Alternative Vacuolar Targeting Mechanisms in Yeast

描述（由申请人提供）：自噬代表了一种复杂的细胞稳态途径，具有细胞保护功能，但如果失调可导致细胞死亡；完整的调控知识对于治疗目的的这一过程的潜在调节至关重要，并增加我们对膜动力学和细胞器生物发生的基本理解。自噬发生在所有真核生物中，自噬机制的蛋白质成分从酵母到哺乳动物都是保守的。这一过程的标志是形成双膜胞质囊泡，即隔离细胞质成分的自噬体。完成后，自噬体与溶酶体/液泡融合，释放被分解的内囊泡，允许进入货物。自噬在多种发育过程中发挥作用，并与一系列病理生理条件相关。本研究的长期目标是了解自噬的机制和调控，以及这如何转化为自噬体的形成。自噬分子领域的研究已经有十多年的历史了，这对于一个与如此广泛的生理过程相联系的途径来说是令人吃惊的。在实际意义上，这也意味着还有许多问题有待回答。例如，我们想要(1)确定环境信号如何被转导成自噬反应，定义控制自噬的激酶、磷酸酶和转录因子；(2)确定隔离囊泡的起源，并确定如何从各种细胞器中征用膜来提供自噬体形成所需的材料；(3)了解哪些调节控制决定了特异性和非特异性自噬类型之间的切换，以及实现货物特异性的方法。我们正在利用酵母研究自噬的分子机制；这是对这一复杂过程进行分子遗传和生化分析的最佳系统。然而，由于高度的保存性，我们从酵母中了解到的信息将适用于高等真核生物。目前，已经鉴定了30多种自噬相关蛋白，但它们的功能和控制它们的调控过程在很大程度上是不明确的。本提案中描述的实验具有重要意义，因为它们将阐明上游调控元件与进行自噬的机制之间的重要联系，为将信号转导元件与功能装置联系起来的综合分析提供下一步，提高我们对基本细胞生物学的认识，并确定最终治疗干预的目标。这项提议的研究是创新的，因为它提供了关于自噬的调节和功能成分的新信息，在某些情况下是范式转换。

DESCRIPTION (provided by applicant): Autophagy represents a complex pathway of cellular homeostasis that functions in cytoprotection, but if dysregulated can cause cell death; a complete knowledge of regulation is critical for the potential modulation of this process for therapeutic purposes, and to increase our basic understanding of membrane dynamics and organelle biogenesis. Autophagy occurs in all eukaryotes and the protein components of the autophagic machinery are conserved from yeast to mammals. The hallmark of this process is the formation of double- membrane cytosolic vesicles, autophagosomes that sequester cytoplasmic components. After completion, the autophagosomes fuse with the lysosome/vacuole to release the inner vesicle that is broken down, allowing access to the cargo. Autophagy plays a role in various developmental processes and is associated with a range of pathophysiological conditions. The long-term goal of this proposal is to understand the mechanism and regulation of autophagy and how this translates into autophagosome formation. The molecular field of autophagy is slightly over ten years old, which is startling for a pathway with connections to such a wide range of physiological processes. In a practical sense, this also means there are many questions remaining to be answered. For example, we want to (1) determine how environmental signals are transduced into an autophagic response, defining the kinases, phosphatases and transcription factors that control autophagy; (2) identify the origin of the sequestering vesicle and determine how membrane from a variety of organelles can be commandeered to provide the material needed for autophagosome formation; and (3) understand what regulatory controls determine the switch between specific and non-specific types of autophagy, and the method of achieving cargo specificity. We are using yeast to investigate the molecular mechanism of autophagy; this is the best system for a molecular genetic and biochemical analysis of this complex process. Because of the high degree of conservation, however, the information we learn from yeast will be applicable to higher eukaryotes. At present, over thirty autophagy-related (Atg) proteins have been identified, but their functions and the regulatory processes that control them are largely undefined. The experiments described in this proposal are significant because they will elucidate important links between upstream regulatory components and the machinery that carries out autophagy, providing the next step in a comprehensive analysis that links the signal transduction elements to the functional apparatus, advancing our knowledge of basic cell biology, and identifying targets for ultimate therapeutic intervention. The proposed research is innovative, because it is providing new, and in some cases paradigm-shifting, information about the regulatory and functional components of autophagy.