Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis.
The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
Marty
I
I
Laboratoire de Biochimie, Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires, Grenoble, France.
Brandolin
G
G
Gagnon
J
J
Brasseur
R
R
Vignais
P V
PV
D000595:Amino Acid Sequence; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001865:Bongkrekic Acid / Q000494:pharmacology; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007425:Intracellular Membranes / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013367:Submitochondrial Particles / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D001865:Bongkrekic Acid; D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; D010450:Endopeptidases; D014357:Trypsin; C044574:lysine-C peptidase; C003853:carboxyatractyloside