Replacements of Basic and Hydroxyl Amino Acids Identify Structurally and Functionally Sensitive Regions of the Mitochondrial Phosphate Transport Protein † Christine Briggs, ‡ Leesa Mincone, and Hartmut Wohlrab* Boston Biomedical Research Institute and Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School, Boston, Massachusetts 02114 ReceiVed December 15, 1998; ReVised Manuscript ReceiVed February 23, 1999 ABSTRACT: The mitochondrial phosphate transport protein (PTP) from the yeast Saccharomyces cereVisiae has been expressed in Escherichia coli, purified, and reconstituted. Basic and hydroxyl residues were replaced to identify structurally and functionally important regions in the protein. Physiologically relevant unidirectional transport from extraliposomal (cytosol) pH 6.8 to intraliposomal (matrix) pH 8.0 was assayed. Replacements that affect transport most dramatically are at Lys42 (matrix end of helix A), Thr79 (helix B), Lys90 (cytosol end of helix B), Arg140 and Arg142 (matrix end of helix C), Lys179 and Lys187 (helix D), Ser232 (helix E), and Arg276 (helix F). The deleterious nature of these mutations was confirmed by the observation that the yeast 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 without affecting growth on glycerol. Alterations in the structure of the transport protein caused by the mutations were characterized by determining the fraction of PTP incorporated into liposomes during reconstitution. The incorporation of all PTPs (wild type and mutant) into liposomes is 15.5 ( 8.4 ng of PTP/25 μL and fairly independent of the amount of PTP in the initial reconstitution mix (49-212 ng of PTP/25 μL). Arg159Ala and Lys295Gln show the smallest incorporation of 2.3 ( 1.6 ng of PTP/25 μL and 2.6 ( 0.2 ng of PTP/25 μL, respectively. Ser145Ala shows the largest incorporation of 37.0 ng of PTP/25 μL. 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 with those from other mutations, suggest that like helix A, the protein segment 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 homodimer. The role of the replacement-sensitive residues in the phosphate or in the coupled proton transport path is discussed. Mitochondria are responsible for providing the bulk of ATP to the cell. The phosphorylation of ADP for this purpose occurs within mitochondria. The P i for this phosphorylation is transported via the PTP (PHT, PHC, PIC, PIT, MIR) (1) as phosphate/proton cotransport from the cytosol into the mitochondrial matrix, following a pH gradient of about 1.2 pH units with a cytosol pH 6.8 and a matrix pH 8.0. We are interested in understanding the function of this protein at the molecular level and have thus replaced several of its basic and hydroxyl residues. Mutant PTPs from yeast mitochondria have already been constructed and character- ized in which the Cys (2, 3), the acidic and His residues (4), Ile141 (5), and other residues (6, 7) have been replaced. Several of these residues have been suggested (a) to be at the interface between the subunits of the homodimer (2), (b) to be primary members of a proton cotransport path (4), or (c) to be at a location where residue replacement uncouples the proton and phosphate transport paths (5). We report now that replacements of basic and hydroxyl residues point toward additional regions in PTP that are structurally and function- ally significant. We have determined the physiologically relevant transport mode of the mutants and the fraction of the purified PTP in the reconstitution mixture that is incorporated into the transport-active proteoliposomes. We discuss the implications of these findings. MATERIALS AND METHODS Preparation of Mutant PTP Plasmids. The yeast shuttle vector pAP-W3 (6) was used to express the mutant PTPs in Saccharomyces cereVisiae (6). The ability of the mutant PTPs to rescue the S. cereVisiae PTP null mutant on glycerol was tested on YPG plates (6). PTP was expressed in Escherichia coli as inclusion bodies with the vector pNYHM131 (5, 8). Mutant PTP genes were prepared according to a PCR pro- tocol (6). Codons of the wild-type and replacement residues are shown in Table 1. † Supported by grants from the NIH (GM33357, GM57563). * Address correspondence to this author at the Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114.Tel: (617) 912-0336. Fax: (617) 912-0308. E-mail wohlrab@bbri.org. ‡ Present address: The Ocular Molecular Genetics Institute, Harvard Medical School, Mass. Eye and Ear Infirmary, 243 Charles Street, Boston, MA. 02114. 1 Abbreviations: M, matrix; C, cytosol; PTP, phosphate transport protein; PHT, phosphate transporter; PHC, phosphate carrier; PIC, phosphate carrier; PIT, phosphate transporter; MIR, mitochondrial import receptor; LIE, liposome incorporation efficiency; Pi, inorganic phosphate; X, any amino acid; HCA, human carbonic anhydrase. 5096 Biochemistry 1999, 38, 5096-5102 10.1021/bi982945n CCC: $18.00 © 1999 American Chemical Society Published on Web 04/02/1999