GENETICS OF ORGANELLE PROTEIN DELIVERY IN YEAST

真核细胞含有多达12个不同的亚细胞 车厢。这些隔室或细胞器中的每一个都携带 产生了一组对细胞存活至关重要的特殊功能。 事实上，人类中的许多遗传性遗传病都是已知的 由正常细胞器功能的改变引起的(例如， 溶酶体储藏疾病)。分子机制 负责将蛋白质准确地输送到正确的位置 细胞器的目的地在很大程度上仍不清楚。我们的研究目标 是为了了解某些线粒体和溶酶体是如何 酵母中的(空泡)蛋白是选择性和高效的 从它们在细胞质中的合成部位运输到 它们在线粒体或线粒体中的独特功能 液泡。 基因融合技术正被用来绘制序列或 核编码线粒体F1-的构象信号 ATPaseβ亚基蛋白和两种空泡蛋白， 羧基肽酶Y和Pep4，使这些蛋白质 适当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 编码这些蛋白质和E.ColiLacZ基因或酵母 SuC2基因(分泌酶转移酶的编码)。数列 在这些线粒体和空泡蛋白中鉴定出 能够重定向大肠杆菌β-半乳糖苷酶或酵母 线粒体或液泡的转移酶将被 定点突变。这些突变序列的影响 不能正确传递融合蛋白和 将对原型蛋白质进行分析。这些研究应该有助于 确定几种具有代表性的蛋白质的信息含量 将信号分类。 酵母突变体改变了他们正确识别这些的能力 蛋白质分选信号的选择是通过利用特定的 基因融合结构赋予酵母的表型。这个 这些突变体中受影响的基因可能编码 解释蛋白质分类的细胞机械 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的方式被克隆 酵母。将会产生针对这些基因产物的抗血清 在细菌中过度表达后。然后这些抗血清将被 被用来分析这些人的蜂窝居住地 酵母中的蛋白质(使用免疫荧光和细胞分级 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.