The mitochondrial phosphate transport protein (PTP) is a critical link in the energy metabolism of the cell and is thus a very important protein. Any one of many mutations within this protein makes it impossible for a mammalian cell to survive. PTP is an excellent protein to study transmembrane transport of metabolites. Its mechanism of transport is easier to understand than that of other metabolite transport proteins because it transports the simple entities inorganic phosphate (Pi) and proton. Previous studies with site-directed mutagenesis have identified regions in the protein that are critical for transport.
The aim now is to identify transmembrane secondary protein structure elements that line the Pi transport path through PTP and to demonstrate that they undergo Pi-induced movements as part of the sequence of events associated with the transport. PTP Pi binding sites on both sides of the membrane will be identified by labeling residues with a competitive inhibitor of PTP, which is a membrane-impermeable photolabel. These sites should be essential for transport and are thus expected to be modified in some PTP mutants inactivated by a conservative mutation. The transport hypothesis implies that PTP functions as a homodimer with two Pi transport paths alternating in the transport of Pi into the mitochondrial matrix. PTP has thus four phosphate binding sites, two on either side of the membrane. Only one of the two sites on either side of the membrane is accessible to Pi at any one time. To support this hypothesis, it will be shown with the help of the photoaffinity label that the Pi-accessible sites are on different subunits and with the help of spin labels that the two sites on the same side of the membrane have different conformations. In addition, spin labels will be attached to PTP to identify the transmembrane secondary structure elements that are associated with the photolabeled residues and that, due to their role in transport, should show Pi-induced movements. Efforts will continue to prepare high quality crystals of PTP to determine its 3D structure. The structure will permit the accurate localization of the functionally relevant residues and regions of PTP and should lead to a good understanding of transmembrane metabolite transport.
