Reconstitution of CHIP E3 ubiquitin ligase activity.

CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone activity to either deliver the substrate to CHIP or to maintain the substrate in a ubiquitination-competent conformation.

Analysis of CFTR folding and degradation in transiently transfected cells.

Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints.

Mechanisms for rescue of correctable folding defects in CFTRDelta F508.

Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear.

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR.

The role of the UPS in cystic fibrosis.

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER).

Chaperone functions of the E3 ubiquitin ligase CHIP.

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone as well as an E3 ubiquitin ligase that protects cells from proteotoxic stress. The abilities of CHIP to interact with Hsp70 and function as a ubiquitin ligase place CHIP at a pivotal position in the protein quality control system, where its entrance into Hsp70-substrate complexes partitions nonnative proteins toward degradation. However, the manner by which Hsp70 substrates are selected for ubiquitination by CHIP is not well understood.

Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis arises from the misfolding and premature degradation of CFTR Delta F508, a Cl- ion channel with a single amino acid deletion. Yet, the quality-control machinery that selects CFTR Delta F508 for degradation and the mechanism for its misfolding are not well defined. We identified an ER membrane-associated ubiquitin ligase complex containing the E3 RMA1, the E2 Ubc6e, and Derlin-1 that cooperates with the cytosolic Hsc70/CHIP E3 complex to triage CFTR and CFTR Delta F508.

Cystic fibrosis transmembrane conductance regulator as a model substrate to study endoplasmic reticulum protein quality control in mammalian cells.

Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum (ER) to select misfolded proteins for degradation. Herein we describe methods that allow for the study of the pathway for proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). The experimental system described employs transiently transfected HEK-293 cells and is utilized to monitor the biogenesis of CFTR by Western blot and pulse-chase analysis.

Adenosine nucleotides and the regulation of GRP94-client protein interactions.

The molecular chaperone heat shock protein 90 (Hsp90) serves essential roles in the regulation of signaling protein function, trafficking, and turnover. Hsp90 function is intimately linked to intrinsic ATP binding and hydrolysis activities, the latter of which is under the regulatory control of accessory factors. Glucose-regulated protein of 94 kDa (GRP94), the endoplasmic reticulum Hsp90, is highly homologous to cytosolic Hsp90.

Distinct roles for the AAA ATPases NSF and p97 in the secretory pathway.

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells.