Orientation of the N-terminal region of the membrane-bound ADP/ATP carrier protein explored by antipeptide antibodies and an arginine-specific endoprotease. Evidence that the accessibility of the N-terminal residues depends on the conformational state of the carrier.
Two peptides corresponding to the amino acid sequences 1-11 (N-terminal peptide) and 288-297 (C-terminal peptide) of beef heart ADP/ATP carrier have been synthesized. After coupling to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificities of the generated antibodies were tested by enzyme-linked immunosorbent assay (ELISA) and (or) Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies reacted specifically with the corresponding peptide. However, only anti-N-terminal antibodies reacted with the isolated ADP/ATP carrier; they also reacted with the membrane-bound carrier in freeze-thawed mitochondria and mitoplasts, indicating that the first 10 amino acid residues of the membrane-bound carrier in mitochondria face the cytosol. On the basis that the ADP/ATP carrier can adopt two conformations, one trapped by carboxyatractyloside (CATR conformation) and the other by bongkrekic acid (BA conformation), the reactivity of the anti-N-terminal antibodies to the ADP/ATP carrier in mitoplasts or freeze-thawed mitochondria was tested for each conformation of the carrier. Only in the CATR conformation was the N-terminal region of the membrane-bound carrier reactive to the N-terminal antibodies; the contrasting weak reactivity of the carrier in the BA conformation suggested that the transition from the CATR conformation to the BA conformation results in a restricted conformation of the peptide chain corresponding to the first 10 amino acid residues or its partial burying in the lipid bilayer. These immunological data were complemented by enzymatic data pertaining to proteolysis of the membrane-bound ADP/ATP carrier by an arginine-specific endoprotease.(ABSTRACT TRUNCATED AT 250 WORDS)
Brandolin
G
G
Département de Recherche Fondamentale, Centre d'Etudes Nucléaires, Grenoble, France.
Boulay
F
F
Dalbon
P
P
Vignais
P V
PV
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010047:Ovalbumin; D011487:Protein Conformation; D012697:Serine Endopeptidases
D000939:Epitopes; D010047:Ovalbumin; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D010450:Endopeptidases; C044499:submandibular proteinase A; D012697:Serine Endopeptidases