Molecular machines: Clathrin Coated Vesicles and Kinetochore Subcomplexes

项目1是一项研究两台复杂分子机器的结构、组装和拆卸的提案： 酿酒酵母动粒的网状蛋白包被小泡和成分。(1)包被网状蛋白的囊泡携带 从质膜和跨高尔基网络到内体的膜运输。这些人的外套 网纹蛋白的结构、晶格、组织膜泡化。拟议工作的出发点 是在前一次授权期内通过拟合7.9A确定的网状蛋白涂层的分子结构 基于X射线原子模型的单粒子低温电子显微镜分辨率图像重建 结晶学。这一努力是本提案的四位政委S中的三位合作的。我们的 现在的目标是：(I)进一步扩大冷冻EM图的分辨率，并分析其他 分子筛晶格；(Ii)确定分子筛分解的结构机制(“去涂层”)；(Iii) 用冷冻电子断层扫描技术研究单个包膜囊泡。(2)动点是多蛋白。 聚集在着丝粒DNA上并中介附着到有丝分裂纺锤体微管的复合体。至 要了解着丝粒组装如何决定染色体的忠实分离，需要分析 动粒结构。发芽酵母的动点含有60多种不同的蛋白质，组织成 超过14个亚复合体。我们已经开始研究其中一些亚复合体的结构 上一个授权期。我们现在建议更详细地分析DASH/DAML复合体，使用冷冻- 电子断层摄影术。DASH是由10种不同蛋白质组成的异十聚体复合体，它组装成环， 包围微管。 相关性：网状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，以及 突触释放神经递质后的膜，所有过程都与人类疾病有关。 着丝点是染色体分离到子代细胞的关键；分离中的错误可能导致 癌症。

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.