PROTON-COUPLED INORGANIC PHOSPHATE TRANSPORT

质子耦合无机磷酸盐传输

• 批准号: 2176996
• 负责人: Hartmut none Wohlrab
• 金额: $ 31.7万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176996
• 关键词: affinity labeling; aminoacid analog; aspartate; autooxidation; chemical substitution; cysteine; electron spin resonance spectroscopy; essential aminoacid; fluorescence spectrometry; gene complementation; gene mutation; histidine; hydrogen transport; ion transport; membrane permeability; membrane transport proteins; mitochondria; mitochondrial membrane; mutant; phenotype; phosphates; polymerase chain reaction; protein purification; protein reconstitution; protein signal sequence; protein structure; protein tyrosine kinase; radiotracer; receptor coupling; serine; site directed mutagenesis; threonine; tryptophan

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.

在这项赠款申请中的研究目的是确定 无机磷跨膜转运的分子机制 磷酸转运蛋白(PTP)催化的线粒体膜 并更明确地证明了其线粒体输入受体 (MIR)函数。人们对其分子机制知之甚少。 以及拟议的研究预计将 产生了许多新的信息。我们的主要方法将利用 与蛋白质互补的定点和随机突变 纯化、重组和转运分析(也是完整的 线粒体)。取代主要感兴趣的氨基酸： 半胱氨酸解释自氧化对转运的可逆性抑制 (因此可能有助于识别PTP同源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 (帮助确定活跃的运输地点和PTP的安排 在膜中)；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 运输路径；作为质子成员的组氨酸和天冬氨酸 共转运途径。找出不那么明显，但很关键的 重要氨基酸，我们将随机诱变酵母菌PTP基因和 通过呼吸缺乏(甘油)确定PTP表型， 糖皮质激素诱导表达，PTP基因互补。因此， 已发现的突变有望聚集在PI结合位点(S)周围， 质子转运氨基酸和二聚体必需氨基酸 形成以及PTP插入到膜中所需的那些 和细胞内蛋白质的稳定性。变种人将被构建成 允许使用自旋标记(EPR)和 色氨酸(本征荧光)。PTP是一种出色的系统，用于 这些研究由于传输底物(PI)是相当简单的 与其他底物相比，如乳糖(乳糖载体)和ADP或ATP (线粒体ADP/ATP转位酶)。这种蛋白质很可能是一种 只有五到六个不同跨膜α-螺旋的同源二聚体， 类似于ADP/ATP转位酶，但不像细菌视紫红质或 十二号紫胶运输船。重要信息可从 周质高亲和力无机磷酸盐的晶体结构 大肠杆菌磷酸特殊转运系统(PST)的结合蛋白： 磷酸盐只通过氢键与蛋白质相互作用，它 可以容纳单价和二价磷酸。突变者 在紫胶渗透酶中糖与质子的偶联 确认身份。同样，PTP突变体，在极端情况下甚至可能是主导的 致命性，可能更容易被定性。我们预计在膜的一侧 具体的方式，线粒体信号序列会影响PTP 运输活性，而核定位信号序列不会。 PTP对于真核细胞的新陈代谢是必不可少的。它的氧气 敏感性可能在心血管疾病中起重要作用 (再灌注)和一些原发肿瘤以外的人类肿瘤的多样性 州政府。