PROTEIN IMPORT INTO YEAST MITOCHONDRIA

线粒体需要数百种核编码蛋白 新陈代谢、生长、分裂和子代细胞的分割，以及 实际上，所有这些蛋白质都必须输入细胞器。 这些进口蛋白质中的许多必须穿过两层膜才能到达 它们的目的地是线粒体。让事情复杂化的是，内心 膜必须保持电化学电位才能驱动 三磷酸腺苷的合成。为了确定蛋白质是如何跨 线粒体内膜，我们已经分离并分析了 酵母中的突变体，酿酒酵母，进口缺陷。 我们的研究已经确定了内膜的两种蛋白质Mas6p 和Sms1p，这是蛋白质转位到 矩阵。我们最近的研究表明，蛋白质可以跨 内膜通过水通道，且Mas6P(和 可能是Sms1p)构成该频道的一部分。这项建议的两个目标 确定Mas6p和Sms1p在进口中的作用。我们有 发现Mas6p和Sms1p相互作用，并与 介导进口的额外蛋白质，增加了Mas6p 和Sms1p是内膜转位复合体的一部分。 这项提议的第三个目标是确定这个组织的新成员 很复杂。 项目1：确定Mas6p在导入中的作用。Mas6P是一种 与前体直接交互的基本导入组件，因为它们 穿过内膜。Mas6p似乎有两个功能 结构域：一个亲水性氨基末端区域，面向 膜间隙和疏水的羧基末端结构域 插入到内膜中。要确定每个组件的功能 作为Mas6p的一部分，我们正在对Mas6p进行具体更改并进行测试 它们对进口的影响。例如，正在询问是否存在基因突变 Mas6p的氨基末端结构域阻止前体与 内膜进口机械。此外，我们正在询问是否 羧基末端结构域构成了蛋白质转位的一部分 内膜中的通道。 项目2：确定Sms1p在进口中的作用。Sms1p，它 对进口也是必不可少的，已被证明与Mas6p相互作用。 为了了解Sms1p在导入中的作用，我们将生成新的 Sms1的等位基因，并检查它们对进口的影响。例如，我们 会问sms1突变体，如mas6突变体，是否存在内在缺陷。 膜通道。 项目3：确定新的膜进口组件并分析其 功能。利用遗传和生物化学方法，我们将 识别新的内膜进口成分并分析其作用 将前体移位到基质中。

Hundreds of nuclear-encoded proteins are required for mitochondrial metabolism, growth, division, and partitioning to daughter cells, and virtually all of these proteins must be imported into the organelle. Many of these imported proteins must cross two membranes to reach their destination inside mitochondria. Complicating matters, the inner membrane must maintain an electrochemical potential to drive the synthesis of ATP. To determine how proteins are translocated across the mitochondrial inner membrane, we have isolated and analyzed mutants in the yeast, Saccharomyces cerevisiae, defective in import. Our studies have identified two proteins of the inner membrane, Mas6p and Sms1p, that are required for the translocation of proteins into the matrix. Our recent studies suggest that proteins are translocated across the inner membrane through an aqueous channel, and that Mas6p (and possibly Sms1p) form part of this channel. Two goals of this proposal are to pinpoint the roles of Mas6p and Sms1p in import. We have found that Mas6p and Sms1p interact with each other and with additional proteins to mediate import, raising the possibility that Mas6p and Sms1p are part of a translocation complex in the inner membrane. A third goal of this proposal is to identify new members of this complex. Project 1: Determine the role of Mas6p in import. Mas6p is an essential import component that directly interact with precursors as they cross the inner membrane. Mas6p appears to have two functional domains: a hydrophilic amino-terminal region that faces the intermembrane space and a hydrophobic carboxyl-terminal domain inserted in the inner membrane. To determine the function of each part of Mas6p, we are making specific alterations in Mas6p and testing their effect on import. For example, are asking if mutations in the amino-terminal domain of Mas6p prevent the binding of precursors to the inner membrane import machinery. In addition, we are asking if the carboxyl-terminal domain forms part of a protein-translocating channel in the inner membrane. Project 2: Determine the function of Sms1p in import. Sms1p, which is also essential for import, has been shown to interact with Mas6p. To understand the role of Sms1p in import, we will generate new alleles of sms1 and examine their effects on import. For example, we will ask if sms1 mutants, like mas6 mutants, are defective in an inner membrane channel. Project 3: Identify new membrane import components and analyze their function. Using both genetic and biochemical approaches, we will identify new inner membrane import components and analyze their role in the translocation of precursors into the matrix.