3-D FINE STRUCTURE OF MULTIVESICULAR BODIES IN SACCHAROMYCES CEREVISIAE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 内吞途径的功能是将注定要在溶酶体中降解的摄入的大分子与再循环回到质膜或被引导到其他细胞内目的地的分子分离。 最易识别的内吞隔室之一是多泡体（MVB），其在其管腔内包含由限制性内体膜向内出芽形成的膜封闭的囊泡。 许多控制细胞增殖的活化的细胞表面受体通过内吞作用和分选到MVB的内腔囊泡中而下调。 这些囊泡及其内容物随后在限制性MVB膜与溶酶体融合后被溶酶体水解酶降解。 初步的断层扫描显示，野生型芽殖酵母含有类似于高等真核细胞中的MVB。一种新的遗传筛选被用来揭示BRO 1基因，该基因编码一种保守的可溶性细胞质蛋白，与内体膜相关。Bro 1蛋白可以从细胞质募集到内体，以协调内体膜的向内内陷和囊泡朝向隔室腔的出芽。 为了验证这一假设，将分析具有结构域特异性突变的Bro 1蛋白的功能和定位。 最初，我们完成了9个双轴断层扫描的BRO 1缺失的细胞，发现这些细胞含有异常的膜室与三种不同的形态。这些包括堆叠池，堆叠池弯曲成C形，弯曲池包围一个球形的隔间。未来的研究将使用遗传和生化分析来鉴定与野生型Bro 1蛋白在MVB囊泡形成中合作的蛋白质。Odorizzi实验室最近聘请了一名全职技术人员Matthew West，他正在利用资源设施研究MVB分选所需的其他基因突变的其他菌株。 总之，这些研究应该确定Bro 1蛋白在MVB囊泡形成中的功能作用，并确定新的酵母蛋白与人类同源物，可以作为人类疾病治疗的目标。

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.