A previous study demonstrated a requirement for the COPII components in the sequestration of the transmembrane ERAD substrate CFTR into ERACs in S. cerevisiae
(Fu and Sztul, 2003
). However, the molecular mechanism by which the COPII machinery contributes to this process is unclear. To achieve a better understanding of the involvement of COPII proteins in ERAC formation, we expanded the previous study by examining the localization of EGFP-CFTR in various temperature-sensitive mutants with a deficiency in ER-to-Golgi transport. The pattern of EGFP-CFTR localization in the sar1D32G
, and sec16-2
strains, all of which were defective in the stage of COPII vesicle budding, showed significant changes at the restrictive temperature: EGFP-CFTR was homogeneously dispersed throughout the ER. In contrast, when the vesicle targeting and fusion mutants sec17-1
were grown at the restrictive temperature, >70% of the mutant cells displayed the ERAC or ERAC and ER pattern of EGFP-CFTR localization, confirming that the sequestration of EGFP-CFTR is independent of ER-to-Golgi trafficking ().
These experiments require a temperature shift from the permissive temperature (23ºC) to the restrictive temperature (37ºC) to evaluate the effect of the mutant COPII component on ERAC formation. Thus one might expect that cytosolic and luminal chaperones that are induced in response to a rapid shift of yeast cells to the restrictive temperature prevent the formation of protein aggregates and/or promote protein refolding, which may act to limit ERAC formation. In fact, others have observed that an elevated temperature markedly stabilizes wild-type CFTR in mammalian cells and its temperature-sensitive folding mutant,
F508-CFTR, from ERAD in response to the induction of heat shock proteins (Strickland et al., 1997
; Loo et al., 1998
; Meacham et al., 1999
; Choo-Kang and Zeitlin, 2001
). However, this is certainly not the case for EGFP-CFTR in yeast, because ERAC formation was not significantly altered upon the temperature shift in either wild-type or sec17-1
mutants (). Alternatively, the accumulation of aberrant proteins in the ER generally leads to the induction of the unfolded protein response (UPR; Walter and Ron, 2011
), which also induces the expression of a variety of genes required for protein folding and ER export (Travers et al., 2000
). Upregulation of COPII-pathway genes may be of significance in view of the data presented here and in previous work (Fu and Sztul, 2003
). However, CFTR fails to induce the UPR (Zhang et al., 2001
); other transmembrane ERAD substrates expressed in yeast also fail to induce the UPR (Ferreira et al., 2002
; Huyer et al., 2004
). Indeed, EGFP-CFTR was normally sequestered into ERACs in cells lacking IRE1
, the master membrane kinase involved in transmitting the unfolded protein signal to activate UPR target genes (Supplemental Figure S1).
Additional evidence for the involvement of a traffic-independent function of COPII components in ERACs formation comes from multicopy suppression analyses. The temperature-sensitive growth defects in many mutants defective in COPII vesicle formation can be suppressed by high-copy expression of other genes involved in this process. However, we observed that such multicopy suppressors were not always able to rescue the ERAC formation defect. In addition, we also found that the overexpression of a number of genes involved in COPII vesicle formation that fail to suppress the ER-to-Golgi transport defect suppresses the ERAC formation defect (). These collective genetic data provide circumstantial evidence that the COPII machinery has at least two different functions in the ER. One function involves ER-to-Golgi transport, and the other involves the sorting of integral membrane ERAD substrates into specialized ER subdomains.
ERAC-like structures that form in response to certain mutant or heterologously expressed proteins have been observed both in yeast and in mammalian cells (Valetti et al., 1991
; Hobman et al., 1992
; Supply et al., 1993
; Nishikawa et al., 1994
; Raposo et al., 1995
; Kamhi-Nesher et al., 2001
; Ferreira et al., 2002
). Because certain misfolded proteins reside only transiently in the ERAC-like structures and eventually enter the secretory pathway, some of these structures have been proposed to represent expanded ER exit sites (Nishikawa et al., 1994
; Hobman et al., 1998
; Kamhi-Nesher et al., 2001
; Ferreira et al., 2002
). However, we could not observe the specific localization of ERES marker proteins within the ERACs induced by EGFP-CFTR. Furthermore, a constitutively inactive form of Sar1p (Sar1p-D32G), which fails to be targeted to ERES (Yorimitsu and Sato, 2012
), was also found to localize to ERACs (). Therefore it is likely that at least the EGFP-CFTR–induced ERACs are not expanded ER exit sites. Instead, some small punctate structures labeled by ERES markers, which closely resemble the structures characteristic of ERES, were often associated within ERACs. Because EGFP-CFTR–induced ERACs are accumulations of tubular membrane extensions connected to the ER (Fu and Sztul, 2003
; Huyer et al., 2004
) and a resident transmembrane ER protein (Sec71p) is also present in ERACs (), we assume that these small puncta represent ERES that had been coincidentally incorporated into the ERAC during its formation. However, we do not have data to indicate that these punctate signals are functionally equivalent to ERES observed within the ER.
The recognition of ERAD substrates is believed to be mediated primarily by molecular chaperones. Integral membrane ERAD substrates with large cytoplasmic domains have been proposed to bind to cytosolic Hsp70s and Hsp40s. In fact, CFTR includes two large cytosolic nucleotide-binding domains, and cytosolically localized Hsp70 and Hsp40s have been shown to facilitate the ERAD of CFTR in both yeast (Zhang et al., 2001
; Youker et al., 2004
) and mammals (Meacham et al., 1999
; Rubenstein and Zeitlin, 2000
). The contributions of Hsp40 proteins in the process of ERAD have been observed to require their interaction with partner Hsp70 proteins. In general, Hsp40 binds directly to substrate polypeptides first and then, in nearly all cases, transfers the bound substrates to its cognate Hsp70 (Craig et al., 2006
; Buck et al., 2007
). We could indeed detect a direct interaction between ER-localized Hsp40, Hlj1p, and EGFP-CFTR (). We demonstrated that the maximal formation of ERAC requires at least two ER-associated Hsp40s, Ydj1p and Hlj1p. However, the function of the Hlj1p/Ydj1p Hsp70 cognate, Ssa proteins, has been shown to be dispensable for CFTR-induced ERAC formation (Zhang et al., 2001
). Although it has been shown that Hlj1p and Ydj1p redundantly function to facilitate CFTR degradation (Youker et al., 2004
), our data showed that deletion of either one caused defects in both sequestration and degradation of EGFP-CFTR (). One possible reason for this discrepancy could be due to a difference in expression level of CFTR: the previous report (Youker et al., 2004
) used the constitutive PGK promoter, whereas we used the inducible CUP1 promoter. When expressed from the relatively weak PGK promoter, the cells with the loss of either HLJ1
may still manage to sequester CFTR into ERACs, whereas CFTR expressed from the strong inducible CUP1 promoter requires full Hsp40 activity. Our finding is the first example in which ER-associated Hsp40s are shown to play a role in a step upstream of ERAC formation.
Several lines of genetic evidence presented here revealed partial overlaps in the functions of COPII proteins and ER-associated Hsp40s during ERAC formation. It is therefore likely that the substrate-binding activities of Hlj1p involved in ERAC formation have a close mechanistic link with COPII functions rather than simply delivering bound substrates to its partner Hsp70. However, we could not detect COPII components in the same Hlj1p-EGFP-CFTR protein complex under detergent-solubilized conditions (). This might be due to several factors, including a breakdown of the interactions in the presence of detergent and/or an inability to maintain the complex under detergent-solubilized membrane-free conditions. Understanding this key step will be important to exploring how COPII might facilitate the sorting of EGFP-CFTR into specialized ER subdomains. Because ERACs are extensive networks of tubulovesicular structures, we speculate that the membrane-deforming ability of Sar1p and COPII coats (Bi et al., 2002
; Bielli et al., 2005
; Lee et al., 2005
; Stagg et al., 2008
; Long et al., 2010
; O'Donnell et al., 2011
) might contribute to drive ERAC formation. Moreover, there is a report that at least some ERACs are cleared from cells through the autophagy-dependent pathway (Fu and Sztul, 2009
). Therefore, taking into account that the requirement for a subgroup of COPII proteins in autophagosome formation has been demonstrated in yeast (Ishihara et al., 2001
; Hamasaki et al., 2003
), it is possible to consider ERACs to be a kind of preautophagosomal-like structure accumulated by COPII components.
In summary, our results demonstrated that the inhibition of ERAC formation in the absence of COPII functions was not due to indirect effects of inactivating ER-to-Golgi trafficking; instead, COPII components played a certain role during ERAC formation. Furthermore, we found that the ER-associated Hsp40s were specifically involved in the substrate sequestration process. We propose that a continued characterization of the underlying mechanisms that govern substrate partitioning to ERACs will yield additional insights into the varied activities of COPII machinery.