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. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRΔF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex

Hiring Criteria in Biology Departments of Academic Institutions

We surveyed faculty in the biology departments of US institutions of higher education to compare the experience and training valued by faculty at hiring institutions with the experience and training most graduate students receive. Our data show that associate, baccalaureate, and master\u27s institutions value teaching experience and skills more highly than research skills. In contrast, doctoral institutions place a higher value on the ability to publish research and obtain outside funding. These findings provide quantitative and qualitative insight into discrepancies between the values of those who train graduate students in biology and the expectations of the institutions likely to hire these individuals

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. We discovered that CHIP possesses an intrinsic chaperone activity that enables it to selectively recognize and bind nonnative proteins. Interestingly, the chaperone function of CHIP is temperature-sensitive and is dramatically enhanced by heat stress. The ability of CHIP to recognize nonnative protein structure may aid in selection of slow folding or misfolded polypeptides for ubiquitination

Mechanisms for Rescue of Correctable Folding Defects in CFTR F508

Premature degradation of CFTRΔF508 causes cystic fibrosis (CF). CFTRΔF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRΔF508 folding efficiency and the identity of CFTRΔF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRΔF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRΔF508 folding to 3–7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRΔF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRΔF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRΔF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRΔF508 after MSD2 synthesis and was ineffective at rescue of ΔF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRΔF508 folding and provide a therapeutic approach for CF

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. Derlin-1 serves to retain CFTR in the ER membrane and interacts with RMA1 and Ubc6e to promote CFTR's proteasomal degradation. RMA1 is capable of recognizing folding defects in CFTR Delta F508 coincident with translation, whereas the CHIP E3 appears to act posttranslationally. A folding defect in CFTR Delta F508 detected by RMA1 involves the inability of CFTR's second membrane-spanning domain to productively interact with amino-terminal domains. Thus, the RMA1 and CHIP E3 ubiquitin ligases act sequentially in ER membrane and cytosol to monitor the folding status of CFTR and CFTR Delta F508