title: Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum-associated degradation.
abstract: The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Matsumura
authors.forename: Larry L
authors.initials: LL
authors.lastname: David
authors.forename: William R
authors.initials: WR
authors.lastname: Skach
mesh_terms: D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D008861:Microsomes / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D051381:Rats; D014157:Transcription Factors / Q000737:chemistry; D054875:Ubiquitination
chemical_list: C091732:BCL2-associated athanogene 1 protein; D004268:DNA-Binding Proteins; D050883:HSC70 Heat-Shock Proteins; D010446:Peptide Fragments; D014157:Transcription Factors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator