Alternative vacuolar targeting mechanisms in yeast

酵母中的替代液泡靶向机制

• 批准号: 6751259
• 负责人: DANIEL J. KLIONSKY
• 金额: $ 52.3万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6751259
• 关键词: Saccharomyces cerevisiae; aminopeptidase; antiserum; autophagy; biological signal transduction; cell component structure /function; fungal proteins; immunoprecipitation; intracellular transport; laboratory rabbit; lysosomes; membrane fusion; membrane proteins; molecular cloning; molecular genetics; posttranslational modifications; protein localization; protein protein interaction; protein structure function; protein transport; radiotracer; receptor binding; vesicle /vacuole; yeast two hybrid system

DESCRIPTION (provided by applicant): Eukaryotic cells maintain organelles that separate many of their essential functions. These compartments contain proteins that must be specifically and efficiently targeted to the correct subcellular location. This segregation of organellar proteins needs to be maintained despite a continual movement of proteins and membranes throughout the cell. How is this achieved? Different signals in combination with sorting machinery allow cytosolic proteins to be delivered to a particular destination. Defects in the localization process have severe physiological consequences. For example, the lysosome is the primary storage site for many hydrolases. Missorting of these enzymes is implicated in a wide range of illnesses. Proper lysoscmal function is not only dependent on the presence of resident hydrolases, but also on the delivery of appropriate substrates. One of the primary pathways for macromolecular turnover and recycling in mammalian cells is autophagy. This process is induced by starvation and results in the delivery of cytoplasmic proteins and organelles to the lysosome via a double membrane vesicle. Under some conditions, this mode of uptake is very specific. The signal transduction pathway by which the nutritional conditions are sensed, the methods of achieving cargo specificity and the mechanism of vesicle formation are largely unknown. Defects in autophagy have been correlated with heart disease, cancer, neurodegenerative disorders such as Parkinson's, Huntington's and Alzheimer's diseases and susceptibility to viral and bacterial infection. The yeast vacuole is analogous to the mammalian lysosome both in terms of its cellular role and its mechanisms of protein delivery. In particular, the autophagic pathway is conserved between yeast and mammalian cells. Due to the ease of genetic approaches, yeast provides a useful model system to study this pathway. In this proposal, we will focus on the elucidation of the molecular components that direct the delivery of cytosolic proteins and organelles to the lysosome/vacuole. We will use molecular genetic and biochemical approaches to determine the signal transduction pathway that allows the nucleation of sequestering vesicles. In addition, we will reconstitute the steps of cargo packaging, vesicle formation and membrane fusion in vitro in order to understand the molecular mechanism that regulates autophagy.

描述（由申请人提供）：真核细胞维持着区分其许多基本功能的细胞器。这些区室含有必须特异性且有效地靶向正确的亚细胞位置的蛋白质。尽管蛋白质和细胞膜在整个细胞中不断运动，但细胞器蛋白质的这种分离仍然需要维持。这是如何实现的？不同的信号与分选机器相结合，可以将胞质蛋白输送到特定的目的地。定位过程中的缺陷会产生严重的生理后果。 例如，溶酶体是许多水解酶的主要储存位点。这些酶的错误分类与多种疾病有关。适当的溶酶功能不仅取决于驻留水解酶的存在，还取决于适当底物的输送。哺乳动物细胞中大分子周转和再循环的主要途径之一是自噬。该过程由饥饿诱导，并导致细胞质蛋白和细胞器通过双膜囊泡递送至溶酶体。在某些条件下，这种吸收模式是非常具体的。感知营养状况的信号转导途径、实现货物特异性的方法以及囊泡形成的机制在很大程度上是未知的。自噬缺陷与心脏病、癌症、神经退行性疾病（如帕金森病、亨廷顿病和阿尔茨海默病）以及对病毒和细菌感染的易感性相关。 酵母液泡在其细胞作用和蛋白质递送机制方面类似于哺乳动物溶酶体。特别是，自噬途径在酵母和哺乳动物细胞之间是保守的。由于遗传方法的简便性，酵母提供了一个有用的模型系统来研究这一途径。在本提案中，我们将重点阐明指导胞质蛋白和细胞器递送至溶酶体/液泡的分子成分。我们将使用分子遗传学和生化方法来确定允许隔离囊泡成核的信号转导途径。此外，我们将在体外重构货物包装、囊泡形成和膜融合的步骤，以了解调节自噬的分子机制。