PHOSPHATE TRANSPORT PATHS WITHIN HOMODIMERIC PTP

线粒体磷酸盐转运蛋白（PTP）是 在细胞的能量代谢，因此是一个非常重要的 蛋白 该蛋白质中的任何一个突变都会使其 哺乳动物细胞是不可能存活的。 PTP是一种很好的蛋白质 研究代谢物的跨膜转运。 机理 转运比其他代谢物更容易理解 因为它运输简单的无机物 磷酸盐（Pi）和质子。 先前的研究， 诱变已经确定了蛋白质中对 运输 现在的目标是鉴定跨膜二级蛋白 通过PTP排列Pi传输路径的结构元素， 证明他们经历了圆周率引起的运动， 与运输相关的事件序列。 PTP Pi结合位点 将通过标记残基来识别膜两侧的 与PTP的竞争性抑制剂，这是一种膜不可渗透的 光标记。这些地点对交通运输必不可少，因此 预期在一些PTP突变体中被修饰，所述突变体被 保守突变 运输假说意味着PTP 作为同二聚体起作用，其中两个Pi传输路径在膜中交替 运输Pi进入线粒体基质。 PTP有四个 磷酸盐结合位点，膜的两侧各有两个。 只有一 膜两侧的两个位点中的一个可接近Pi， 任何一次。 为了支持这一假设，将使用 借助Pi可接近位点所在的光亲和标记 在自旋标记的帮助下， 在膜的同一侧具有不同的构象。 在 此外，将在PTP上贴上自旋标签， 跨膜二级结构元件，其与 由于其在迁移中的作用， 显示圆周率引起的运动。 将继续努力准备高 质量晶体的PTP，以确定其3D结构。 结构 将允许准确定位功能相关的 PTP的残基和区域，并应导致良好的理解 跨膜代谢物转运

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.