Requirements for cytosolic chaperones in the de novo folding of septin proteins

细胞功能需要组装由不同多肽组成的大分子复合体 以精确的方式相互作用的亚基。蛋白质序列往往不足以保证在拥挤的 细胞环境每个新生亚基只进行正确的蛋白质-蛋白质相互作用。不正确 构象和相互作用会导致细胞缺陷和人类疾病。细胞如何确保新的- 人工合成的多肽组装成功能复合体仍然知之甚少。Septin蛋白 组装成高度保守的细胞骨架杂低聚物，已成为许多 细胞过程。所有的Septin亚基在结构上都是相关的，但杂寡聚体的组织结构是紧密的 由尚不清楚的机制控制。影响Septin蛋白折叠的突变会导致男性不育， 野生型间隔蛋白的错误折叠可能会导致其他疾病(如阿尔茨海默氏症)。分子伴侣 促进许多蛋白质的正确折叠和组装，但尚不清楚其中是否有隔膜蛋白。这个 长期的目标是了解正确从头组装Septin杂合的分子要求。 齐聚物。这个应用程序的目标是确定在死亡过程中与酵母间隔蛋白结合的伴侣。 新的生物发生，以确定介导这些相互作用的隔膜的分子特征，并 确定它们对从新开始的Septin折叠动力学的影响。一种酵母模型已经被开发出来 测量Septin折叠的动力学，以及快速和灵敏地定位Septin-伴侣的新工具 并确定它们在Septin折叠途径中的发生顺序。中心假说 胞质伴侣蛋白网络参与新生的隔膜蛋白，促进天然折叠和功能 Septin齐聚。这一模型是根据在 申请人的实验室。这个项目的基本原理是，Septin错误折叠和 人类疾病表明了Septin折叠在Septin功能中的重要性，但细胞对 Septin折叠是未知的。对这一过程的更深入理解可能直接转化为对以下方面的洞察 与Septin相关的人类疾病的分子基础。该模型将通过以下两个问题进行测试 制定为具体目标：(1)伴侣如何识别新生的隔膜蛋白？(2)哪些伴侣是 从头开始折叠Septin所需的？在第一个目标中，使用最先进的活体方法来鉴定 并确定它们在Septin生物发生过程中出现的顺序。 强大的遗传学被用来剖析单个间隔蛋白的特定区域在这些关联中的作用。 第二个目的是利用四种平行的、独立的七叶素折叠试验来确定哪一种伴侣-- Septin关联在功能上很重要。这种创新的方法首次探索了角色的折叠 对于与Septin相互作用的伴侣，使用以前从未使用过的方法来解决这个问题。该提案的 意义在于它有可能解释Septin突变或伴侣功能障碍是如何导致疾病的。

Cellular function requires the assembly of macromolecular complexes composed of distinct polypeptide subunits that interact in precise ways. Protein sequence is often insufficient to guarantee that in the crowded cellular environment each nascent subunit makes only the correct protein-protein interactions. Incorrect conformations and interactions lead to cellular defects, and human disease. How cells ensure that newly- synthesized polypeptides assemble into functional complexes remains poorly understood. Septin proteins assemble into highly conserved cytoskeletal hetero-oligomers that have emerged as central players in many cellular processes. All septin subunits are structurally related, yet the organization of hetero-oligomers is tightly controlled by mechanisms that remain unclear. Mutations that affect septin protein folding cause male infertility, and misfolding of wildtype septins may contribute to other diseases (e.g. Alzheimer's). Molecular chaperones promote the proper folding and assembly of many proteins, but it is not known if septins are among them. The long-term goal is to understand the molecular requirements for proper de novo assembly of septin hetero- oligomers. The goals of this application are to identify the chaperones that engage yeast septins during de novo biogenesis, to define the molecular features in the septins that mediate these interactions, and to determine their effects on the kinetics of de novo septin folding. A yeast model has been developed to measure the kinetics of septin folding, as well as new tools to rapidly and sensitively map septin-chaperone interactions and identify the order in which they occur along the septin folding pathway. The central hypothesis is that a network of cytosolic chaperones engages nascent septins to promote native folding and functional septin oligomerization. This model was generated from extensive preliminary observations made in the applicant's laboratory. The rationale for this project is that emerging links between septin misfolding and human disease point to the importance of septin folding in septin function, yet the cellular requirements for septin folding are unknown. Deeper understanding of this process may translate directly to insights regarding the molecular basis of septin-associated human diseases. This model will be tested by pursuing two questions formulated as specific aims: (1) How do chaperones recognize nascent septins?; (2) Which chaperones are required for de novo septin folding? In the first aim, cutting-edge in vivo methods are employed to identify septin-chaperone interactions and determine the sequential order in which they occur during septin biogenesis. Powerful genetics are used to dissect the roles of specific regions of individual septins in these associations. The second aim exploits four parallel, independent assays for septin folding to determine which chaperone- septin associations are functionally important. This innovative approach explores for the first time folding roles for septin-interacting chaperones, using methods never before applied to this question. The proposal's significance lies in its potential to explain how septin mutations or chaperone dysfunctions cause disease.