Deletion of IRE1 or Expression of Misfolded Proteins Causes Cell Wall Defects
To test the hypothesis that ER quality control mechanisms affect cell wall biosynthesis, we compared the growth of an ire1
Δ mutant to WT in the presence of the cell wall perturbing agents CFW and Congo red. ire1
Δ (W303 background) is hypersensitive to CFW (A) and Congo red (Supplemental Figure S1A). Because the phenotypes of ire1
Δ and cell wall-related mutants can vary with strain background (Larson et al., 2002
), we also tested an ire1
Δ mutant constructed in the S288c background. Although the S288c-derived ire1
Δ strain was less sensitive to CFW than the W303 ire1
Δ mutant, it showed a growth defect when exposed to the combined stress of CFW and elevated growth temperature (B).
Figure 1. Null mutation of IRE1 or expression of CPY* causes cell wall defects. (A) ire1Δ mutants in both W303 and S288c backgrounds are hypersensitive to CFW by spot dilution assay. (B) In the S288c background, ire1Δ and ssd1Δ show synthetic (more ...)
A well known difference between the W303 and S288c strain backgrounds is a polymorphism at the SSD1
is involved in cell wall integrity (Kaeberlein and Guarente, 2002
) and W303 has a hypofunctional, truncated allele (ssd1-d
), whereas S288c has a functional, full-length allele (ssd1-v
). As shown in B, the S288c-derived ssd1
Δ mutant recapitulates the CFW phenotype of W303-derived ire1
Δ, suggesting that the background dependence of ire1
Δ CFW sensitivity is related to differences in the functionality of SSD1
. The hypersensitivity of ire1
Δ mutants to cell wall perturbation and the genetic interaction of IRE1
, a gene involved in cell wall integrity, support our hypothesis that IRE1
is required for proper yeast cell wall biosynthesis.
Because Ire1p is required for the cell to effectively degrade unfolded proteins during periods of stress (Casagrande et al., 2000
), it seemed likely that the cell wall defects associated with ire1
Δ mutants may be due to the accumulation of misfolded or misprocessed proteins. If that was correct, then expression of a misfolded protein would be expected to also cause cell wall defects. To test this hypothesis, we examined the growth of strains expressing the well-studied misfolded protein, carboxypeptidase Y (CPY*) from a galactose-inducible promoter on plates containing CFW. Spear and Ng have shown that expression of CPY* at this level saturates ER quality control mechanisms and causes severe ER stress (Spear and Ng, 2003
). In wild-type (WT) cells, high level expression of CPY* causes hypersensitivity to CFW (C), indicating that saturation of ER quality control mechanisms with a misfolded protein compromises cell wall integrity. It is important to note that, although ire1
Δ mutants in some strain backgrounds grow poorly on galactose medium, the growth of ire1
Δ on YPG is only slightly reduced relative to WT cells and that its growth is clearly decreased by the addition of CFW to YPG, indicating that the phenotype is due to the presence of CFW (Larson et al., 2002
To confirm the effect of misfolded protein accumulation on cell wall integrity, we also tested a second misfolded protein, the ERAD substrate referred to as KWW. KWW is a chimeric fusion of the luminal portion of the simian virus 5 hemagglutinin-neuraminidase ectodomain (KHN) with the transmembrane and cytosolic domains of Wsc1p (KWW = K
sc1p transmembrane domain/W
sc1p cytosolic domain; Vashit and Ng, 2004
). Indeed, expression of KWW from the ACT1
promoter also increased the CFW sensitivity of both WT and ire1
Δ cells, demonstrating that this effect is not unique to CPY* (Supplemental Figure S1B). These results suggest that uncompensated ER stress generated by either loss of UPR or the accumulation of a misfolded protein leads to defects in cell wall integrity.
Next, we examined the stability of ire1
Δ and CPY*-expressing strains to treatment with the cell wall-degrading enzyme, zymolyase to confirm the cell wall effects of ER stress by using a second standard cell wall phenotype (de Nobel et al., 2000
). Consistent with the spot dilution assays, ire1
Δ, GAL-CPY*, and ire1
Δ GAL-CPY* are more easily lysed by zymolyase than WT (D). We note that ire1
Δ GAL-CPY* strains do not grow on galactose-containing agar plates (Spear and Ng, 2003
) but, in our hands, slowly grow for four to five doublings in liquid medium, permitting the assays shown here and below.
Although the ER is involved in a variety of cellular processes that could, in principle, affect cell wall integrity, it seemed likely that cell wall protein homeostasis might be most directly affected by uncompensated ER stress. To test this hypothesis, we carried out a set of experiments to characterize the protein fraction of the cell wall in ER-stressed strains. As shown in A, the total amount of protein per milligram of cell wall is increased in these mutants; indeed, twice as much protein is present in the cell wall of the ire1
ΔGAL-CPY* strain relative to WT. Consistent with this finding, ire1
ΔGAL-CPY* also stained much more intensely than WT when treated with fluorescein isothiocyanate-labeled concanavalin A, a lectin that binds mannoprotein (Supplemental Figure S2A). Furthermore, ER-stressed strains formed large cellular aggregates (B) and displayed slightly increased flocculation (Supplemental Figure S2B). Because intracellular interactions responsible for aggregation are mediated by the outermost, mannoprotein layer of the cell wall (Ferreira et al., 2006
), this result suggests that the surface properties of ER-stressed cells are quite different from WT cells.
Figure 2. Null mutation of IRE1 and expression of CPY* causes alterations in cell wall composition and architecture. (A) Cell wall protein composition of cell walls from the indicated strains was determined (micrograms of protein per milligram of cell wall dry (more ...)
To begin to characterize the cell walls of ER-stressed mutants, we extracted whole cells with 5 mM DTT, a procedure that liberates noncovalently and dithiol-linked cell wall proteins and that yields increased amounts of extractable proteins when the cell wall is defective (Hagen et al., 2004
). SDS-PAGE fractionation of DTT extracts from equivalent numbers of cells (20 OD600
equivalents/lane) showed that increased amounts of protein are released by DTT in ER-stressed mutants as detected by silver staining (C). In addition, the spent media from cultures of ire1
Δ, GAL-CPY*, and ire1
ΔGAL-CPY* contains more protein than that from WT cultures (D). Increased release of protein into the culture medium is also a phenotype of strains with mutations that affect cell wall proteins (Richard et al., 2002
). Thus, these two experiments indicate that uncompensated ER stress causes changes to the protein component of the cell wall that are similar to those observed when cell wall proteins are mutated, supporting the idea that ER quality control mechanisms may be required for the proper function of cell wall proteins.
To further explore the effects of uncompensated ER stress on the protein component of the cell wall, we examined the fraction of proteins covalently attached to the cell wall through an alkali-sensitive ester linkage with 1,3-β-glucan (Kapteyn et al., 1999
) in ER-stressed strains. In contrast to the noncovalently bound cell wall protein fraction, ER stress seems to decrease the amount of protein that is released from cell walls by alkali extraction (E). An important caveat to the cell wall extraction and protein release experiments is that no internal protein loading control is available; thus, the samples were normalized by cell number or cell wall weight. Consequently, we cannot rule out the possibility that differences in the ratio of cell wall material to cell number may contribute to some of the observed changes in protein levels. With these limitations in mind, our results are consistent with similar experiments performed with cell wall mutants (Kapteyn et al., 1999
; Hager et al.
, 2004). Therefore, the data support the notion that ER stress has a variety of affects on cell wall protein homeostasis. Furthermore, the fact that multiple fractions of cell wall protein are altered by ER stress suggests that these cell wall defects are unlikely to be due to misprocessing of a single class of cell wall proteins or components.
As a final means to characterize the cell wall of ER-stressed mutant strains, we examined the cells by electron microscopy (). WT, ire1
Δ, GAL-CPY*, and ire1
ΔGAL-CPY* strains all displayed the characteristic bilayer cell wall with a translucent, carbohydrate region sandwiched between two electron-dense mannoprotein layers (Osumi, 1998
). Although no gross, general morphological defects were observed in the mutant strains, two more subtle differences between WT and ER-stressed strains are apparent. First, the outer mannoprotein layer of ire1
Δ and ire1
ΔGAL-CPY*, in particular, seems less electron dense. Additionally, fewer electron dense mannoprotein fibrils emanate from the cell surface of these strains ().
Uncompensated ER stress causes alterations in cell wall ultrastructure. The indicated strains were grown to stationary phase in YPG, harvested, and processed for electron microscopy as described in Materials and Methods. Bars, 0.5 μm.
Second, the distance between the electron-dense layers of the cell wall of the ire1
Δ and ire1
* cells is decreased in comparison with either WT or GAL-CPY
*. The distance between electron-dense layers in WT cells was similar to that reported for the same W303-WT strain (105 nm [SD 20], Wright et al., 2007
), whereas the translucent layer in the ire1
Δ and ire1
* strains was thinner (75 nm [SD 19] and 46 nm [SD 5], respectively). Because the translucent layer of the cell wall is mainly carbohydrate, we measured the total carbohydrate content of cell walls isolated from the mutants and, consistent with the morphological observations, there is less carbohydrate in the cell wall of ire1
Δ (25% reduced) and ire1
ΔGAL-CPY* (48% reduced) relative to WT and GAL-CPY* (Supplemental Figure S2C). Thinning of the cell wall has also been observed in Candida albicans
mutants lacking glycosylphosphatidylinositol (GPI)-linked cell wall proteins, indicating that our findings are likely to represent significant alterations to cell wall structure (Plaine et al., 2008
). Together, our data support the notion that uncompensated ER stress causes a variety of changes to the cell wall and, hence, seems to affect several processes involved in cell wall biosynthesis.
ire1Δ and ERAD Pathway Mutants Display Synthetic Cell Wall Phenotypes
The genes involved in ERAD are up-regulated by UPR and are also required for maintaining ER quality control during unstressed, vegetative growth. We, therefore, wondered whether mutations of ERAD components would cause cell wall defects. Because ERAD and ire1
Δ mutants display synthetic phenotypes (Friedlander et al., 2000
; Travers et al., 2000
), we also constructed ERAD/UPR double mutants to determine whether loss of both quality control mechanisms would cause cell wall defects. ERAD has three subtypes that are distinguished by whether the lesion is in the luminal (ERAD-L), membrane (ERAD-M), or cytosolic (ERAD-C) portion of the misfolded protein (Carvalho et al., 2006
; Ismail and Ng, 2006
). To test the effects of these different subtypes, we constructed three ERAD single and ERAD/ire1
Δ double mutants in the S288c background: hrd1
Δ (ERAD-L and -M); doa10
Δ (ERAD-C), and ubc7
Δ (common to ERAD-L, -M, and -C).
As shown in , A and B, none of the single ERAD mutants was hypersensitive to Congo red or zymolyase digestion. However, ire1Δhrd1Δ and ire1Δubc7Δ were hypersensitive to all three conditions, with ire1Δhrd1Δ being affected most severely. hrd1Δ ire1Δ and ubc7Δ ire1Δ also showed increased DTT-extractable cell wall protein (C) in a pattern similar to either CPY* expression or ire1Δ mutation in the W303 background (see C). ire1Δdoa10Δ, in contrast, showed no increased sensitivity to cell wall perturbation and no changes in DTT-extractable protein.
Figure 4. Double mutants of ire1Δ and ERAD mutants cause cell wall defects. (A) ire1Δ hrd1Δ and ire1Δ ubc7Δ mutants are hypersensitive to CFW and Congo red by spot dilution assay. (B) Zymolyase sensitivity of ire1Δ (more ...)
We also examined the cell wall ultrastructure of the ire1Δhrd1Δ mutant (Supplemental Figure S3). Although the cell wall thickness was not decreased appreciably, the outer cell wall layer of ire1Δhrd1Δ cells is less electron dense than WT, and, similar to the mutants shown in , the hair-like mannoprotein fibrils decorating the outer surface of the cells are less prominent in the ire1Δhrd1Δ cell wall. Together, the cell wall defects shown by double mutants involving specific ERAD components and ire1Δ provide additional evidence that ER quality control mechanisms are required to preserve cell wall integrity. Ire1p, however, seems to be more important for cell wall integrity than any of the single ERAD components examined.
Cell Wall Stress Activates UPR
Because our data clearly demonstrate UPR is involved in the maintenance of cell wall integrity, it seemed plausible that cell wall stress may induce UPR. Although ER stress is known to activate the CWI pathway (Bonilla and Cunningham, 2003
; Chen et al., 2005
), to our knowledge, cell wall stress has not been shown to trigger UPR. To test this hypothesis, we used a sensitive reporter of UPR-mediated transcription that contains four copies of the unfolded protein response element (UPRE
) fused to the E. coli
β-galactosidase gene (lacZ
) on a centromeric plasmid (Cox and Walter, 1996
). Treatment of WT cells harboring the 4X-UPRE
reporter with cell wall perturbing agents CFW and caffeine induced reporter activity six- and ninefold, respectively (A). Although this level of UPR activation is three- to fourfold below that induced by DTT (a compound that generates misfolded proteins; Back et al., 2005
), UPR is clearly induced by exposure of cells to cell wall stress. The UPR reporter activity induced by both CFW and caffeine is abolished by null mutation of IRE1
(A), indicating that cell wall stress activates the reporter through UPR and not via an alternate, Ire1p-independent pathway.
Figure 5. Cell wall stress activates UPR. (A) WT, ire1Δ, gas1Δ, or ire1Δ gas1Δ cells (S288c background) containing the UPR reporter plasmid pJC104 (CEN, URA, 4XUPRE-lacZ; Cox and Walter, 1996 ) were grown to logarithmic phase and (more ...)
To confirm these observations, we next examined the effect of other cell wall stressors on UPR activity. Incubation at 37°C is a mild cell wall stress that activates CWI signaling, and, accordingly, causes a modest induction of UPR. gas1
Δ is a mutant with significant cell defects that result in constitutive activation CWI signaling pathway as a compensatory response (de Nobel et al., 2000
; Popolo and Vai, 1999
); the fact that gas1
Δ also shows increased basal UPR, strongly supports the notion UPR is induced by cell wall stress.
The cellular effects of cell wall stress are frequently suppressed by the addition of an osmotic support such as 1 M sorbitol to the growth medium of cell–wall-stressed cells (Levin, 2005
). Indeed, CFW treatment of cells in YPD + 1 M sorbitol induced less UPR activity compared with standard YPD, providing further support for the notion that cell wall damage, and not some other cellular CFW effect, triggers UPR (B).
To confirm that cell wall stress activates UPR, we next examined the effect of CFW on HAC1u
splicing by using a sensitive RT-PCR assay (Bicknell et al., 2007
). As shown in C, CFW treatment results in an increase in spliced HAC1i
, the key molecular event mediated by Ire1p. CFW treatment also leads to the translation of hemagglutinin-tagged Hac1p as demonstrated by the Western blot shown in D. Together, these data demonstrate for the first time that cell wall stress induces UPR.
UPR Activation during Cell Wall Stress and Vegetative Growth Is Dependent on Mpk1p
The CWI MAP kinase pathway is the most important regulator of the cell wall stress response in Saccharomyces cerevisiae
; therefore, it seemed a likely candidate to mediate cell wall stress-induced UPR (Levin, 2005
). Consistent with this hypothesis, treatment of a strain lacking MPK1
, the MAP kinase of the CWI pathway, with sublethal CFW (20 μg/ml) did not increase UPR activity (A). In addition, the basal level of UPR activity during unstressed vegetative growth was decreased twofold. To confirm that Mpk1p contributes to the regulation of basal UPR activity, we compared the extent of HAC1u
splicing in unstressed, logarithmic phase WT and mpk1
Δ cells by RT-PCR (B). HAC1
splicing under these conditions was reduced by one-half in mpk1
Δ cells compared with WT. Bicknell et al. (2007)
recently showed that vegetative cells express low levels of basal UPR activity and that it is required for efficient cytokinesis. The CWI pathway is activated during new bud formation; hence, a low level of Mpk1p activity is present during vegetative growth (Zarzov et al., 1996
). Thus, the partial dependence of basal UPR on Mpk1p provides further support for the notion that the CWI pathway regulates UPR.
Figure 6. CFW-induced UPR activity is dependent on Mpk1p. (A) Wt and mpk1Δ (S288c) cells containing pJC104 were exposed to CFW (25 μg/ml) and processed for β-galactosidase activity as described in A. Bars indicate the mean of two-three (more ...)
The fact that basal and cell wall stress-activated UPR requires both Mpk1p and Ire1p suggested that these two proteins may function in a common regulatory pathway. Consistent with this model, the ire1Δmpk1Δ double mutant is as sensitive to CFW as the mpk1Δ single mutant, indicating that the ire1Δ and mpk1Δ mutations are epistatic (C). Furthermore, these results also indicate that a linear pathway may connect CWI signaling to UPR during cell wall stress and vegetative growth.
Mid2p and Swi6p Are Involved in CWI Pathway-mediated UPR Activation
To determine which components of the CWI pathway are involved in cell wall stress-induced UPR, we measured CFW-induced 4XUPRE-lacZ
reporter activity in a set of CWI-pathway mutants (). Initially, we tested strains lacking the putative cell wall stress receptors Mid2p and Wsc1p (A). One difference between Mid2p and Wsc1p is that Mid2p mediates CFW-induced CWI pathway activity, whereas Wsc1p does not (de Nobel et al., 2000
). As shown in , CFW-induced UPR activation was decreased in the mid2
Δ mutant, but WT activity was retained in the wsc1
Δ mutant. RT-PCR assays confirmed that CFW-induced splicing was decreased in the mid2
Δ mutant but not in wsc1
Δ (B). That Mid2p mediates both CFW-induced cell wall gene expression and UPR supports the idea that these two transcriptional responses represent separate outputs of CWI signaling.
UPR induced by CFW in cell wall integrity MAP kinase signaling pathway mutants
Figure 7. CFW-induced UPR activity is dependent on components of the CWI pathway. (A) Schematic of the CWI MAPK signaling cascade. (B) Null mutation of mid2Δ but not wsc1Δ decreases CFW-induced HAC1 splicing. (C) swi6Δ cells transformed (more ...)
Three transcription factors/regulators are controlled in part by the CWI pathway: Rlm1p, Swi4p, and Swi6p (Levin, 2005
). Basal UPR activity was decreased twofold in the absence of Rlm1p (). In contrast, CFW treatment of rlm1
Δ induced the same relative increase in UPR as observed with WT (~5-fold), indicating that Rlm1p may play a role in CWI-dependent basal UPR, but that it does not seem to mediate cell wall–stress-induced UPR. Swi4p and Swi6p form a dimeric transcription factor called SBF that plays a role in CWI and cell cycle signaling (Levin, 2005
; Madden et al., 1997
). The swi4
Δ mutant displayed basal and CFW-induced UPR activity similar to WT, whereas UPR activation was significantly lower in the swi6
Δ mutant under both conditions ( and C). In addition, introduction of a multicopy plasmid expressing SWI6
from its endogenous promoter into a swi6
Δ strain complemented the HAC1 splicing defect and, in fact, increased basal and CFW-induced UPR by RT-PCR (C). These results strongly suggest that Swi6p is required for basal and cell wall stress-induced UPR in a manner that is independent of the Swi4p and hence SBF.
In addition to Swi4p, Swi6p has two other well-characterized binding partners, Mbp1p and Stb1p. Mb1p interacts with Swi6p to form a second dimeric transcription factor known as the MBF complex (Koch et al., 1993
), whereas Stb1p regulates the timing of transcription at Start through its interaction with Swi6p (Ho et al., 1999
). As shown in , CFW treatment of either mbp1
Δ or stb1
Δ activated the UPR reporter to the same extent as WT strains, indicating that the role of Swi6p in cell wall stress-induced UPR is independent of its three best characterized binding partners (Swi4p, Mbp1p, and Stb1p).