Quality control of protein biogenesis within the secretory pathway is a fundamentally important process; the impaired folding and trafficking of proteins is the proximal cause of a number of human diseases. We sought to better understand the interplay between protein folding and forward transport through the secretory pathway by examining how the stability of a misfolded protein within the ER influences uptake into ER-derived COPII vesicles. We explored these pathways by investigating the biogenesis of the yeast ABC transporter Yor1p, a plasma membrane-localized pleiotropic drug pump that is homologous to CFTR with respect to domain arrangement and topology. An aberrant form of Yor1p with an analogous phenylalanine deletion within NBD1, Yor1p-ΔF670, is retained in the ER and degraded by ERAD (Katzmann et al., 1999
). We used several in vitro assays to demonstrate that Yor1p-ΔF fails to enter into COPII vesicles and that this aberrant protein is improperly assembled.
We used limited proteolysis to demonstrate that Yor1p-ΔF showed enhanced susceptibility to proteolytic attack, consistent with a partially folded or conformationally destabilized structure similar to that seen for the ΔF form of CFTR (Du et al., 2005
). Biochemical and structural analyses of CFTR NBD1 suggest that the ΔF508 mutation does not dramatically alter the fold of the isolated domain but that it may instead disrupt surface topology. This in turn may perturb an interaction between NBD1 and MSD1, indirectly affecting the interactions between transmembrane helices (Lewis et al., 2005
; Thibodeau et al., 2005
). Furthermore, F508 in CFTR also contributes to the posttranslational folding of NBD2, likely through interdomain interactions (Du et al., 2005
). Because the epitope tag with which we detected Yor1p is located at the C terminus of the protein, trypsinolysis specifically probes the conformational state of the second half of the protein. Thus, the stable ~65-kDa proteolytic fragment observed for wild-type Yor1p likely corresponds to a cleavage event between TM8 and TM9, with the ΔF670 mutation likely destabilizing NBD2, similar to the situation observed for CFTR. We also used cysteine substitution and cross-linking analysis to probe the arrangement of transmembrane domains in Yor1p. The inability of Yor1p-ΔF to form cross-linkable interactions between membrane spanning domains suggests that, like CFTR, multiple interdomain interactions are impaired by this misfolding lesion (Cui et al., 2007
Together, these in vitro assays suggest that Yor1p-ΔF presents multiple assembly defects: improperly arranged transmembrane segments as well as a destabilized cytoplasmic domain (NBD2). The presence of multiple misfolding lesions is consistent with its redundant use of at least two disposal pathways. Both the Hrd1p-dependent pathway, which likely disposes of proteins with membrane-localized lesions in addition to ERAD-L clients (Carvalho et al., 2006
), and the Doa10p-dependent pathway, which degrades ERAD-C substrates, can divert Yor1p-ΔF for degradation; only when both pathways are disabled is Yor1p-ΔF stabilized. Because this stabilization is only partial, the possibility remains that additional machinery also contributes in vivo to the efficient destruction of aberrant Yor1p. The specific misfolding lesions detected by each of these degradation systems remain to be defined, but they likely result from the exposure of multiple domain interfaces that are usually buried in the native protein. Our evidence that both NBD2 and the transmembrane domains of Yor1p-ΔF are improperly assembled suggests that both cytosolic and intramembrane lesions are potential signals for degradation by the ERAD-C and ERAD-L/M pathways, respectively. The dual disposal mechanism we see for Yor1p-ΔF is similar to that used by the amino acid permease, Gap1p, which is rapidly degraded in the absence of its chaperone, Shr3p (Kota et al., 2007
). Again, the precise nature of the conformational abnormality presented by Gap1p in the absence of Shr3p is not known, but based on our data for Yor1p-ΔF, it seems likely that Gap1p must present multiple folding defects to access both the ERAD-C and ERAD-L/M pathways.
Although misfolded Gap1p and Yor1p-ΔF share the same redundant degradation pathways, these two proteins differ in their ability to be rescued by inhibiting degradation. Blocking ERAD of Yor1p-ΔF did not permit capture into COPII vesicles, nor did it restore oligomycin resistance. However, when misfolded Gap1p was stabilized by introducing ERAD mutations into an shr3
Δ strain, partial recovery of amino acid transport function was observed (Kota et al., 2007
). This difference in protein recovery may stem from the nature of the specific folding defects associated with each individual protein. Aberrant Gap1p produced in the absence of its chaperone may represent a mixed population of folding intermediates, some of which can attain a folding state compatible with COPII engagement if the degradation pathways are impaired. Conversely, the specific genetic lesion that causes misfolding of Yor1p-ΔF may render it incapable of proper assembly even given prolonged residence time in the ER. Thus, the ability of an aberrant protein to be rescued by impeding its destruction seems likely to be dependent on the specific nature of the misfolding defect, which will in turn influence the folding, degradative and trafficking pathways that each specific protein can access. Indeed, different misfolding variants of a single protein may behave distinctly; a variety of transthyretin mutants showed distinct secretion phenotypes that were not entirely correlated with the degree of global protein stability, even in the absence of stabilization through ERAD inhibition (Sekijima et al., 2005
). Furthermore, even when considering a single protein, different mechanisms of stabilization may result in different outcomes. Attempts to rescue CFTR-ΔF by inhibiting its destruction have met with mixed results; early experiments using proteasome inhibitors did not result in rescue of CFTR-ΔF (Kopito, 1999
) but more recent experiments in lung epithelial cells suggest that inhibition of either the proteasome or the protein extraction apparatus, p97/VCP/Cdc48p, can rescue delivery of CFTR-ΔF to the plasma membrane (Vij et al., 2006
). Indeed, the ability for a misfolded protein to be rescued by simple stabilization may also depend on the cell type, with the specific complement of cellular chaperones associated with a given tissue likely to play an important role. This seems particularly true for polytopic membrane proteins such as Yor1p and CFTR, which are subject to complex quality control processes involving multiple chaperone machineries (Younger et al., 2006
One additional mechanism that may contribute to the differing abilities of misfolded proteins to be rescued by inhibition of ERAD is the presentation of an appropriate ER export signal. Such signals tend to be small peptides with limited structural features that may be obscured or improperly presented in the context of a misfolded protein. The failure to present an appropriate export motif could account for the distinct intracellular fates of misfolded variants of transthyretin; the different mutants may display or obscure their ER export signals independently of the thermodynamics of the particular protein fold (Sekijima et al., 2005
). In membrane proteins, which interact directly with Sec24p, the presentation of an appropriate export motif in the context of a misfolded protein is likely to be a more complicated affair. For CFTR, the diacidic ER export signal is located in the same domain as the misfolding lesion, suggesting that the structural destabilization induced by the ΔF508 lesion may directly impair the presentation of the signal to Sec24p (Wang et al., 2004
). In Yor1p, the diacidic export motif is located in a different cytosolic domain from the misfolding lesion, suggesting the misfolding event has a more indirect effect on protein traffic. One key question that remains unanswered from our study is whether the ΔF lesion impacts the interaction between Sec24p and Yor1p directly. We attempted to address this issue by isolating “prebudding complexes” that contain cargo proteins bound to Sar1p/Sec23p/Sec24p and represent an early cargo–capture phase of vesicle biogenesis (Kuehn et al., 1998
). However, we were unable to reproducibly detect wild-type Yor1p in these complexes, perhaps due to the difficulty in solubilizing a protein with so many transmembrane domains (Kung and Miller, unpublished data). Alternative approaches such as reconstitution of Yor1p into detergent micelles or synthetic liposomes may allow us to more directly assess the binding between Yor1p and Sec24p and ask how the ΔF lesion impacts this interaction. In addition to directly impeding interaction with Sec24p, another mechanism by which a misfolding lesion may impact forward traffic is by indirectly preventing capture into vesicles through the recruitment of cytoplasmic chaperones at early stages of protein synthesis. We have demonstrated that cytoplasmic HSP90s play a role in productive biogenesis of wild-type Yor1p. One can imagine a model whereby correctly folded Yor1p no longer interacts with HSP90, release of which in turn exposes the Sec24p interaction motif. Thus, action of Sec24p would be sterically inhibited by HSP90, and a molecular handoff would occur between the folding chaperones and the forward transport machinery. Detailed coimmunoprecipitation experiments, in combination with kinetic analysis of these interactions during Yor1p biogenesis, will be required to fully map the sequential and competitive interactions between the various cytoplasmic chaperone machinery, the ER export apparatus and newly synthesized Yor1p. Furthermore, the action of these chaperones may be very complex: recent evidence suggests that HSP90 can influence CFTR biogenesis in both a positive and negative manner, likely modulated by distinct cochaperones (Wang et al., 2006
In addition to HSP90s, the ER-localized HSP40s, Ydj1p and Hlj1p, are also candidates for interfering with ER export of Yor1p. Through recruitment of cytoplasmic HSP70s, these chaperones may impair maturation of misfolded proteins either by sequestering the aberrant protein in a distinct ER subdomain or by triggering the degradation pathway. In our experiments, the action of these HSP40s promoted the trypsin-sensitive conformation of Yor1p-ΔF, suggesting an active role in unfolding the cytoplasmic domains before exposure to the ubiquitination and retrotranslocation machinery. A similar role in actively unfolding cytosolic domains of misfolded proteins (both soluble and membrane-bound) before proteasomal degradation has been suggested for the Ssa1p–Ydj1p chaperone complex (Taxis et al., 2003
; Park et al., 2007
). This action may be required for tightly folded domains that could not be denatured solely by the 19S cap of the proteasome (Taxis et al., 2003
). We note that despite some restoration of a more wild-type conformation of the C-terminal domain, Yor1p-ΔF still failed to enter into COPII vesicles in the HSP40 mutant, likely because the arrangement of its transmembrane segments was still aberrant.
In summary, we have developed Yor1p-ΔF as a model yeast misfolded protein, and we have defined some of the chaperones, ER export machinery, and degradation apparatus that engage both the wild-type and aberrant forms of the protein. It is clear from our data that aberrant Yor1p accesses multiple degradation pathways and that stabilization of this misfolded protein by blocking its degradation does not confer transport competence. This observation has important implications for potential treatments for diseases caused by improper ER retention and degradation. A growing body of evidence suggests that different misfolded proteins are handled differently by the multiple processes that comprise ER quality control. It seems likely that, like the multiple pathways of protein degradation from the ER, diverse mechanisms that prevent forward transport are likely to impact the efficiency of ER export in the context of protein misfolding. Such mechanisms may include active ER retention, steric hindrance of interaction with Sec24p, prevention of capture of prebudding complexes into a vesicle proper, or failure to engage additional sorting machinery. Given the wide array of ER export signals and binding sites on Sec24p, it is easy to imagine that cells use a variety of mechanisms to ensure that aberrant proteins are not deployed within the secretory pathway. A deeper understanding of the interplay between the degradative pathways and the ER export machinery will be required to fully elucidate how misfolded proteins are subjected to quality control within the secretory pathway.