IRON TRAFFICKING IN SACCHAROMYCES CEREVISIAE

描述：（申请人的摘要）在该提议中，基因和 蛋白质参与运输铁之间的质膜和 将定义酿酒酵母线粒体内的蛋白质。 一 将研究一种新的酵母突变体， 组成性高细胞铁摄取、铁敏感性和积累 线粒体内的铁。 这些变种人将被组织成 互补组，基因将被克隆，基因产物 表征了 这种类型的突变体，如ma 15突变体， 铁作为铁蛋白质复合物与线粒体，这种复合物将 纯化并根据其蛋白质和铁组成进行表征。 此外，还发现了一种从线粒体中分离的铁蛋白 ma 15突变体（33 kDa铁蛋白）缺陷。 这种蛋白质将 纯化和鉴定。 这些突变体的分子缺陷可能是 直接或间接地与这种蛋白质有关， 确定这种铁蛋白的缺乏是否代表一种普遍的缺陷， 铁传递到线粒体内的蛋白质或更窄的缺陷。 的 第二个具体目标是确定铁输入的生化特征 转化为线粒体。 将确定输入线粒体的速率 活细胞和/或分离的线粒体，寻找异常 可能是变种人特有的 制定的测量方法 将蛋白质输入到纯化的线粒体中将用于帮助 这些研究的发展。 最后，具体目标3旨在 确定MA 15突变体中铁毒性的性质。 的基础 将评估小菌落表型和铁敏感性， 证据表明，线粒体DNA代表了毒性铁损伤的目标， 将使用酵母特有的方法评估突变体。 在线粒体内以致密沉积物形式积累铁的酵母突变体 虽然不能正确合成铁蛋白质， 一组称为铁粒幼细胞的人类疾病的红系祖细胞 贫血 铁粒幼细胞性贫血的获得性形式通常代表一种 白血病前期状态，以及疾病的基础和向 白血病是未知的，因此，酵母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.