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

细胞功能需要由不同多肽组成的大分子复合物的组装 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 在细胞环境中，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 构象和相互作用导致细胞缺陷和人类疾病。细胞如何确保新的- 合成的多肽组装成功能性复合物仍然知之甚少。Septin蛋白 组装成高度保守的细胞骨架异源寡聚体，这些寡聚体在许多肿瘤中作为核心参与者出现。 细胞过程所有septin亚基在结构上都是相关的，但异源寡聚体的组织是紧密相关的。 由尚不清楚的机制控制影响Septin蛋白折叠的突变导致男性不育， 野生型septins的错误折叠可能导致其他疾病（如阿尔茨海默氏病）。分子伴侣 促进许多蛋白质的正确折叠和组装，但尚不清楚septins是否在其中。的 长期目标是了解septin异源蛋白正确从头组装的分子要求， 低聚物本申请的目标是确定在降解过程中参与酵母胞苷的分子伴侣。 新的生物发生，以确定介导这些相互作用的septins的分子特征， 确定它们对从头septin折叠动力学的影响。已经开发了一种酵母模型， 测量Septin折叠的动力学，以及快速灵敏地绘制Septin-伴侣蛋白的新工具 相互作用，并确定它们沿隔蛋白折叠途径沿着发生的顺序。核心假设 胞质分子伴侣网络与新生的胞隔蛋白结合，以促进天然折叠和功能性结合， Septin寡聚化。该模型是根据在1999年进行的广泛的初步观察而产生的。 申请人的实验室这个项目的基本原理是，septin错误折叠和 人类疾病指出了septin折叠在septin功能中的重要性，然而， septin折叠未知。对这一过程的深入理解可能直接转化为对以下方面的见解： 败血素相关人类疾病的分子基础。这个模型将通过两个问题来检验 制定为具体的目标：（1）如何伴侣识别新生septins？(2)哪些监护人是 是否需要重新分隔折叠？在第一个目标中，采用尖端的体内方法来鉴定 septin-chaperone相互作用，并确定它们在septin生物发生过程中发生的顺序。 强大的遗传学被用来剖析在这些协会个别septins的特定区域的作用。 第二个目标是利用四种平行的、独立的Septin折叠试验来确定哪种分子伴侣- 隔蛋白缔合在功能上是重要的。这种创新的方法首次探索了折叠角色 对于septin相互作用的伴侣，使用以前从未应用于这个问题的方法。该提案的 其意义在于它有可能解释septin突变或伴侣蛋白功能障碍如何导致疾病。

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.