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.
The studies proposed in this application are relevant to human health because the understanding of chaperone roles in septin protein folding and septin filament assembly are ultimately expected to provide fundamental insights into the etiologies of human diseases in which septins misfold, such as male infertility and familial Parkinson's. These studies are therefore relevant to the part of the NIH's mission that pertains to developing basic understanding that will help alleviate human suffering due to disease.
