title: Import of precursor proteins into mitochondria: site of polypeptide unfolding.
abstract: The matrix-targeting signal of mitochondrial preornithine carbamyl transferase has been fused to either murine dihydrofolate reductase (pODHFR) or bacterial chloramphenicol acetyltransferase (pOCAT). Loosening of the tightly folded "native" structure of the two proteins following their synthesis in a rabbit reticulocyte lysate was assayed by the acquisition of protease sensitivity (pODHFR and pOCAT) or by the loss of enzyme activity (pOCAT). By these criteria, the bulk population of both precursor proteins was tightly folded following release from the ribosome, even in the presence of ATP and excess reticulocyte lysate. Neither protein unfolded as a consequence of binding to the surfaces of anionic liposomes or intact mitochondria. However, a non-native form of full-length pOCAT, exhibiting a loss of enzymatic activity and an enhanced protease sensitivity, was detected in association with a submitochondrial fraction that banded between the inner and outer mitochondrial membrane fractions on sucrose density gradients. Delivery of the precursor molecule to this position required ATP and a proteinaceous component on the surface of the organelle.
authors.affiliation: Department of Biochemistry, McGill University, McIntyre, Montreal, Canada.
authors.forename: I S
authors.initials: IS
authors.lastname: Skerjanc
authors.forename: W P
authors.initials: WP
authors.lastname: Sheffield
authors.forename: S K
authors.initials: SK
authors.lastname: Randall
authors.forename: J R
authors.initials: JR
authors.lastname: Silvius
authors.forename: G C
authors.initials: GC
authors.lastname: Shore
mesh_terms: D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D019286:Endopeptidase K; D007425:Intracellular Membranes / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D009954:Ornithine Carbamoyltransferase; D015500:Chloramphenicol O-Acetyltransferase; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K