TY - JOUR
T1 - Mitochondrial proton/phosphate transporter
T2 - An antibody directed against the COOH terminus and proteolytic cleavage experiments provides new insights about its membrane topology
AU - Ferreira, Gloria C.
AU - Pratt, Raymond D.
AU - Pedersen, Peter L.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1990/12/5
Y1 - 1990/12/5
N2 - Molecular cloning and sequencing of a full-length cDNA encoding the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has revealed its primary structure (Ferreira, G. C., Pratt, R. D., and Pedersen, P. L. (1989) J. Biol. Chem. 264, 15628-15633). To date, no experimental data pertinent to the membrane topology of this transporter are available. For this reason, four different peptides which represent different regions of the H+/Pi symporter were synthesized and used to raise polyclonal antibodies. Each of the antipeptide antibodies exhibits immunoreactivity with its synthetic peptide antigen, but only antiserum against a COOH-terminal peptide reacts with the native transporter, suggesting that the other peptides are either conformally restricted or located in the interior of the protein. Competitive radioimmunoassays, using intact "mitoplasts" (outer membrane-free mitochondria) and inverted inner membrane vesicles, show that the COOH-terminal antibodies bind only to the cytoplasmic surface of the inner membrane, indicating that the COOH terminus of the protein is normally exposed to the mitochondrial intermembrane space. In support of this conclusion, tryptic digestion of mitoplasts but not of the inside-out vesicles, cleaves the antigenic site for the COOH-terminal antibodies. In other experiments, it was shown that N-ethylmaleimide, a sulfhydryl alkylating agent known to inhibit the mitochondrial phosphate transporter, markedly reduces the accessibility of the COOH terminus to trypsin. These studies provide the first direct experimental data relevant to the membrane topology of the mitochondrial H+/Pi symporter. In addition, they support the view that alkylation of a reactive cysteine residue induces a significant conformational change in the transporter.
AB - Molecular cloning and sequencing of a full-length cDNA encoding the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has revealed its primary structure (Ferreira, G. C., Pratt, R. D., and Pedersen, P. L. (1989) J. Biol. Chem. 264, 15628-15633). To date, no experimental data pertinent to the membrane topology of this transporter are available. For this reason, four different peptides which represent different regions of the H+/Pi symporter were synthesized and used to raise polyclonal antibodies. Each of the antipeptide antibodies exhibits immunoreactivity with its synthetic peptide antigen, but only antiserum against a COOH-terminal peptide reacts with the native transporter, suggesting that the other peptides are either conformally restricted or located in the interior of the protein. Competitive radioimmunoassays, using intact "mitoplasts" (outer membrane-free mitochondria) and inverted inner membrane vesicles, show that the COOH-terminal antibodies bind only to the cytoplasmic surface of the inner membrane, indicating that the COOH terminus of the protein is normally exposed to the mitochondrial intermembrane space. In support of this conclusion, tryptic digestion of mitoplasts but not of the inside-out vesicles, cleaves the antigenic site for the COOH-terminal antibodies. In other experiments, it was shown that N-ethylmaleimide, a sulfhydryl alkylating agent known to inhibit the mitochondrial phosphate transporter, markedly reduces the accessibility of the COOH terminus to trypsin. These studies provide the first direct experimental data relevant to the membrane topology of the mitochondrial H+/Pi symporter. In addition, they support the view that alkylation of a reactive cysteine residue induces a significant conformational change in the transporter.
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M3 - Article
C2 - 2250020
AN - SCOPUS:0025648976
SN - 0021-9258
VL - 265
SP - 21202
EP - 21206
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -