THE ROLE OF CYTOPLASMIC CHAPERONES IN THE BIOGENESIS, MATURATION, AND DEGRADATION OF CYTOPLASMIC AND INTEGRAL MEMBRANE PROTEINS

I have characterized chaperone requirements for the biogenesis, maturation, and degradation of a cytosolic substrate, firefly luciferase (FFLux), in yeast, and of an integral membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), in yeast and in mammals. It was previously demonstrated that the cytoplasmic Hsp40, Ydj1p, is required for efficient expression of FFLux in yeast. This raised the question whether two Ydj1p-interacting molecular chaperones, the yeast Hsp70, Ssa1p, and the yeast Hsp90, Hsp82, also impact FFLux expression. The possible influence of a nucleotide exchange factor for Ssa1p, Fes1p, was also investigated. I found that the chaperone requirements for FFLux biogenesis are distinct but overlapping. Whereas Ssa1p and Fes1p likely collaborate to fold FFLux, Ssa1p, independent of its nucleotide exchange factor, was necessary for stabilizing FFLux protein and message, and for efficient induction of FFLux mRNA. Therefore, Fes1p impacts only a subset of Ssa1p¡¦s actions. Although FFLux folding progresses independent of Hsp82, efficient expression of FFLux depends on Hsp82, mainly due to Hsp82¡¦s contribution to FFLux translation. To identify the complete spectrum of chaperones that affect ER associated degradation (ERAD) of CFTR, I took a genomic approach in yeast. Transcriptional profiles between yeast expressing CFTR and control strains were examined by microarray analysis. Among the genes up-regulated in strains expressing CFTR was one encoding a small heat shock protein (sHsp), HSP26. Therefore, I investigated CFTR degradation in yeast strains lacking HSP26 and found that the protein was stabilized; stabilization was enhanced in a strain lacking both HSP26 and another sHsp-encoding gene, HSP42. In contrast, degradation of a soluble ERAD substrate and of another transmembrane protein proceeded with equal efficiency in wild type and in hsp26hsp42 mutant yeast. Next, I examined whether sHsps regulate CFTR biogenesis in mammalian cells. I found that ƒ´F508-CFTR degradation was enhanced when ƒÑA-crystallin was over-expressed in HEK293 cells, although wild type CFTR biogenesis was unaffected. To examine why this sHsp accelerated degradation of ƒ´F508-CFTR, ƒÑA-crystallin was purified and I found that it was able to suppress aggregation of CFTR¡¦s first nucleotide binding domain. Together, these results suggest that sHsps increase ƒ´F508-CFTR¡¦s accessibility during proteasome-mediated degradation