Alternative vacuolar targeting mechanisms in yeast

真核细胞维持分离其许多基本功能的细胞器。这些隔室 含有必须特异性和有效地靶向正确的亚细胞位置的蛋白质。这 尽管蛋白质持续运动，但细胞器蛋白质的分离需要维持， 整个细胞的膜。这是如何实现的？不同的信号与排序相结合 机器允许胞质蛋白被递送到特定的目的地。本地化的缺陷 过程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 与多种疾病有关正常的溶细胞功能不仅依赖于 的常驻水解酶，但也对适当的基板的交付。其中一个主要途径是 哺乳动物细胞中的大分子周转和再循环是自噬。这个过程是由 饥饿并导致细胞质蛋白和细胞器通过双- 膜泡在某些情况下，这种摄取方式是非常具体的。的信号转导 感测营养状况的途径、实现货物特异性的方法以及 囊泡形成的机制尚不清楚。自噬的缺陷与心脏 疾病、癌症、神经变性疾病如帕金森病、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。 酵母液泡在其细胞作用和其生物学功能方面都类似于哺乳动物溶酶体。 蛋白质传递的机制。特别是，自噬途径在酵母和 哺乳动物细胞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点 通路在这个建议中，我们将集中在阐明的分子组成，指导 将胞质蛋白和细胞器递送至溶酶体/液泡。我们将使用分子遗传学和 生物化学方法来确定信号转导途径，允许成核的 隔离囊泡此外，我们将重组货物包装，囊泡形成和 为了了解调节自噬的分子机制，我们在体外研究了细胞膜融合。

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 lagely 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 provide 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.