PROTON-COUPLED INORGANIC PHOSPHATE TRANSPORT

质子耦合无机磷酸盐传输

• 批准号: 3282975
• 负责人: Hartmut none Wohlrab
• 金额: $ 28.79万
• 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/3282975
• 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.

这是本基金申请研究的目的，以确定