A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites.
Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.
Vestweber
D
D
University of Basel, Department of Biochemistry, Switzerland.
Schatz
G
G
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D001692:Biological Transport; D004220:Disulfides; D003576:Electron Transport Complex IV / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship
D004220:Disulfides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000426:Alcohol Dehydrogenase; D003576:Electron Transport Complex IV; D006180:Proton-Translocating ATPases