The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide.
Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp60src docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp60src NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60src binding protein. An analysis of pp60v-src binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp60src NH2 terminus suggests alternative strategies for uncovering pp60src membrane binding species.
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Department of Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
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D000595:Amino Acid Sequence; D000818:Animals; D003411:Arvicolinae; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D009227:Myristic Acids; D009842:Oligopeptides; D010446:Peptide Fragments; D019814:Myristic Acid; D000226:Mitochondrial ADP, ATP Translocases; D015688:Oncogene Protein pp60(v-src)