MECHANISMS OF ENDOSOME TO LYSOSOME TRANSPORT IN YEAST

酵母内体到溶酶体的转运机制

• 批准号: 6637231
• 负责人: THOMAS A VIDA
• 金额: $ 23.77万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637231
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; binding proteins; cytoplasm; eukaryote; gene expression; hydrolase; intracellular transport; lipid transport; lysosomes; membrane fusion; membrane transport proteins; mutant; organelles; polymerase chain reaction; protein degradation; protein purification; protein structure function; protein transport; secretory protein; vesicle /vacuole

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.

分泌和内吞途径的机制确保真核细胞中的每个细胞器获得其正确分配的常驻蛋白。特定细胞器蛋白运输的适当维持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。两种完整的方法，遗传学和不充分的理解仍然在蛋白质从前溶酶体室（PLC）转移到溶酶体。常驻溶酶体蛋白从反式高尔基网络的分泌蛋白中分离出来后通过PLC。同样，在细胞表面内吞作用后降解的蛋白质在其在溶酶体中周转之前通过PLC（或后期内体）。有了这些多种蛋白质运输方向，真核细胞必须结合许多事件来正确分类、靶向和递送来自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运送到溶酶体，或者PLC是否经历成熟过程，变成溶酶体。结合遗传、分子和生化方法的研究将导致对这种细胞间蛋白质运输问题的完全理解。酵母（Saccharomyces cerevisiae）不仅含有溶酶体样液泡，还含有plc样液泡前室（PVC）。该提案描述了体外无细胞重构分析、体内酵母突变分析和形态学技术的使用，以阐明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特定的载体囊泡将蛋白质货物从PVC运输到液泡的概念。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。提出以下具体目标来证明或反驳这些假设。(1)通过泡前室的生物发生和功能，阐明参与将常驻水解酶传递到液泡/溶酶体的细胞质蛋白的功能。(3)纯化和表征酵母中包含高尔基/液泡系统细胞器的膜结合区室，以发现新的标记蛋白。这些细胞器包括泡前室、pvc衍生的中间囊泡或小室和液泡。

项目成果

A cell-free system for reconstitution of transport between prevacuolar compartments and vacuoles in Saccharomyces cerevisiae.

用于重建酿酒酵母液泡前区室和液泡之间运输的无细胞系统。

• DOI: 10.1007/978-1-59745-261-8_4
• 发表时间: 2008
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Vida,ThomasA

Isolation of yeast mutants defective for localization of vacuolar vital dyes.

• DOI: 10.1073/pnas.95.20.11721
• 发表时间: 1998-09
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Bing Zheng;Jennifer N. Wu;Wendy Schober;Dorothy E. Lewis;Thomas A. Vida

Flow cytometry/cell sorting for isolating membrane trafficking mutants in yeast.

流式细胞术/细胞分选用于分离酵母中的膜运输突变体。

• DOI: 10.1016/s0076-6879(02)51872-3
• 发表时间: 2002
• 期刊: Methods in enzymology
• 作者: Vida,Thomas;Wendland,Beverly
• 通讯作者: Wendland,Beverly

mVps24p functions in EGF receptor sorting/trafficking from the early endosome.

mVps24p 在早期内体的 EGF 受体分选/运输中发挥作用。

• DOI: 10.1016/j.yexcr.2004.11.003
• 发表时间: 2005
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: Yan,Qing;Hunt,PiperReid;Frelin,Laurence;Vida,ThomasA;Pevsner,Jonathan;Bean,AndrewJ
• 通讯作者: Bean,AndrewJ

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

无细胞测定允许重建 Vps33p 依赖性运输至酵母液泡/溶酶体。

• DOI: 10.1083/jcb.146.1.85
• 发表时间: 1999
• 期刊: The Journal of cell biology
• 作者: Vida,T;Gerhardt,B
• 通讯作者: Gerhardt,B