / Q000032:analysis; D011817:Rabbits; D051381:Rats; D014158:Transcription, Genetic
chemical_list: D000596:Amino Acids; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D009954:Ornithine Carbamoyltransferase; D008025:Ligases; D001217:Aspartate-Ammonia Ligase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)