MITOCHONDRIAL PI (H+) AND PI DICARBOXYLATE TRANSPORTER

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Membrane transport proteins are essential to living organisms. The mechanism of solute transport is not well understood for any transport protein and only very few transport proteins have been purified to such an extent as to make studies of the catalytic mechanism possible. The purpose of this research project is to investigate the molecular mechanism of the mitochondrial phosphate transport protein (PTP), the dicarboxylate transport protein (DTP), as well as their relationship to the adenine nucleotide translocase (ANT). Both bind phosphate with similar affinity. PTP most likely cotransports H+ with Pi- while DTP exchanges divalent anions, including Pi-2. These studies will help us understand phosphate binding sites of transport proteins, molecular mechanisms of transport protein catalysis in general, and possibly the differential/coordinated expression of these transport proteins in various tissues. PTP has been purified. DTP has not been identified. We plan (PTP): (a) to eliminate ANT and minor low mw proteins from the transport-active PTP; (b) to finish determining its primary sequence by protein biochemical techniques and cDNA sequencing (also ANT); this will yield leader sequence(s) and predictions for its secondary/tertiary structure in the membrane; permit us to prepare PTP cDNA from different organisms (and organs) to identify partial protein sequences of high functional importance on the basis of common homologies; attempts will be made to generate in vitro point mutations to generate additional critical functional information; PTP (and ANT) from rat heart and liver have different mobilities in SDS gels due to differences in: their structural genes or signal protease processing by the heart/liver mitochondria; (c) to identify amino acid residues at the transport site(s), at the C and M side of the membrane, and at protein regions interacting with lipids or other proteins with detailed kinetic studies, with chemical reagents, and (monoclonal) antibodies; we expect to detect membrane PsipH and/or phosphate induced protein conformational changes; (d) to identify the oligomeric state of PTP and minimum size peptide able to catalyze transport; (e) to prepare two or three dimensional PTP crystals for structural studies. We will use photoaffinity substrate analogues to identify DTP, purify, reconstitute, and characterize it with methods minimally modified from those developed for PTP. We will use the cDNA's prepared for the transport proteins to isolate and characterize genomic genes to help us explain coordinated/differential expression of these proteins in different tissues.