Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport.

The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path.

Replacements of basic and hydroxyl amino acids identify structurally and functionally sensitive regions of the mitochondrial phosphate transport protein.

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.

Mitochondrial phosphate transport protein. replacements of glutamic, aspartic, and histidine residues affect transport and protein conformation and point to a coupled proton transport path.

The homodimeric mitochondrial phosphate transport protein (PTP), which has six transmembrane helices per subunit, catalyzes inorganic phosphate transport in an electroneutral and pH gradient-dependent manner across the inner membrane. We have replaced the Glu, Asp, and His residues of the yeast PTP to assess their role in the transport mechanism. Mutants with physiologically relevant transport activity were identified by their ability to rescue the PTP null mutant yeast from glycerol medium.