Molecular machines: Clathrin Coated Vesicles and Kinetochore Subcomplexes

项目1是研究两个复杂分子机器的结构、组装和拆卸的建议： 网格蛋白包被的囊泡和S.酿酒酵母动粒。(1)网格蛋白包被的囊泡携带 从质膜和trans-Golgi网络到内体的膜运输。这些外套 结构，网格蛋白的晶格，组织膜泡。拟议工作的出发点 是网格蛋白外壳的分子结构，在上一个资助期通过拟合a7.9A 用从X射线导出的原子模型从单粒子cryoEM重建分辨率图像 结晶学这项工作是四个私人侦探中的三个合作完成的。的当前提案。我们 现在的目标是：（i）进一步扩展cryoEM图的分辨率，并分析额外的 网格蛋白晶格;（ii）确定网格蛋白分解（“脱包衣”）的结构机制;（iii） 通过冷冻电子断层扫描研究单个包被囊泡。(2)动粒是多蛋白质 在着丝粒DNA上组装并介导与有丝分裂纺锤体微管连接的复合物。到 了解动粒组装如何决定染色体的忠实分离需要分析 动粒结构芽殖酵母的动粒含有超过60种不同的蛋白质， 超过14个子复合体。我们已经开始研究其中一些亚复合物的结构， 上一个补助期。我们现在提出更详细的分析DASH/Daml复合物，使用低温， 电子断层摄影术DASH是十种不同蛋白质的异十聚体复合物，组装成环， 环绕着微管。 相关性：网格蛋白介导的途径包括转铁蛋白、低密度脂蛋白和病毒的内吞作用， 突触释放神经递质后，细胞膜上的细胞分裂，所有这些过程都与人类疾病有关。 着丝粒对于染色体分离到子细胞中是至关重要的;分离中的错误可导致染色体分裂。 癌

Project 1 is a proposal to study structure, assembly, and disassembly of two intricate molecular machines: clathrin coated vesicles and components of the S. cerevisiae kinetochore. (1) Clathrin coated vesicles carry membrane traffic to endosomes from the plasma membrane and the trans-Golgi network. The coats of these structures, lattices of clathrin, organize membrane vesiculation. The point of departure for the proposed work is the molecular structure of clathrin coats, determined during the previous grant period by fitting a7.9A resolution image reconstruction from single-particle cryoEM with atomic models derived from x-ray crystallography. This effort was a collaboration among three of the four P.I.'s of the present proposal. Our aims are now: (i) to extend further the resolution of the cryoEM map and to analyze designs of additional clathrin lattices; (ii)to determine the structural mechanism of clathrin disassembly ("uncoating"); (iii)to study individual coated vesicles by cryo-electron tomography. (2) Kinetochores are the multiprotein complexes that assemble on centromeric DNA and mediate attachment to mitotic spindle microtubules. To understand how kinetochore assembly determines faithful segregation of chromosomes requires analysis of kinetochore structure. Kinetochores of budding yeast contain over 60 distinct protein species, organized into over 14 subcomplexes. We have begun to study the structures of some of these subcomplexes during the previous grant period. We now propose more detailed analysis of the DASH/Daml complex, using cryo- electron tomography. DASH, a heterodecameric complex of ten distinct proteins, assembles into rings that encircle microtubules. Relevance: Clathrin-mediated pathways include endocytosis of transferrin, LDL, and viruses, and reuptake of membrane after release of neurotransmitter at synapses, all processes relevant to human disease. Kinetochores are critical for chromosome segregation into daughter cells; errors in segregation can lead to cancer.