title: Replacements of basic and hydroxyl amino acids identify structurally and functionally sensitive regions of the mitochondrial phosphate transport protein.
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 microL and fairly independent of the amount of PTP in the initial reconstitution mix (49-212 ng of PTP/25 microL). Arg159Ala and Lys295Gln show the smallest incorporation of 2.3 +/- 1.6 ng of PTP/25 microL and 2.6 +/- 0.2 ng of PTP/25 microL, respectively. Ser145Ala shows the largest incorporation of 37.0 ng of PTP/25 microL. 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.
authors.affiliation: Boston Biomedical Research Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Massachusetts 02114, USA.
authors.forename: C
authors.initials: C
authors.lastname: Briggs
authors.forename: L
authors.initials: L
authors.lastname: Mincone
authors.forename: H
authors.initials: H
authors.lastname: Wohlrab
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D008081:Liposomes; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011510:Proteolipids; C020485:proteoliposomes; D017665:Hydroxyl Radical