首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Mitochondrial proton/phosphate transporter. An antibody directed against the COOH terminus and proteolytic cleavage experiments provides new insights about its membrane topology
Authors:G C Ferreira  R D Pratt  P L Pedersen
Institution:Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract: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-ethylmalemide, 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.
Keywords:
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号