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

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Abstract
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 freeeze-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. Enzymatic cleavage of the carrier occurred in inside-out submitochondrial particles, but not in right-side-out particles, yielding a large fragment of Mr .simeq. 25 000 that was immunodetected on Western blot by anticarrier antibodies but not by anti-N-terminal antibodies. These results demonstrated that the arginine-specific endoprotease had access to the matrix face of the inner mitochondrial membrane, at Arg 30 or at Arg 59. Thus, it appears that in intact mitochondria the sequence of the ADP/ATP carrier including Arg 30 or Arg 59 protrudes into the matrix space, whereas the N-terminal segment corresponding to the first 10 amino acid residues protrudes into cytosol, and the intermediate, rather hydrophobic, sequence spanning residues 9-28 transverses the lipid bilayer of the inner mitochondrial membrane.