GENETICS OF ORGANELLE PROTEIN DELIVERY IN YEAST

真核细胞含有多达12个不同的亚细胞 隔间 每一个隔室或细胞器 一组对细胞生存力至关重要的特殊功能。 事实上，已知许多人类遗传性遗传疾病是 由正常细胞器功能的改变引起（例如，的 溶酶体贮积病）。 的分子机制 负责将蛋白质准确地运送到正确的位置， 细胞器的目的地仍然是未知的。 我们的研究目标 是了解某些线粒体和溶酶体 酵母中的（液泡）蛋白质选择性地和有效地 从细胞质中的合成位点运输到 它们的独特功能部位， 空泡 基因融合技术正被用于绘制序列或 在核编码线粒体F1构象信号- ATP酶β亚基蛋白和两种液泡蛋白， 羧肽酶Y和Pep 4，使这些蛋白质 正确地分类到它们各自的细胞器目的地。 基因 已经在酵母基因之间构建了融合体， 编码这些蛋白质，E.大肠杆菌lacZ基因或酵母 SUC 2基因（编码分泌型转化酶）。 序列 在这些线粒体和液泡蛋白中发现， 能够重定向E.大肠杆菌β-半乳糖苷酶或酵母 转化酶对胞体或液泡的作用将被修饰， 定点诱变。 这些突变序列 对融合蛋白和融合蛋白的适当递送有影响。 将分析原型蛋白质。 这些研究有助于 定义了几个代表性蛋白质的信息含量 分类信号 酵母突变体改变了正确识别这些 蛋白质分选信号已经通过利用某些 通过基因融合构建体赋予酵母的表型。 的 这些突变体中受影响的基因可能编码 解释蛋白质分选的细胞机制 信号并将这些蛋白质引导到它们适当的细胞器 目的地. 这些基因将通过互补克隆， 酵母 将针对这些基因的产物产生抗血清 在细菌中过度表达后。 这些抗血清将被 用于分析这些细胞居住的网站， 酵母中的蛋白质（使用免疫荧光和细胞分级分离 技术）及其生物发生机制。

Eukaryotic cells contain as many as a dozen distinct subcellular compartments. Each of these compartments or organelles carries out a specialized set of functions that are crucial to cell viability. Indeed, many inherited genetic disorders in man are known to be caused by alterations in normal organelle functioning (e.g., the lysosomal storage diseases). The molecular mechanisms responsible for the accurate delivery of proteins to their correct organelle destinations remain largely unknown. Our research goal is to understand how certain mitochondrial and lysosomal (vacuolar) proteins in yeast are selectively and efficiently transported from their site of synthesis in the cell cytoplasm to their unique site of function in either the mitochondrion or the vacuole. The technique of gene fusion is being used to map the sequence or conformational signals in the nuclear encoded mitochondrial F1- ATPase beta-subunit protein and two vacuolar proteins, carboxypeptidase Y and Pep4, that allow these proteins to be properly sorted to their respective organelle destinations. Gene fusions have been constructed between the yeast genes that encode these proteins and either the E. coli lacZ gene or the yeast SUC2 gene (codes for the secreted enzyme invertase). Sequences identified in these mitochondrial and vacuolar proteins that are capable of redirecting either E. coli beta-galactosidase or yeast invertase to the mitochondrion or vacuole will be modified by site-directed mutagenesis. The effect these mutant sequences have on proper delivery of both the fusion proteins and the prototypic proteins will be analyzed. These studies should help to define the information content of a few representative protein sorting signals. Yeast mutants altered in their ability to properly recognize these protein sorting signals have been selected by exploiting certain phenotypes conferred to yeast by the gene fusion constructs. The genes affected in these mutants may code for components of the cellular machinery that function to interpret the protein sorting signals and direct these proteins to their proper organelle destinations. These genes will be cloned by complementation in yeast. Antisera will be raised against the products of these genes after overexpression in bacteria. These antisera will then be employed to analyze the site of cellular residence of these proteins in yeast (using immunofluorescence and cell fractionation techniques) and their mechanism of biogenesis.