PROTON-COUPLED INORGANIC PHOSPHATE TRANSPORT

本研究申请的目的是确定 无机磷跨膜转运的分子机制 磷酸盐转运蛋白催化的线粒体膜 并更明确地证明其线粒体输入受体 (mir)功能 相对而言，我们对这种疾病的分子机制知之甚少。 运输蛋白的一般和拟议的研究预计将 产生了很多新的信息。 我们的主要方法是利用 定点突变和随机突变，辅以蛋白质 纯化、重构和转运测定（也在完整的 线粒体）。 用于取代的主要感兴趣的氨基酸： 半胱氨酸解释了通过自氧化可逆抑制转运 (and因此可能有助于鉴定PTP同源二聚体亚基上的氨基酸 界面）及N-乙基马来酰亚胺和汞水杨酸对转运抑制作用 (to有助于表征活跃的转运位点和PTP的排列 羟基氨基酸，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 运输途径;组氨酸和天冬氨酸作为质子的成员 共转运途径 去识别那些不太明显但很关键的 重要的氨基酸，我们将随机诱变酵母PTP基因， 通过呼吸缺陷（甘油）鉴定PTP-表型， 糖皮质激素诱导表达和PTP基因互补。 因此 预期鉴定的突变聚集在Pi结合位点周围， 质子转运氨基酸和二聚体必需氨基酸 形成以及PTP插入膜所需的那些 和细胞内蛋白质稳定性。 突变体将被构造成 允许利用自旋标记（EPR）进行膜内排列研究， 内荧光（intrinsic fluorescence）。 PTP是一个优秀的系统， 这些研究，因为传输基板（Pi）是相当简单的 与其他底物如乳糖（乳糖载体）和ADP或ATP相比 （线粒体ADP/ATP转位酶）。 这种蛋白质很可能是 只有5或6个不同跨膜α-螺旋同二聚体， 类似于ADP/ATP移位酶，但不像细菌视紫红质的7或 12号运载火箭。 重要信息可从 周质高亲和性无机磷酸盐的晶体结构 E.大肠杆菌磷酸盐特异性转运系统（Pst）： 磷酸盐仅通过氢键与蛋白质相互作用， 可以容纳单价和二价磷酸盐。 突变体 在乳糖通透酶中糖与质子的偶联中， 鉴定 同样，PTP突变体，在极端情况下甚至可能是显性的， 致命的可能更容易描述 我们希望在膜侧 线粒体信号肽序列会以特定的方式影响PTP 而核定位信号序列将不会。 PTP对于真核细胞的代谢是必需的。 其氧 敏感性可能在心血管疾病中起重要作用 （再灌注）和一些人类肿瘤的多样性超出了原发性 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.