Molecular and Genetic Analysis of Potassium Transporters

钾转运蛋白的分子和遗传分析

• 批准号: 9406577
• 负责人: Richard Gaber
• 金额: $ 33.25万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-15 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9406577&HistoricalAwards=false
• 关键词: Molecular; Genetic; Analysis; Potassium; Transporters

9406577 Gaber The PI, Dr. Richard Gaber, proposes to conduct genetic and molecular analyses of K+ transporters in the yeast Saccharomyces cerevisiae. The proposal has four aims. These are to investigate by molecular and genetic methods: 1) the potassium transporting glucose transporters. A detailed analysis of potassium uptake by one of these transporters, HXT3, will be carried out. An extensive collection of spontaneous suppressor mutations in HXT3 will be obtained and the nature of each suppressor mutation will be determined. Their effects on potassium/glucose stoichiometry, glucose transport kinetics and substrate specificity will be determined. 2) Genetic and molecular analysis of potassium transporting amino acid transporters will be carried out. Some trk1d trk2d suppressors do not show dependency on glucose. DNA sequence analysis of one of these, RPD4-1, revealed that it encodes a new member of a family of genes that encode amino acid transporters. Further experiments will determine whether this mutant transporter mediates potassium uptake, and if so, whether potassium uptake is dependent on co-transport of the permeant amino acid. 3) Genetic and molecular analysis of additional trk1d and trk2d suppressors will be carried out. Genetic analysis of the collection of these suppressors will be completed and a representative of each genetically distinct locus will be cloned and sequenced. The collection contains at least two more suppressors of interest, RPD7, represented by 10 independent dominant alleles, and rpd6-1, which is allelic to VPS16, a gene encoding a vacuolar sorting protein. 4) Suppressor mutations will be targeted to specific transporters. %%% Simple mutations in sugar transporters can confer upon these proteins the ability to transport potassium ions thus suppress the potassium-uptake-defective phenotype of Saccharomyces cerevisiae cells deleted for their normal potassium transporters. These suppressor mutations alter single amino acids within putative membrane spanning domains to confer potassium/sugar co-transport. A similar suppressor mutation in a putative amino acid transporter has also been found. These novel transporters offer a unique opportunity to study the molecular basis of solute transport. ***

9406577 PI Gaber Richard Gaber博士提议在酵母糖酵母中对酿酒酵母进行K+转运蛋白的遗传和分子分析。 该提案有四个目标。 这些是通过分子和遗传方法研究：1）转运葡萄糖转运蛋白的钾。 将对这些转运蛋白之一HXT3对钾摄取的详细分析进行。 将在HXT3中获得广泛的自发抑制突变，并确定每个抑制突变的性质。 将确定它们对钾/葡萄糖化学计量，葡萄糖转运动力学和底物特异性的影响。 2）将对转运氨基酸转运蛋白的遗传和分子分析进行。 某些TRK1D TRK2D抑制器不会显示对葡萄糖的依赖性。 其中一种RPD4-1的DNA序列分析表明，它编码了编码氨基酸转运蛋白的基因家族的新成员。 进一步的实验将确定该突变转运蛋白是否介导钾的摄取，如果是，钾的摄取是否取决于渗透氨基酸的共转移。 3）将对其他TRK1D和TRK2D抑制器进行遗传和分子分析。 这些抑制剂收集的遗传分析将完成，并将每个遗传学基因座的代表克隆和测序。 该集合包含至少有两个感兴趣的抑制剂RPD7，由10个独立的主要等位基因代表，RPD6-1（RPD6-1）是等位基因的VPS16，这是一个编码液泡分类蛋白的基因。 4）抑制突变将针对特定转运蛋白。 糖转运蛋白中的简单突变可以赋予这些蛋白质运输钾离子的能力，从而抑制糖含量的酿酒酵母细胞的钾 - 摄取缺陷表型被删除的正常钾转运蛋白。 这些抑制突变改变了假定的膜跨度域内的单个氨基酸，以赋予钾/糖共转移。 还发现了推定氨基酸转运蛋白中类似的抑制突变。 这些新型的转运蛋白为研究溶质传输的分子基础提供了独特的机会。 ***