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。特定细胞器蛋白质转运的适当维持的缺陷是许多人类疾病如癌症和阿尔茨海默氏综合征的基础。两个完整的方法，遗传学和不充分的理解仍然在蛋白质从前溶酶体区室（PLC）转移到溶酶体。驻留的溶酶体蛋白质在从transGolgi网络中的分泌蛋白质中分选出来后通过PLC。类似地，从细胞表面胞吞后降解的蛋白质在其在溶酶体中周转之前通过PLC（或晚期内体）。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来正确分选、靶向和递送来自PLC的蛋白质。关于PLC最大的困惑来源是关于载体囊泡是否将物质转运到溶酶体，或者PLC是否经历了成熟过程，变成了溶酶体。联合收割机的遗传，分子和生物化学方法的研究，将导致这间室的蛋白质运输问题的完整理解。酿酒酵母（Saccharomyces cerevisiae）不仅含有溶酶体样液泡，而且含有PLC样前液泡区室（PVC）。该提案描述了使用无细胞重建试验在体外，酵母突变体分析在体内，和形态学技术，以澄清PVC和液泡之间的运输机制。这些简单的方法已经产生了证据来支持这样的观点，即特定的载体囊泡起着将蛋白质货物从PVC运输到液泡的作用。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液泡系统与哺乳动物细胞的溶酶体系统更加相似。下面提出的具体目标是证明或反驳这些假设。(1)阐明胞质蛋白质的功能，这些蛋白质参与通过液泡前区室的生物发生和功能将常驻水解酶递送到液泡/溶酶体。(3)纯化和表征包含酵母中高尔基体后/液泡系统细胞器的膜结合区室，以发现新的标记蛋白。这些细胞器包括前液泡室、PVC衍生的中间囊泡或亚室和液泡。

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.