Zinc Transporters and Intracellular Zinc Trafficking

说明(申请人提供)：锌对所有生物体都是必需的。锌是300多种酶的催化辅因子，是锌指等结构基序的关键成分。因此，细胞需要有效的机制来积累锌。许多锌依赖蛋白要么分泌，要么定位于细胞内细胞器内部，如分泌途径和线粒体。因此，还需要有效的机制来将锌分布到细胞内的隔室。相比之下，过量的锌可能会对细胞产生毒性。这种毒性可能是由于锌与蛋白质或辅因子中不适当的位置结合所致。例如，过量的锌会干扰线粒体乌头酸酶的活性，损害呼吸。锌的本质是有毒的，需要精确的体内平衡机制来控制细胞内“游离”的水平，即松散结合或不稳定的锌。目前，我们对细胞内锌的转运和锌的封存知之甚少。我们正在使用酿酒酵母作为真核细胞的模型来研究这些过程。我们的研究提出了四个核心假设，它们是这一提议的基础：1)酵母细胞的溶酶体样室--酵母液泡，在缺锌条件下将锌储存在充足的细胞中以供以后使用的过程中发挥着重要作用。2)当细胞内不稳定的锌库因锌稳态的扰动而发生变化时，液泡作为锌的缓冲器来控制细胞质和其他隔室中的锌水平。3)Msc2蛋白是无处不在的CDF金属离子转运蛋白家族的成员，负责将锌运输到内质网，为分泌途径中的分泌和驻留蛋白提供金属。4)酵母的内质网中也存在额外的转运蛋白，将锌运输到分泌途径中。为了验证这些假说，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，来定义酵母细胞内锌运输的机制和作用。

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.