BUDDING YEAST SEPTIN FILAMENTS: CONFORMATION AND KINETICS OF FILAMENT FORMATION

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。 Septin蛋白组装成一种细胞骨架聚合物。Septin细胞骨架是细胞分裂的最后一步，胞质分裂是必不可少的。在萌芽酵母中，芽颈上可见丝状的隔质环，并将其收缩，以分隔母细胞和子细胞。我们使用的是在体外自组装的酵母隔膜蛋白。有丝分裂的酿酒酵母细胞表达5种Septin：CDc3、CDc10、CDc11、CDc12和Shs1/Sep7。除Shs1外，所有基因都是细胞分裂所必需的。我们用电子显微镜的标记技术确定了最小复合体中四种基本的隔膜蛋白的分子组织。除CDC10外，所有的间隔蛋白都有一个预测的卷曲卷曲，可以促进间隔蛋白组装成成对的细丝。当离子强度降低到200 mM一价盐时，八聚体最小亚基组装成细丝。小角X射线散射将用于野生型间隔蛋白和去除卷曲的突变间隔蛋白，以能够定位这些灵活的结构域。此外，还将使用停流混合器来探索细丝形成的动力学。这些动力学实验将在各种核苷酸存在的情况下进行，因此可以测试它们对聚合过程的影响。

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. Septin proteins assemble into a cytoskeletal polymer. Septin cytoskeleton is essential for cytokinesis, the last step in cell division. In budding yeast, filamentous rings of septins have been visualized at the bud neck and constrict it in order to separate the mother and daughter cells. We are using yeast septins that self-assemble in vitro. Mitotic Saccharomyces cerevisiae cells express five septins: Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7. All, but Shs1, are essential for cell division. We have determined the molecular organization of the four essential septins within the minimal complex by labeling techniques using electron microscopy. All of the septins but Cdc10 have a predicted coiled coil which promotes septin assembly into paired filaments. Octameric minimal subunits assemble into filaments when the ionic strength is lowered to 200 mM monovalent salt. Small angle x-ray scattering will be used on both wild type septins and on mutant septins deprived of coiled coils to be able to locate those flexible domains. In addition to that, using a stopped flow mixer, the kinetics of filament formation will be probed. Those kinetics experiments will be performed in the presence of various nucleotides so their effect on the polymerization process can be tested.