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.

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.The ErbB2 gene (also known as Her2/neu), a homolog of the rat neu gene, encodes a 185-kDa receptor-like glycoprotein that is a member of the ErbB family of receptor tyrosine kinases that also include the epidermal growth factor (EGF) 1 receptor (ErbB1) (1), ErbB3 (2), and ErbB4 (3). ErbB receptors are single transmembrane proteins with an extracellular domain (ECD) that bears two cysteine-rich clusters and is responsible for interaction with polypeptide ligands and an intracellular domain (ICD) that contains a tyrosine kinase motif and a long hydrophilic segment at the C-terminal end (4). Binding of the ECD with ligands causes hetero-and/or homodimerization of ErbB proteins, followed by stimulation of their intrinsic kinase activity, leading in turn to the phosphory...

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. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATPhydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion. INTRODUCTIONMembrane fusion is an essential step in all forms of vesicle trafficking and organelle assembly. Fusion is driven by a series of regulated protein-protein interactions. Many participating proteins have been identified, and their specific roles are gradually coming to light (reviewed in Jahn et al., 2003). Among these proteins are a number of ATPases.N-ethyl maleimide sensitive factor (NSF) was one of the first proteins specifically linked to membrane fusion (Wilson et al., 1989). It belongs to a family of chaperone-like ATPases known as AAA (ATPases associated with a variety of cellular activities) proteins (Neuwald et al., 1999). NSF together with ␣-SNAP (soluble NSF attachment protein) dissociates the SNARE (SNAP receptor) complexes that promote association and fusion of cellular membranes. More recently, NSF has also been implicated in other cellular processes on the basis of its ability to bind the AMPA receptor GluR2, ␤-arrestin 1, and GATE-16 (reviewed in Whiteheart et al., 2001). However, its role in SNARE disassembly and membrane fusion remains its best understood function.Not all ATP-requiring steps that lead to membrane fusion can be attributed to NSF (Goda and Pfeffer, 1991;Latterich and Schekman, 1994;Rodriguez et al., 1994;Wilson, 1995).Other ATPases must therefore be involved. One AAA protein thought to be an alternate to NSF is known as p97 (also referred to as valosin-containing protein, VCP). p97 is an abundant and highly conserved protein (Peters et al., 1990) whose cellular function has been the subject of much debate. A break in the mystery of p97's function came when it turn...

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.Cells must constantly monitor the folding status of nascent polypeptides and repair or degrade misfolded proteins during protein denaturing stress. To accomplish these tasks, the cell relies on an intricately regulated protein quality control (QC) 5 system. The cytosolic QC system consists of molecular chaperones, such as Hsp70 and Hsp40, that promote the proper folding and refolding of nonnative proteins and the ubiquitin proteasome system, which degrades misfolded or stress-damaged proteins (1-4). Maintenance of cellular homeostasis requires a delicate balance between that activity of protein folding and ubiquitin proteasome systems. Under circumstances where the molecular chaperone system is unable to promote proper folding of a protein substrate to its native state, it is necessary for the substrate protein to be selected for degradation, a process that is often referred to as protein triage (5, 6). Protein triage needs to be tightly regulated, since the escape of toxic proteins from QC systems or overactivity of protein degradation pathways leads to a variety of human diseases (7).Partitioning of nonnative polypeptides between folding and degradation pathways appears to be influenced by the folding kinetics of individual proteins as well as by a network of cochaperones that bind and regulate polypeptide binding and release by Hsp70 family members (8 -13). CHIP is a co-chaperone that functions as an E3 ubiquitin ligase that links the polypeptide binding activity of Hsp70 to the ubiquitin proteasome system. CHIP binds Hsp70 through interactions between its N-terminal TPR domains and the C-terminal EEVD motif found on Hsp70. The binding of CHIP to Hsp70 can stall the folding of Hsp70 client proteins (14 -16) and concomitantly facilitate the U-box dependent ubiquitination of Hsp70-bound substrates (16,17). CHIP appears to play a central role in cell stress protection (18 -20) and is responsible for the degradation of disease-related proteins that include cystic fibrosis transmembrane conductance regulator (15), p53 (21), huntingtin (22), ataxin-3 (22), Tau protein (23-25), and ␣-synuclein (26).The domain ...

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 CFTR⌬F508 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. INTRODUCTIONCystic fibrosis transmembrane conductance regulator (CFTR) is a membrane glycoprotein that is localized to the apical surface of epithelial cells that line ducts of glands and airways. CFTR functions as an ATP-gated Cl Ϫ channel that is critical for proper hydration of the mucosal layer that lines lung airways (Welsh and Smith, 1993). Individuals who inherit two mutant forms of CFTR have exceedingly viscous mucous and, due to chronic lung infections, develop cystic fibrosis and often die from lung failure. CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (Hyde et al., 1990) and is a 1480-amino acid protein that contains two membrane spanning domains (MSDs), MSD1 and MSD2; two cytosolic nucleotide binding domains (NBDs), NBD1 and NBD2; and a regulatory (R) domain (Riordan et al., 1989). The proper folding and assembly of CFTR subdomains in the endoplasmic reticulum (ER) is required for CFTR to engage the COPII machinery and be packaged into vesicles for transport to the plasma membrane (Kopito, 1999;Wang et al., 2004). The folding pathway of this complex polytopic membrane protein has been a topic of great interest, because misfolding results in premature recognition of CFTR by the ER quality control system (ERQC) and degradation by the ubiquitin proteasome system (Skach, 2000). In fact, the most common disease-causing mutation of CFTR, ⌬F508CFTR, results in almost complete degradation of the protein by the ERQC system, which gives rise to a loss of function phenotype and lung disease (Ward and Kopito, 1994).The assembly of CFTR into an ion channel is complicated because it requires the coordinated folding and assembly of its membrane and cytoplasmic domains into a functional unit (Du et al., 2005;Riordan, 2005;Cui et al., 2007). CFTR is a modular protein, and its domains can collapse to a protease-resistant conformation i...