3-D FINE STRUCTURE OF MULTIVESICULAR BODIES IN SACCHAROMYCES CEREVISIAE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。内吞途径的功能是将注定在溶酶体中降解的摄入大分子与被循环回质膜或被输送到其他细胞内目的地的分子分离开来。多泡体（MVB）是最常见的内吞区室之一，其腔内含有由限制性内体膜向内出芽形成的膜封闭囊泡。许多控制细胞增殖的活化细胞表面受体通过MVBs的内吞作用和分选进入管腔囊泡而下调。在限制性MVB膜与溶酶体融合后，这些囊泡及其内容物随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与高等真核细胞相似的MVBs。一种新的遗传筛选方法被用来发现BRO1基因，该基因编码一种与内体膜相关的保守的可溶性细胞质蛋白。Bro1蛋白可能从细胞质被募集到核内体，以协调核内体膜向内内陷和囊泡向腔室出芽。为了验证这一假设，将分析具有结构域特异性突变的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。其中包括堆叠的蓄水池，弯曲成c形的堆叠蓄水池，以及包围球形隔间的弯曲蓄水池。未来的研究将利用遗传和生化分析来确定与野生型Bro1蛋白合作的蛋白在MVB囊泡形成中的作用。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），他正在利用资源实验室的设施研究MVB分类所需的其他基因突变的其他菌株。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，并确定具有人类同源物的新酵母蛋白，这些蛋白可以作为治疗人类疾病的靶点。

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.