Chaperones and membrane protein biogenesis

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DESCRIPTION (provided by applicant): The objective of the proposed work is to define how cytosolic molecular chaperone proteins function in protein quality control to facilitate the folding and degradation of membrane proteins. The approach to taken to solve this problem is to study the biogenesis of normal and disease causing mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR). Our previous studies which utilized cultured cells and cell free systems defined roles for the cytosolic Hsc70 chaperone system in facilitating both the folding and degradation of nascent endoplasmic reticulum (ER) forms of CFTR. Data generated in the last funding period indicate that Hsc70 acts in complex that contains the ER localized Hsp40 Hdj-2 to promote CFTR folding/assembly. In addition, we found Hsc70 to interact with a new type of co-chaperone termed CHIP that contains an E3 ubiquitin ligase domain termed the U-box, to facilitate CFTR ubiquitination and degradation. These data demonstrate that cytosolic Hsc70 functions in both the folding and degradation of membrane proteins and provide evidence that co-chaperones help mediate the partitioning of nascent membrane proteins between folding and degradation pathways. We now seek additional funding to extend these studies and propose a series of experiments that are designed to further elucidate the pathways for CFTR folding and degradation. These studies will be carried out in 3 specific aims. Aim 1. Investigate roles that cytosolic and lumenal ER chaperones systems play in CFTR folding. Aim 2. Determination of the mechanism by which the Hsp70/CHIP complex controls the partitioning of nascent CFTR between folding and degradation pathways. Aim 3. Identify the components that facilitate the delivery of ubiquitinated CFTR to the proteasome. Overall, these studies will provide a comprehensive view of how the cell mediates the folding and degradation of CFTR. The long term objective of these studies is to provide information that can identify targets for development of therapeutic to treat CF.