Alternative vacuolar targeting mechanisms in yeast

描述(申请人提供)：真核细胞维持细胞器，这些细胞器将它们的许多基本功能分开。这些隔室包含的蛋白质必须特异性和有效地定位于正确的亚细胞位置。尽管蛋白质和细胞膜在整个细胞内不断运动，这种细胞器蛋白质的分离仍然需要保持。这是如何实现的？不同的信号与分选机制相结合，使胞浆蛋白能够被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部位。这些酶的错误分类与一系列疾病有关。正确的溶菌谱功能不仅取决于驻留水解酶的存在，而且还取决于适当底物的输送。哺乳动物细胞中大分子周转和循环的主要途径之一是自噬。这一过程是由饥饿诱导的，导致细胞质蛋白和细胞器通过双层膜小泡输送到溶酶体。在某些情况下，这种吸收模式是非常特殊的。感知营养状况的信号转导途径、获得货物专一性的方法和囊泡形成的机制在很大程度上尚不清楚。自噬缺陷与心脏病、癌症、帕金森氏症、亨廷顿病和阿尔茨海默氏症等神经退行性疾病以及病毒和细菌感染的易感性有关。 酵母液泡在细胞功能和蛋白质输送机制方面与哺乳动物的溶酶体相似。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，酵母为研究这一途径提供了一个有用的模型系统。在这项提案中，我们将重点阐明直接将胞浆蛋白和细胞器输送到溶酶体/液泡的分子成分。我们将使用分子遗传学和生物化学方法来确定允许隔离小泡成核的信号转导途径。此外，我们还将在体外重构货物包装、囊泡形成和膜融合的步骤，以了解调控自噬的分子机制。

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.