Zinc Transporters and Intracellular Zinc Trafficking

描述（由申请人提供）：锌对所有生物体都是必需的。锌是超过300种酶的催化辅因子，是锌指等结构基序的关键组分。因此，细胞需要有效的机制来积累锌。许多锌依赖性蛋白质被分泌或定位于细胞内细胞器的内部，例如分泌途径和线粒体的细胞器。因此，还需要有效的机制将锌分配到细胞内隔室中。相反，过量的锌可能对细胞有毒。这种毒性可能是由于锌与蛋白质或辅因子中的不适当位点结合所致。例如，过量的锌可以干扰线粒体顺乌头酸酶活性并损害呼吸。锌的基本但有毒的性质需要精确的稳态机制来控制细胞内的“游离”水平，即，松散的或不稳定的，锌。我们目前对细胞内锌转运和锌螯合知之甚少。我们正在研究这些过程中使用酵母酿酒酵母作为模型真核细胞。我们的研究提出了四个中心假设，这是这个建议的基础：1）酵母液泡，酵母细胞的溶酶体样区室，在储存锌在充满细胞，供以后在缺锌条件下使用的重要作用。2)当细胞内不稳定的锌池响应于锌稳态的扰动而改变时，液泡作为锌缓冲器来控制细胞溶质和其他隔室中的锌水平。3)Msc2蛋白是金属离子转运蛋白的普遍存在的CDF家族的成员，负责将锌转运到内质网中，以将金属供应到分泌途径中的分泌蛋白和驻留蛋白。4)另外的转运蛋白也存在于酵母的ER中，其将锌转运到分泌途径中。为了验证这些假设，我们提出了一个多方面的方法，使用遗传学，分子生物学，细胞生物学，生物化学和生物无机化学工具的协同组合，以定义酵母细胞内锌转运的机制和作用。

DESCRIPTION (provided by applicant): Zinc is essential for all organisms. Zinc is a catalytic cofactor of over 300 enzymes and is a critical component of structural motifs such as zinc fingers. Therefore, cells require efficient mechanisms for the accumulation of zinc. Many zinc-dependent proteins are either secreted or localized to the interior of intracellular organelles such as those of the secretory pathway and mitochondria. Therefore, efficient mechanisms are also needed to distribute zinc into intracellular compartments. In contrast, excess zinc can be toxic to cells. This toxicity may be due to the binding of zinc to inappropriate sites in proteins or cofactors. For example, excess zinc can interfere with mitochondrial aconitase activity and impair respiration. The essential yet toxic nature of zinc necessitates precise homeostatic mechanisms to control the intracellular levels of "free", i.e., loosely bound or labile, zinc. We currently know little about intracellular zinc transport and zinc sequestration. We are studying these processes using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Our studies have raised four central hypotheses that are the foundation of this proposal: 1) The yeast vacuole, the lysosome-like compartment of the yeast cell, plays an important role in storing zinc in replete cells for later use under conditions of zinc deficiency. 2) The vacuole acts as a zinc buffer to control zinc levels in the cytosol and other compartments when the intracellular labile zinc pool changes in response to perturbations in zinc homeostasis. 3) The Msc2 protein, a member of the ubiquitous CDF family of metal ion transporters, is responsible for transporting zinc into the endoplasmic reticulum to supply the metal to secreted and resident proteins in the secretory pathway. 4) Additional transporters are also present in the ER of yeast that transport zinc into the secretory pathway. To test these hypotheses, we propose a multifaceted approach, using a synergistic combination of genetic, molecular biology, cell biology, biochemistry, and bioinorganic chemistry tools, to define the mechanisms and roles of intracellular zinc transport in yeast.