3-D FINE STRUCTURE OF MULTIVESICULAR BODIES IN SACCHAROMYCES CEREVISIAE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 内吞途径的功能是将溶酶体中注定要降解的大分子与被循环回质膜或路由到其他细胞内目的地的分子分开。最容易辨认的内细胞室之一是多囊泡体(MVB)，它的管腔内含有由限制的内胚体膜向内萌发形成的封闭囊泡。许多激活的控制细胞增殖的细胞表面受体通过内吞作用和分选进入MVB的管腔小泡而下调。当限制性MVB膜与溶酶体融合时，这些囊泡及其内容物随后被溶酶体水解酶降解。初步断层扫描显示，野生型萌芽酵母含有与高等真核细胞中类似的MVB。一种新的遗传筛选被用来揭示BRO1基因，它编码一种保守的、与内体膜相关的可溶性细胞质蛋白。Bro1蛋白可以从细胞质招募到内体，以协调内体膜的内陷和小泡向腔腔的萌发。为了验证这一假设，我们将分析具有结构域特定突变的Bro1蛋白的功能和定位。最初，我们完成了9个BRO1缺失细胞的双轴断层扫描，发现这些细胞含有三种不同形态的异常膜室。这些包括堆叠的脑池、弯曲成C形的堆叠的脑池和包围球形隔室的弯曲的脑池。未来的研究将使用遗传和生化分析来确定在MVB囊泡形成过程中与野生型Bro1蛋白合作的蛋白质。奥多里齐实验室最近聘请了一名全职技术人员马修·韦斯特(Matthew West)，他正在使用资源设施研究其他菌株，这些菌株在MVB分选所需的其他基因中存在突变。总之，这些研究应该确定Bro1蛋白在MVB囊泡形成中的功能作用，并确定新的具有人类同源物的酵母蛋白，这些蛋白可以作为人类疾病治疗的靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The endocytic pathway functions to segregate ingested macromolecules that are destined to be degraded in the lysosome from molecules that are either recycled back to the plasma membrane or routed toward other intracellular destinations. One of the most recognizable endocytic compartments is the multivesicular body (MVB), which contains within its lumen membrane-enclosed vesicles that are formed by inward budding of the limiting endosomal membrane. Many activated cell-surface receptors that control cell proliferation are down-regulated through endocytosis and sorting into the lumenal vesicles of MVBs. These vesicles and their contents are subsequently degraded by lysosomal hydrolases upon fusion of the limiting MVB membrane with the lysosome. Preliminary tomograms have shown that wild-type budding yeasts contain MVBs that are similar to those in higher eukaryotic cells. A novel genetic screen was used to uncover the BRO1 gene, which encodes a conserved, soluble cytoplasmic protein that associates with endosomal membranes. The Bro1 protein may be recruited from the cytoplasm to the endosome in order to coordinate inward invagination of the endosomal membrane and budding of vesicles toward the compartment's lumen. To test this hypothesis, the function and localization of Bro1 proteins that have domain-specific mutations will be analyzed. Initially, we completed 9 dual-axis tomograms of BRO1-deleted cells and found that these cells contained aberrant membrane compartments with three distinct morphologies. These included stacked cisternae, stacked cisternae that were curved into a C-shape, and curved cisternae enclosing a spherical compartment. Future studies will use genetic and biochemical analysis to identify proteins that cooperate with the wild-type Bro1 protein in MVB vesicle formation. The Odorizzi lab has recently hired a full time technician, Matthew West who is using the Resource facilities to work on additional strains that have mutations in other genes required for MVB sorting. Together, these studies should define the functional role of the Bro1 protein in MVB vesicle formation and identify new yeast proteins with human homologues that could serve as targets for therapeutics in human diseases.