Abstract
The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.
Highlights
Chemistry and Molecular Pharmacology, The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candidaparapsilosis
The N-ethylmaleimide-reactive of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl reacts with another cysteine to inhibit the transport
The results reported in this paper clearly show that the phosphate transport catalyzed by the proteoliposomes is due to the phosphate transport protein
Summary
Of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl reacts with another cysteine to inhibit the transport. Terns are sensitive to mercurials whereas only the transport catalyzed by the phosphate transport protein is N-ethylmaleimide-sensitive Definitive separation of these two transport functions was obtained by the purification of the active form of PTP’ from beef heart mitochondria [3, 4]. Incorporated into liposomes it catalyzes phosphate:phosphate exchange that is sensitive to N-ethylmaleimide but not to the dicarboxylate carrier- specific inhibitor n-butylmalonate [3]. A novel method is described for removing this AAC from the 5’. cerevkiae PTP preparation
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