The three Cys of the yeast (Saccharomyces cerevisiae) mitochondrial phosphate transport protein (PTP) subunit were replaced with Ser. The seven mutants (single, double, and complete Cys replacements) were expressed in yeast, and the homodimeric mutant PTPs were purified from the mitochondria and reconstituted. The pH gradient-dependent net phosphate (Pi) transport uptake rates (initial conditions: 1 mM [Pi]e, pHe 6.80; 0 mM [Pi]i, pHi 8.07) catalyzed by these reconstituted mutants are similar to those of the wild-type protein and range from 15 to 80 micromol Pi/min mg PTP protein. Aerobic media inhibit only the Pi uptake rates catalyzed by PTPs with the conserved (yeast and bovine) Cys28. This inhibition in the proteoliposomes is 84-95% and can be completely reversed by dithiothreitol. Transport by the wild type as well as by all mutant proteins with Cys28 is more than 90% inhibited by mersalyl. Transport catalyzed by mutant proteins with only Cys300 or only Cys134 is less sensitive, and that catalyzed by the no Cys mutant shows 40% inhibition by mersalyl. When dithiothreitol is removed from purified single Cys mutant proteins, only the mutant protein with Cys28 appears as a homodimer in a nonreducing SDS polyacrylamide gel. Thus, the function relevant transmembrane helix A, with Cys 28 about equidistant from the two inner membrane surfaces, is in close contact with parts of transmembrane helix A of the other subunit in the functional homodimeric PTP. The results identify for the first time not only a transmembrane helix contact site between the two subunits of a homodimeric mitochondrial transport protein but also a contact site that if locked into position blocks transport. The results are related to two available secondary transporter structures (lactose permease, glycerol-3-phosphate transporter) as well as to a low resolution projection structure and a high resolution structure of monomers of inhibitor ADP/ATP carrier complexes.

• PMID: 15147204
• DOI full text
• PubMed

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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Phelps A, Wohlrab H

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