IRON TRAFFICKING IN SACCHAROMYCES CEREVISIAE

描述：(申请人摘要)在这份提案中，基因和 参与铁在质膜和细胞膜之间运输的蛋白质 将定义酿酒酵母线粒体内的蛋白质。一个 将研究新的酵母突变株集合，展示 细胞铁摄取、铁敏感性和蓄积性高 线粒体中的铁。这些突变体将被组织成 互补组，将克隆的基因(S)和基因产物(S) 特色化的。这种类型的突变体，如MA15突变体， 铁作为一种含有线粒体的铁-蛋白质复合体，这个复合体将是 根据其蛋白质和铁的组成进行提纯和表征。 此外，还出现了一种从线粒体中分离出来的特殊铁蛋白。 缺乏MA15突变体(33 kDa铁蛋白)。这种蛋白质将是 提纯和鉴定。这些突变体中的分子缺陷可能是 与这种蛋白质直接或间接相关，这将是重要的 确定这种铁蛋白的缺乏是否代表着 铁向线粒体或更窄的缺陷内的蛋白质输送。这个 第二个具体目的是确定进口铁的生化特征 变成线粒体。线粒体的输入速率将被确定。 活细胞和/或分离的线粒体，寻找异常 这可能是变种人特有的。为测量而开发的方法 将蛋白质导入纯化的线粒体将用于帮助 这些研究的进展。最后，具体目标3是针对 确定MA15突变体中铁毒性的性质。的基础 将对小菌落表型和铁敏感性进行评估，并 有证据表明mtDNA是有毒铁损伤的靶点 突变株将使用酵母特有的方法进行评估。 在线粒体内的致密沉积物中积累铁的酵母突变体 虽然不能正确地合成铁蛋白，但与 一组称为铁粒母细胞的人类疾病的红系祖细胞 贫血。获得性铁粒母细胞性贫血通常代表一种 白血病前期状态，以及疾病和过渡到 白血病是未知的，因此，酵母菌工作的临床相关性将是 通过寻找保守的基因和功能来寻找。

DESCRIPTION: (applicant's abstract) In this proposal, the genes and proteins involved in trafficking of iron between the plasma membrane and protein within mitochondria of Saccharomyces cerevisiae will be defined. A novel collection of yeast mutants will be studied that exhibit constitutively high cellular iron uptake, iron sensitivity and accumulation of iron within mitochondria. These mutants will be organized into complementation groups, the genes(s) will be cloned and the gene products(s) characterized. Mutants of this type, such as the ma15 mutant, accumulate iron as an iron-protein complex with mitochondria, and this complex will be purified and characterized in terms of its protein and iron composition. Furthermore, a specific iron protein isolated from mitochondria appeared deficient in the ma15 mutant (33 kDa iron protein). This protein will be purified and identified. The molecular defect in these mutants may be directly or indirectly related to this protein, and it will be important to ascertain if the lack of this iron protein represents a general defect in iron delivery to proteins within mitochondria or a more narrow defect. The second specific aim is to determine the biochemical features of iron import into mitochondria. The rates of import into mitochondria will be determined for living cells and/or isolated mitochondria, looking for abnormalities that might be specific for the mutants. Methods developed for measuring protein import into purified mitochondria will be used to aid in developments of these studies. Finally, specific aim 3 is aimed at determining the nature of iron toxicity in the ma15 mutant. The basis of the small colony phenotype and iron sensitivity will be evaluated and the evidence that mtDNA represents a target for toxic iron damage in these mutants will be evaluated using methods unique to yeast. The yeast mutants that accumulate iron in dense deposits within mitochondria while failing to properly synthesize iron proteins bear a resemblance to the erythroid progenitors of a group of human disorders called sideroblastic anemia. The acquired form of sideroblastic anemia often represents a preleukemic state, and the basis for the disease and for the transition to leukemia is unknown, Thus, clinical relevance of the yeast work will be sought by looking for conserved genes and function.