Yeast mitochondrial phosphate transport protein expressed in Escherichia coli. Site-directed mutations at threonine-43 and at a similar location in the second tandem repeat (isoleucine-141).
Yeast mitochondrial phosphate transport activity has been reconstituted from the import receptor (MIR) expressed as inclusion bodies in Escherichia coli. This result undermines the suggestion [Murakami, H., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3358-3362] that the MIR has been misidentified as the phosphate transport protein (PTP). PTP was solubilized with N-lauroylsarcosinate and Triton X-100 and purified with a yield of about 2 mg/L of induced bacterial culture. This PTP, reconstituted in liposomes, catalyzes phosphate uptake with a Vmax [24.5 degrees C, net (zero trans), pHi = 8.0, pHe = 6.8] of 0.61 mmol of phosphate min-1 (mg of PTP)-1 and a Km of 1.30 mM. This Vmax is higher and the Km about the same as that obtained with PTP purified from mitochondria. Replacement of Thr43 and Ile141 by other amino acids results in three types of PTP: (a) 2.5-5.0% Vmax of wild-type PTP (PTPwt) (Thr43Cys; Thr43Ser; Ile141Cys), (b) < 0.1% Vmax (detection limit of assay) of PTPwt (Thr43Ala; Thr43Asp), and (c) proton transport uncoupled from phosphate transport (Ile141Cys). Km changes are not significant. Activity of Thr43Cys confirms results obtained with mitochondrially expressed protein. Thus, yeast PTP requires Thr43 and mammalian PTP the similarly located Cys42 for high transport activity. Thr43 and Ile141 are each situated between two basic residues (LysThrArg vs ArgIleArg). Cys substitutions in either of these positions confer the same high N-ethylmaleimide sensitivity to the yeast PTPwt as displayed by the mammalian PTP.(ABSTRACT TRUNCATED AT 250 WORDS)
Wohlrab
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Boston Biomedical Research Institute, Harvard Medical School, Massachusetts 02114.
Briggs
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D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000235:genetics; D008928:Mitochondria / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013912:Threonine / Q000235:genetics
D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D007532:Isoleucine; D013912:Threonine; D005033:Ethylmaleimide