MECHANISMS OF ENDOSOME TO LYSOSOME TRANSPORT IN YEAST

分泌和内吞途径中存在的机制确保真核细胞中的每个细胞器获得其正确分配的驻留蛋白质。特定细胞器蛋白运输的适当维持缺陷是许多人类疾病的基础，如癌症和阿尔茨海默氏症。在蛋白质从溶酶体前间隔室(PLC)到溶酶体的转移过程中，仍然存在两种完整的方法，即遗传学和不充分的理解。驻留的溶酶体蛋白在从反式高尔基体网络中的分泌蛋白中分离出来后，通过PLC。类似地，从细胞表面吞噬后将被降解的蛋白质在其在溶酶体中周转之前通过PLC(或晚期内涵体)。有了这些多个蛋白质运输方向，真核细胞必须结合许多事件才能正确地分选、靶向和运送来自PLC的蛋白质。关于PLC最大的困惑来源是载体囊泡是否将物质输送到溶酶体，或者PLC是否经历了成熟过程，变成了溶酶体。结合遗传、分子和生化方法的研究将导致对这一间隔间蛋白质运输问题的全面了解。酿酒酵母不仅含有溶酶体样空泡，还含有类似PLC的泡前隔室(PVC)。本提案描述了使用体外无细胞重组试验、体内酵母突变分析和形态技术来阐明聚氯乙烯和液泡之间的运输机制。这些简便的方法已经产生了证据，支持特定的载体小泡将蛋白质货物从聚氯乙烯运送到液泡的观点。此外，最近的数据表明，这些细胞器可能不是酵母中的单一实体。这种假定的复杂性将使酵母空泡系统与哺乳动物细胞的溶酶体系统更加相似。为了证明或反驳这些假说，本文提出了以下具体目标。(1)阐明胞质蛋白通过泡前小室的生物发生和功能参与将滞留的水解酶运送到液泡/溶酶体的功能。(3)纯化和鉴定酵母中高尔基体后/液泡系统细胞器的膜结合室，以发现新的标记蛋白。这些细胞器包括泡前室、聚氯乙烯衍生的中间囊泡或亚室以及液泡。

Mechanisms within the secretory and endocytic pathways exist to ensure that each organelle in eukaryotic cells obtain its correct allotment of resident proteins. Defects in proper maintenance of specific organellar protein transport are the basis for many human diseases such as cancer and Alzheimer's syndrome. Two integral approaches, genetics and inadequate understanding remains in transfer of proteins from the pre- lysosomal compartment (PLC) to the lysosome. Resident lysosomal proteins pass through the PLC after being sorted away from secretory proteins in the trans Golgi network. Similarly, proteins destined for degradation after endocytosis from the cell surface pass through the PLC (or late endosome) before their turnover in the lysosome. With these multiple protein traffic directions, eukaryotic cells must combined many events for proper sorting, targeting, and delivery of proteins from the PLC. The greatest source of confusion about the PLC concerns whether carrier vesicles transport material to lysosomes or if the PLC undergoes a maturation process, changing into a lysosome. Studies that combine genetic, molecular, and biochemical approaches will lead to the complete understanding of this intercompartmental protein transport problem. The yeast, Saccharomyces cerevisiae, contains not only a lysosome-like vacuole, but also a PLC-like prevacuolar compartment (PVC). This proposal describes the use of cell-free reconstitution assays in vitro, yeast mutant analyses in vivo, and morphological techniques to clarify transport mechanisms between the PVC and the vacuole. These facile approaches have produced evidence to support the notion that specific carrier vesicles act to transport protein cargo from the PVC to the vacuole. Moreover, recent data suggests that these organelles may not be single entities in yeast. This putative complexity would give the yeast vacuolar system even more similarity to the lysosomal system of mammalian cells. The following specific aims are proposed to prove or disprove these hypotheses. (1) Elucidate the function of cytosolic proteins involved in delivery of resident hydrolases to the vacuole/lysosome via the biogenesis and function of the prevacuolar compartment. (3) Purify and characterize membrane-bound compartments that comprise organelles of the post- Golgi/vacuolar system in yeast to discover novel marker proteins. These organelles include prevacuolar compartments, PVC-derived intermediate vesicles or subcompartments, and vacuoles.