Mechanisms of septin assembly that shape cellular function

项目总结 大多数细胞功能是由多亚单位蛋白质复合体执行的。尽可能多地转录和翻译 你想，但如果你的蛋白质不能与其他蛋白质正确组装，基因也可能是 脱下来。作为大多数基因表达的最后一步，蛋白质复合体组装的研究还很少，而且它已经 直到最近才清楚地发现，教科书上关于单个分子之间扩散受限碰撞的模型 是不够的。Septin蛋白在许多真核细胞中以细胞骨架细丝的形式存在，并参与一种 各种各样的细胞功能。隔膜细丝的构件是棒状的隔膜复合体。 由不同的隔膜蛋白亚基组成。多个间隔蛋白“竞争”占据相同的位置，并授予 Septin复合体的特殊性质，但还不完全清楚特定的亚基是如何被“选择”到 组装功能上合适的复合体。不同的间隔蛋白如何占据 复合体是Septin生物学中最古老的问题之一。致病的Septin突变突显了 回答这个问题的重要性。我们的实验室在这一领域处于领先地位，在接下来的五年里，我们将 希望使用我们在萌芽酵母中开发的强大工具来解决特定的知识差距。为 其他细胞骨架蛋白，要获得能够进行复杂组装的构象，需要 分子伴侣。伴侣辅助的从头开始的Septin折叠如何与Septin的途径相适应 复杂的装配？我们最近的工作建立了Septin异八聚体组装的逐步途径，并 已鉴定的与Septin相互作用的伴侣分子，它们参与Septin-Septin相互作用界面，是 高效的隔膜折叠。我们将结合遗传学、细胞生物学和生物化学来确定 伴侣行为为在复杂的组装过程中Septin-Septin相遇奠定了基础。最近的研究表明 许多复合体的组装是以协同翻译的方式进行的，我们发现高效的伴侣要求 赛普丁翻译。Septin复合体组装在多大程度上是共翻译的？我们将调查Septin- 在主动翻译的背景下，伴侣和Septin-Septin的相互作用。我们之前确定了关键 Septin-Septin相互作用界面中的残基在组装和口述过程中介导“伙伴识别” 络合物内的亚基组成。一个永恒的谜团是酵母“末端”的两个亚基是如何 Septin杂八聚体总是匹配的。跨Septin-Septin相互作用的变构构象如何变化 界面直接决定了Septin复合体亚单位组成的特异性？我们将确定其机理基础 并确定不适当的Septin亚基“混合”的表型后果 在建筑群内。最后，还不知道一旦被制造出来，Septin复合体是如何被改造以结合新的 细胞分化过程中的亚基。我们将确定酵母Septin的分子机制 复合体在配子发生过程中发生重塑。这些研究将为细胞如何 组装和修饰Septin复合体，对一般的多亚基组装具有更广泛的影响。

PROJECT SUMMARY Most cellular functions are carried out by multisubunit protein complexes. Transcribe and translate as much as you want, but odds are if your protein doesn’t assemble properly with other proteins, the gene might as well be off. As the final step in expression of most genes, protein complex assembly is hugely understudied, and it has only recently become clear that the textbook model of diffusion-limited collisions between individual molecules is inadequate. Septin proteins are found as cytoskeletal filaments in many eukaryotic cells and participate in a wide variety of cellular functions. The building blocks of septin filaments are rod-shaped septin complexes composed of distinct septin subunits. Multiple septins “compete” to occupy the same position in, and confer specialized properties to, septin complexes, but it is not fully understood how specific subunits are “chosen” to assemble the functionally appropriate complexes. How individual septins occupy specific positions within complexes is one of the oldest questions in septin biology. Disease-causing septin mutations highlight the importance of answering this question. Our lab is among the leaders in this field and in the next five years we want to use the powerful tools we have developed in budding yeast to address specific knowledge gaps. For other cytoskeletal proteins, achieving the conformation competent for complex assembly requires help from molecular chaperones. How does chaperone-assisted de novo septin folding fit into the pathway of septin complex assembly? Our recent work established the step-wise pathway of septin hetero-octamer assembly, and identified septin-interacting chaperones that engage a septin-septin interaction interface and are necessary for efficient septin folding. We will use a combination of genetics, cell biology and biochemistry to determine how chaperone action sets the stage for septin-septin encounters during complex assembly. Recent studies show that assembly of many complexes occurs co-translationally, and we find chaperone requirements for efficient septin translation. To what extent is septin complex assembly co-translational? We will investigate septin- chaperone and septin-septin interactions in the context of active translation. We previously identified key residues in septin-septin interaction interfaces that mediate “partner recognition” during assembly and dictate the subunit composition within complexes. An enduring mystery is how the two subunits at the “ends” of yeast septin hetero-octamers always match. How do allosteric conformational changes across septin-septin interaction interfaces direct the specificity of septin complex subunit composition? We will determine the mechanistic basis of this phenomenon and determine the phenotypic consequences of inappropriate “mixing” of septin subunits within complexes. Finally, it is not known how, once made, a septin complex is remodeled to incorporate new subunits during cellular differentiation. We will determine the molecular mechanism by which yeast septin complexes are remodeling during gametogenesis. These studies will provide valuable insights into how cells assemble and modify septin complexes, with broader implications for multisubunit assembly in general.