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.

Functional and Structural Role of Amino Acid Residues in the Odd-numbered Transmembrane α-Helices of the Bovine Mitochondrial Oxoglutarate Carrier

“…The amount of reconstituted wild-type and mutant proteins varied between 16% and 28% of the added protein, which is similar to values found for other recombinant reconstituted mitochondrial carrier proteins. 17,[22][23][24][25][26][27] For transmembrane α-helices H1, H3 and H5, Figure 2 shows the initial transport rates of 2-oxoglutarate for each recombinant OGC as a percentage of the value for the cysteine-less carrier. The effects of the mutations on the initial transport rate were mapped on the comparative model of OGC ( Figure 3).…”

“…The proline residues of the signature motif induce sharp kinks in the odd-numbered α-helices, closing the central pore of the α-helical bundle at the matrix side, whereas the charged residues form a salt bridge network at the bottom of the cavity. 10 Some residues of the signature motif have been shown to be important for function in the ADP/ ATP carrier, [13][14][15] the phosphate carrier 16,17 and the citrate carrier. 18 The structure of the mitochondrial ADP/ATP carrier in the cytoplasmic state is known, 10 but the structural changes required for the translocation of the substrate are not.…”

The Yeast Mitochondrial Citrate Transport Protein

“…Lys 179 and Lys 187 ) that reside within its fourth ␣-helical transmembrane domain (27). These residues FIG.…”

“…Accordingly, the CTP was incorporated into liposomes both in the (25,26,27), depicts six transmembrane ␣-helical segments. These segments are connected by five hydrophilic loops.…”

Specific Labeling of the Bovine Heart Mitochondrial Phosphate Carrier with Fluorescein 5-Isothiocyanate

“…The same P i transport inhibitions by FITC with and without DTT treatment clearly showed that the possible labeling of cysteine residues with FITC (24) was not associated with P i transport inhibition by FITC. This conclusion is supported by the finding that replacement by Ala or Arg of Lys 187 of the yeast carrier, which corresponds to Lys 185 of the bovine carrier, resulted in complete loss of the transport activity (33).…”