Replacements of basic and hydroxyl amino acids identify structurally and functionally sensitive regions of the mitochondrial phosphate transport protein

The mitochondrial phosphate transport protein (PTP) from the yeast Saccharo myces cerevisiae has been expressed in Escherichia coli, purified, and reco nstituted. Basic and hydroxyl residues were replaced to identify structural ly and functionally important regions in the protein. Physiologically relev ant unidirectional transport from extraliposomal (cytosol) pH 6.8 to intral iposomal (matrix) pH 8.0 was assayed. Replacements that affect transport mo st dramatically are at Lys42 (matrix end of helix A), Thr79 (helix B), Lys9 0 (cytosol end of helix B), Arg140 and Arg142 (matrix end of helix C), Lys1 79 and Lys187 (helix D), Ser232 (helix E), and Arg276 (helix F). The delete rious nature of these mutations was confirmed by the observation that the y east PTP null mutant transformed with any one of these mutant genes cannot grow or has difficulties growing with glycerol as the primary carbon source . More than 90% of transport activity can be blocked by various mutations w ithout affecting growth on glycerol. Alterations in the structure of the tr ansport protein caused by the mutations were characterized by determining t he fraction of PTP incorporated into liposomes during reconstitution. The i ncorporation of all PTPs (wild type and mutant) into liposomes is 15.5 +/- 8.4 ng of PTP/25 mu L and fairly independent of the amount of PTP in the in itial reconstitution mix (49-212 ng of PTP/25 mu L). Arg159Ala and Lys295Gl n show the smallest incorporation of 2.3 +/- 1.6 ng of PTP/25 mu L and 2.6 +/- 0.2 ng of PTP/25 mu L, respectively. Ser145Ala shows the largest incorp oration of 37.0 ng of PTP/25 mu L. These three mutants show near wild-type reconstituted transport activity. Two of these three mutations are located in the loop connecting the matrix ends of helices C and D, Ser145 at its N- terminal (the matrix end of helix C) and Arg159 near its center. Lys295 is located at the C-terminal of PTP beyond helix F. These results, together wi th those from other mutations, suggest that Like helix A, the protein segme nt consisting of the loop connecting helices C and D and helix D as well as the C-terminal of PTP beyond helix F faces the subunit interface of this h omodimer. The role of the replacement-sensitive residues in the phosphate o r in the coupled proton transport path is discussed.