Herein we provide new insights into how CFTR and CFTRΔF508 biogenic intermediates are partitioned between folding and degradation pathways. The data presented suggest a model for quality control in which newly synthesized CFTR and CFTRΔF508 initiate folding, but intermediates of each accumulate in a kinetically trapped conformation that is maintained in a soluble state by Hsc70. CHIP then interacts with Hsc70 and functions via a two-step mechanism to attract UbcH5a into Hsc70–CFTR complexes. Then the Hsc70–CHIP–UbcH5a E3 formed acts to polyubiquitinate CFTR.
Interestingly, inhibition of Hsc70–CHIP E3 activity caused the accumulation of a nonaggregated and ER localized CFTRΔF508 biogenic intermediate that was bound by Hsc70. Addition of chemical chaperones to growth media and reduction of cell growth temperatures permitted this nonubiquitinated CFTRΔF508 degradation intermediate to fold, exit the ER, and accumulate on the cell surface. These are the first data that describe a nonubiquitinated CFTRΔF508 biogenic intermediate and they demonstrate that it can be maintained in a nonaggregated and foldable state. This new information suggests that the development of drug cocktails that contain ubiquitination blockers and chemical chaperones could increase the cell surface expression of CFTRΔF508 and provide a treatment for CF.
The nature of the folding defect that arrests the progression of CFTRΔF508 through its folding cascade and what causes it to be selected for proteasomal degradation is not entirely clear. One school of thought is that CFTRΔF508 is highly prone to misfolding and aggregation and is therefore selected for ERAD. Such a notion is supported by the observation that inhibition of the proteasome blocks CFTRΔF508 degradation and drives the accumulation of ubiquitinated forms of CFTRΔF508 in Triton X-100–insoluble aggregates (Ward and Kopito, 1998
). However, because the inactivation of the Hsc70–CHIP E3 ligase leads to the accumulation of a soluble ER localized CFTRΔF508 biogenic intermediate, the data we present support a different view. It appears that inhibition of the proteasome leads polyubiquitinated CFTRΔF508 to aggregate because it can be extracted from the ER membrane by the p97–UFD1–NPL4 complex (Ye et al., 2003
), and because it cannot be degraded, polyubiquitinated CFTRΔF508 accumulates in aggresomes (Ward and Kopito, 1998
). On the other hand, the nonubiquitinated CFTRΔF508 that accumulates in response to inhibition of the Hsc70–CHIP E3 does not aggregate because it is inserted into the ER membrane and is bound by cytosolic Hsc70. Thus, while CFTRΔF508 has a folding defect that prevents it from passing quality control and escaping the ER, it does not appear to be overly aggregation prone and cellular chaperones can maintain it in a foldable state.
Because the cellular activity of CHIP and UbcH5a influence the partitioning of CFTR biogenic intermediates between folding and degradation pathways, we were interested in investigating whether inhibition of the Hsc70–CHIP E3 would influence the processing efficiency of CFTR and CFTRΔF508. In pulse-chase experiments UbcH5a C85A overexpression increased the half-life of the B form of CFTR and CFTRΔF508 from two- to threefold. Therefore, UbcH5a C85A overexpression increased the steady-state levels of CFTR and CFTRΔF508 severalfold. However, the processing efficiency of CFTR from its B form to its C form remained at around 25% whether or not the Hscp70–CHIP E3 complex was active. Thus, while the elevation of cellular Hsc70–CHIP E3 activity can divert the B form of CFTR away from its folding pathway, the ability of full-length CFTR to stay on pathway and collapse to the native state appears to be limited by its intrinsic folding pathway and/or additional quality control factors.
We conclude that the E2 UbcH5a is a cytosolic factor that functions with Hsc70 and CHIP to mediate CFTR ubiquitination. This conclusion is supported by three lines of experimental evidence. First, purified CHIP and UbcH5a cooperated to facilitate the polyubiquitination of CFTR. Second, when CHIP and UbcH5a were coexpressed together they appeared to act synergistically to reduce the steady-state levels of CFTRΔF508. Third, the coexpression of UbcH5a C85A with CHIP, blocked CHIPs ability to degrade CFTR and converted it into a protein that behaved like the CHIP U box mutant P269A.
UbcH5a is a member of a family of conserved E2 proteins that include UbcH5b and UbcH5c that are nearly 90% identical to each other (Scheffner et al., 1994
; Jensen et al., 1995b
). In addition to UbcH5a, purified CHIP can interact with UbcH5b and UbcH5c, and mRNAs for each of these E2 proteins is present in all tissues tested (Jiang et al., 2001
; Jensen et al., 1995b
). Thus, we propose that CHIP functions with an UbcH5 E2 family member to ubiquitinate CFTR and other Hsc70 substrates, but we are not able to state whether it prefers one family member to the other. At this point, it is interesting to note that UbcH5 proteins are related to the yeast Ubc4/5 proteins that function to target misfolded proteins for degradation and protect cells from protein denaturing physiological stress (Seufert and Jentsch, 1990
). In fact, one member of the Ubc4/5 family, Ubc1, has been shown to function on the ER surface to ubiquitinate ERAD substrates (Bays et al., 2001
). Thus, it is logical that UbcH5 is a component of an E3 complex, which contains molecular chaperones, that serves to prevent the accumulation of toxic protein aggregates.
A potential caveat to the interpretation that Hsc70 and CHIP interact with a UbcH5 family member to select CFTR for degradation is that the overexpression of UbcH5a C85A may nonspecifically inhibit the action of other cytosolic quality control factors that function on the ER surface to mediate ERAD. Though possible, data from the control studies with the ERAD substrates TCRα and ApoB48, whose degradation relies on cytsolic E2s, demonstrate that their degradation was not delayed by overexpression of UbcH5a C85A. Thus, it appears that the reduced rates of CFTR degradation caused by UbcH5a C85A overexpression are due to specific inactivation of the Hsc70–CHIP–UbcH5 E3 complex.
The E2s Ubc6 and Ubc7 function with E3s such as gp78 and Doa10 on the cytoplasmic face of the ER to ubiquitinate a variety of substrates (Cyr et al., 2002
). Hence, it is plausible that E2–E3 complexes that contain Ubc6 and/or Ubc7 function to select CFTR and CFTRΔF508 for degradation. Sommer and colleagues have explored this concept and found that overexpression of Ubc6, but not Ubc7, modulates the rate of CFTRΔF508 degradation (Lenk et al., 2002
). When we compared the effect that dominant negative forms of Ubc6, Ubc7, and UbcH5a had on CFTR and CFTRΔF508 expression, UbcH5a C85A and Ubc6 C91S drove the accumulation of the B form of CFTR, whereas Ubc7 had no apparent effect. The influence that UbcH5a C85A had on the accumulation of the B form of CFTR and CFTRΔF508, was markedly more dramatic than that of Ubc6 C91S, yet Ubc6 clearly plays a role in CFTR quality control. Studies in yeast demonstrate that Ubc6 cooperates with the transmembrane E3 Doa10 to degrade membrane and cytosolic proteins (Swanson et al., 2001
) and Doa10 is required for efficient CFTR turnover in yeast (Gnann et al., 2004
). Thus, the Doa10–Ubc6 E3 may function alongside the Hsc70–CHIP–UbcH5 E3 to mediate quality control of CFTR. The Hsc70–CHIP–UbcH5 E3 recognizes cytosolic regions of CFTR, whereas the Doa10/Ubc6 E3 may recognize unassembled transmembrane regions. This scenario would explain why turnover of the B form of CFTR and CFTRΔF508 is delayed, but not completely blocked, by the inactivation of the Hsc70–CHIP E3 complex. A critical question pertaining to the function of CHIP as a quality control factor is related to the mechanism by which it regulates Hsc70 polypeptide binding and protein folding activity. The data presented suggest that CHIP functions via a two-step mechanism to determine the fate of Hsc70 clients such as CFTR. The first step involves the binding of CHIP to the COOH-terminal EEVD motif in the lid domain of Hsc70 (Ballinger et al., 1999
; Scheufler et al., 2000
). This event alters the Hsc70 polypeptide binding and release cycle to arrest CFTR folding and may involve the transient stabilization of Hsc70–CFTR complexes. This putative event would give the U box on CHIP the time required to attract UbcH5 to Hsc70–CFTR complexes and facilitate CFTR ubiquitination.
Interestingly, the ability of CHIP to ubiquitinate Hsc70 clients can be modified by other cochaperones. Data presented herein demonstrate that Hdj-2 cooperates with Hsc70 and CHIP to mediate CFTR ubiquitination. However, the cochaperone HspBP1, which is a member of a family of nucleotide exchange factors that promote substrate release from Hsc70, blocks the ability of CHIP to ubiquitinate CFTR (Alberti et al., 2004
). Thus, the fate of proteins that are bound to Hsc70 is regulated by its interactions with multiple cochaperones. To understand this process, the temporal relationship and driving force for interactions between Hsc70 and its folding or degradatory cochaperones needs to be determined.