PROTON-COUPLED INORGANIC PHOSPHATE TRANSPORT

本拨款申请中的研究目的是确定 无机磷酸盐跨内运输的分子机制 由磷酸转运蛋白（PTP）催化的线粒体膜 并更明确地证明其线粒体输入受体 （米尔）功能。 人们对分子机制知之甚少 运输蛋白的总体情况和拟议的研究预计 产生很多新信息。 我们的主要方法将利用 定点和随机诱变，辅以蛋白质 纯化、重构和运输测定（也以完整形式进行） 线粒体）。 主要用于替换的氨基酸： 半胱氨酸解释自氧化对运输的可逆抑制 （因此可能有助于识别 PTP 同二聚体亚基上的氨基酸 界面）以及 N-乙基马来酰亚胺和 mersalyl 对转运的抑制 （帮助表征主动运输站点和 PTP 的安排 在膜中）；羟基氨基酸，例如苏氨酸和丝氨酸 磷酸盐-蛋白质相互作用中可能的氢键供体 运输路径；组氨酸和天冬氨酸作为质子的成员 共转运途径。 识别不太明显但具有批判性的 重要的氨基酸，我们将随机诱变酵母 PTP 基因并 通过呼吸缺陷（甘油）识别 ptp- 表型， 糖皮质激素诱导表达和 PTP 基因互补。 因此 预计已识别的突变会聚集在 Pi 结合位点周围， 质子传输氨基酸和二聚体必需的氨基酸 形成以及 PTP 插入膜所需的那些 和细胞内蛋白质的稳定性。 突变体将被构建为 允许利用自旋标签 (EPR) 进行膜内排列研究 色氨酸（固有荧光）。 PTP 是一个优秀的系统 这些研究是因为传输底物 (Pi) 相当简单 与乳糖（乳糖载体）和 ADP 或 ATP 等其他底物相比 （线粒体 ADP/ATP 转位酶）。 该蛋白质很可能是 仅具有五或六个不同跨膜α螺旋的同二聚体， 类似于 ADP/ATP 转位酶，但不像细菌视紫红质或细菌视紫红质的 7 lac载体的12。 重要信息可从 周质高亲和力无机磷酸盐的晶体结构 大肠杆菌磷酸盐特异性转运系统 (Pst) 的结合蛋白： 磷酸盐仅通过氢键与蛋白质相互作用 可以容纳一价和二价磷酸盐。 突变体 在紫胶通透酶中糖与质子的偶联 确定。 再说一次，PTP 突变体，在极端情况下甚至可能占主导地位 致命，可能更容易描述。 我们期望在膜侧 具体方式，线粒体信号序列会影响PTP 转运活性，而核定位信号序列则不会。 PTP 对于真核细胞的新陈代谢至关重要。 它的氧气 敏感性可能在心血管疾病中发挥重要作用 （再灌注）和一些人类肿瘤超出原发性的多样性 状态。

It is the aim of the research within this grant application to identify the molecular mechanism of inorganic phosphate transport across the inner mitochondrial membrane catalyzed by the phosphate transport protein (PTP) and to demonstrate more definitively its mitochondrial import receptor (mir) function. Relatively little is known about the molecular mechanism of transport proteins in general and the proposed studies are expected to yield much new information. Our primary approach will utilize site-directed and random mutagenesis, complemented with protein purification, reconstitution, and transport assays (also in intact mitochondria). Amino acids of primary interest for substitutions: cysteines to explain reversible inhibition of transport by autoxidation (and thus possibly help identify amino acids at the PTP homodimer subunit interface) and inhibition of transport by N-ethylmaleimide and mersalyl (to help characterize active transport sites and the arrangement of PTP in the membrane); hydroxyl amino acids such as threonine and serine as possible hydrogen bond donors in phosphate-protein interaction in the transport path; histidine and aspartate as members of a proton cotransport pathway. To identify the less obvious, yet critically important amino acids, we will random mutagenize the yeast PTP gene and identify ptp- phenotypes by respiratory deficiency (glycerol), glucocorticoid induced expression, and PTP gene complementation. Thus identified mutations are expected to cluster around Pi binding site(s), proton-transport amino acids and amino acids essential for dimer formation as well as those required for PTP insertion into the membrane and intracellular protein stability. Mutants will be constructed to permit intramembrane arrangement studies utilizing spin labels (epr) and tryptophans (intrinsic fluorescence). The PTP is an excellent system for these studies since the transported substrate (Pi) is rather simple compared to other substrates like lactose (lac carrier) and ADP or ATP (mitochondrial ADP/ATP translocase). The protein is most likely a homodimer with only five or six different transmembrane alpha-helices, like the ADP/ATP translocase, but not like the 7 of bacteriorhodopsin or the 12 of the lac carrier. Important information is available from the crystal structure of the periplasmic high affinity inorganic phosphate binding protein of the E. coli phosphate specific transport system (Pst): the phosphate interacts with the protein only via hydrogen bonds and it can accommodate both the monovalent and the divalent phosphate. Mutants in the coupling of sugars with protons in the lac permease have been identified. Again, PTP mutants, that may in the extreme even be dominant lethal, may be easier to characterize. We expect that in a membrane-side specific manner, mitochondrial signal sequences will affect PTP transport-activity while nuclear localization signal sequences will not. The PTP is essential for the metabolism of eukaryotic cells. Its oxygen sensitivity may play an important part in cardiovascular diseases (reperfusion) and the diversity of some human tumors beyond the primary state.