Mitochondrial Phosphate Transport

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candidaparapsilosis. 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 antisera 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 V ,,,Bx of 170 pmol of phosphate min-’ (mg PTP)-’ (22 “C) by increasing the pHi of the proteoliposomes from 6.8 (same as pH,) to 8.0. The K, for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 pM) 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 Cys4’ 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 CYSTS has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.