Our results show that membrane expansion in response to ER stress involves the generation of large ER sheets, is restricted to the peripheral ER, and is impaired by disruption of the UPR. ER stress also induces lipid synthesis enzymes through the UPR and the Ino2/4 transcription factor complex. In the absence of Ino2/4, stress-induced membrane expansion is diminished, likely because of reduced lipid biosynthesis. Conversely, activation of Ino2/4 causes constitutive ER membrane expansion. Importantly, membrane expansion by activation of Ino2/4 occurs without a concomitant increase in ER chaperone levels and is independent of Hac1, showing that ER expansion and the UPR can be uncoupled. ER membrane expansion on its own alleviates ER stress, indicating that enlarging the ER is an integral part of an effective UPR. Furthermore, the predominantly cisternal expanded ER is converted to mainly tubular by overexpression of Rtn1, suggesting that ER shape is determined by reticulon capacity. However, changing the ratio of tubules to sheets does not affect the alleviation of ER stress by membrane expansion. These findings reveal an important role of ER size control in the maintenance of ER homeostasis.
The discovery that a larger ER alone alleviates ER stress argues that the role of membrane expansion during a normal UPR, when ER size and chaperone levels increase simultaneously, goes beyond merely providing space to accommodate newly synthesized folding machinery. One possibility is that a larger ER can tolerate more misfolded proteins before essential functions break down. Moreover, a larger ER could promote protein folding itself. During folding, proteins expose hydrophobic residues that are buried in their final conformations. This makes folding intermediates vulnerable to aggregation should they encounter one another. Increasing ER volume lowers the concentration of folding intermediates, which may give proteins more time to fold by avoiding aggregate formation. This idea is consistent with in vitro experiments, showing that the unassisted folding of proteins is more efficient at low concentrations because aggregation is reduced (Apetri and Horwich, 2008
). A similar mechanism might apply for membrane-associated proteins, whose dilution caused by an increase in membrane area could help avoid detrimental interactions. Finally, membrane-associated processes that support protein folding or remove misfolded proteins, such as protein glycosylation or ERAD, could operate more efficiently when a larger membrane area is available. None of these possibilities are mutually exclusive.
The observation that the shape of the expanded ER can be shifted from cisternal to tubular by simple overexpression of a reticulon suggests that ER shape depends on the balance between the amount of ER membrane and the reticulons’ capacity to generate tubules. According to this view, the sheet morphology of the expanded ER during the UPR results from membrane growth without a corresponding increase in reticulon activity so that their tubulation capacity is exceeded and sheets form either by default or through the action of sheet-stabilizing proteins. A similar model invoking limiting reticulon capacity has been proposed recently to explain the formation of an appropriately sized nuclear envelope at the end of mitosis (Webster et al., 2009
). We note that raising reticulon levels may only be a crude experimental substitute for how their capacity is normally regulated. Reticulon capacity could be controlled posttranslationally, perhaps by changes in oligomerization behavior (Shibata et al., 2008
). The physiological significance of the transition from a tubular to a cisternal ER during the UPR remains an open question. It is not obvious whether the tubule to sheet conversion contributes to the up to fivefold increase in ER volume, and accurate measurements of the dimensions of sheets and tubules before and after ER stress by electron tomography are likely needed to answer this question. Also, it is unknown whether sheets and tubules have different functions relevant for mitigating ER stress. In any event, forcing a tubular morphology onto the expanded ER by reticulon overexpression did not affect membrane expansion or sensitivity to ER stress. Therefore, the main benefit of ER remodeling during the UPR appears to lie in the increase in ER size rather than the conversion of tubules into sheets.
The appearance of tangles of smooth tubular ER in UPR-deficient cells exposed to ER stress is intriguing. These tangles could reflect disruption of ER structure by misfolded proteins. Alternatively, they could arise from the lack of a sheet-stabilizing protein. A candidate for such a protein is Sec61, which forms the translocation channel for protein import into the ER. Sec61 is also needed for the binding of ribosomes to the ER membrane, and ribosome binding has been suggested to stabilize ER sheets (Shibata et al., 2006
; Puhka et al., 2007
). In addition, Sec61 is induced by ER stress in a UPR-dependent manner (Travers et al., 2000
). However, opi1
mutants and cells expressing ino2(L119A) have expanded rough ER sheets despite normal Sec61 protein levels (Fig. S4
), indicating that Sec61 is not limiting for the generation of new ER sheets.
We have proposed previously that the Hac1 transcription factor coordinates the induction of chaperone genes and membrane biogenesis (Cox et al., 1997
). The finding that Hac1-dependent Ino2/4 activity is needed for proper ER membrane expansion strengthens this model. In fact, the relationship between Hac1 and Ino2/4 is remarkably similar to that between the UPR and ERAD. The ERAD machinery operates at a basal level at all times but is activated during ER stress by Hac1-dependent induction of ERAD components. ERAD-deficient yeast are hypersensitive to ER stress and show constitutive activation of the UPR. Deletion of either IRE1
or an ERAD component is tolerated well, but combined deletion causes severe synthetic phenotypes (Travers et al., 2000
). Likewise, Ino2/4 activity is stimulated during ER stress through Hac1, and ino2
mutants show increased sensitivity to tunicamycin, have elevated ER chaperone levels indicative of constitutive UPR signaling, and display synthetic sickness upon additional deletion of HAC1
. Thus, Ino2/4 is another functional module that is recruited by Hac1 to help cells mount an effective UPR.
Nevertheless, many questions remain concerning the cascade of events that culminates in ER membrane expansion. First, it is unclear how UPR signaling activates Ino2/4-dependent transcription. A plausible mechanism is that Hac1 inhibits Opi1, thereby derepressing Ino2/4 (Cox et al., 1997
; Brickner and Walter, 2004
). There are several ways in which Hac1 could inhibit Opi1, e.g., by directly binding to Opi1 to promote dissociation from Ino2 or by inducing the transcription of an Opi1 inhibitor. Interestingly, Opi1 translocates from the nucleus to the peripheral ER after inositol depletion (Loewen et al., 2004
) but not after DTT treatment (unpublished data), indicating different mechanisms of Ino2/4 derepression. Second, we do not know which Ino2/4 target genes are critical for ER membrane expansion. Given that the requirement for Ino2/4 is bypassed when lipids are provided exogenously, lipid synthesis genes are probably a key. We tested several Ino2/4-regulated lipid synthesis genes, including INO1
, and OPI3
, but no single deletion phenocopied the ER expansion defect seen in ino2
mutants (unpublished data). Third, it remains to be determined whether deletions of INO2
are truly equivalent. It is generally accepted that neither transcription factor can function without the other, but some gene promoters appear to be bound by only one of the two proteins (Lee et al., 2002
), and a previous study concluded that membrane proliferation after expression of the canine ribosome receptor in yeast required INO2
but not INO4
(Block-Alper et al., 2002
). Fourth, the residual expansion seen in hac1
mutants and the residual increase in Opi3 and Ino1 protein levels in HAC1
-deficient cells suggest that another signaling pathway exists in yeast that can sense ER stress and induce membrane expansion. This putative second pathway, which may correspond to the recently described super–UPR pathway (Leber et al., 2004
), could also help explain why overexpression of ER transmembrane proteins can still trigger ER expansion in IRE1
-deficient yeast (Menzel et al., 1997
; Larson et al., 2002
). Perhaps this alternative pathway is sufficient to allow long-term adaptation of ER size but is overwhelmed by the acute ER stress caused by DTT or tunicamycin. Finally, it is intriguing that the size of the nuclear envelope does not change during UPR. Unlike mammalian cells, yeast do not have nuclear lamins that could act as a scaffold to restrict nuclear size during ER membrane expansion. However, it has been found that at least part of the yeast nuclear envelope can resist expansion by an unknown mechanism (Campbell et al., 2006
Similar to yeast, full ER expansion in mammals requires the Hac1 homologue XBP1, but some residual expansion still seems possible in its absence (Lee et al. 2005
). This points to additional signaling pathways that can regulate ER size, and the ATF6 pathway has recently been suggested to play such a role (Bommiasamy et al., 2009
). Also, similar to yeast, UPR signaling activates lipid biosynthesis in fibroblasts, and experimentally activating phosphatidylcholine synthesis leads to ER membrane expansion without an accompanying increase in ER chaperone levels (Sriburi et al., 2004
). Although the expansion elicited by increased phosphatidylcholine production was modest compared with that achieved by expression of active XBP1, these results indicate that ER membrane expansion may be driven by lipid biosynthesis also in mammalian cells. Because there is no known mammalian master regulator of lipid biosynthesis analogous to the yeast Ino2/4 complex, it is difficult to test whether a more comprehensive activation of lipid biosynthesis would recapitulate UPR-mediated ER membrane expansion, as is the case in yeast. Nevertheless, it would be interesting to further explore the poorly understood regulation of mammalian lipid biosynthesis by the UPR (Acosta-Alvear et al., 2007
In summary, ER stress induces membrane expansion through UPR-mediated activation of lipid biosynthesis, and the subsequent increase in ER size on its own is sufficient to alleviate stress. Thus, the UPR maintains ER homeostasis by two intimately connected but distinct mechanisms: by providing new ER-folding machinery and by providing more ER surface area and lumenal space.