Crh2p is a cell wall-localized GPI-linked protein that is involved in the cross-linking of chitin to 1,6-β-glucan (7
). The laboratories of Arroyo and Cabib have extensively studied this protein (7
) and, in the course of that work, utilized two fusion alleles derived from Crh2p containing HA and GFP fused within the C-terminal region of the protein. While both HA and GFP show identical localizations to the cell wall of the budneck (25
), the Crh2-HA fusion is stable at 38°C, whereas the Crh2-GFP construct is not (7
). Consistent with the instability of the GFP construct, it failed to complement a crh2
Δ mutant, while the overexpression of the HA allele did complement the strain (7
). Therefore, it appears that the insertion of the GFP moiety within this region affects both the stability and the function of Crh2p, possibly by interfering with folding or processing. However, note that at least a fraction of the total protein produced by this construct is functional, since both the GFP and HA alleles show similar localizations, and the overexpression of the HA-tagged allele complements a crh2
Based on those previously reported observations, it is therefore not surprising that the overexpression of Crh2-HA/GFP induces ER stress, as our data clearly indicate. However, the ER stress generated by these constructs is not entirely the result of the internal epitope tags, because the overexpression of an untagged allele also induces ER stress (). The tag plays a role in the amount of ER stress, since the HA allele triggers 2-fold-higher levels of UPR activation than does the GFP allele (). It is, however, somewhat counterintuitive that the apparently less stable GFP-tagged allele generates less ER stress than does the more stable HA allele. The Western blots of the GFP allele show extensive degradation at the steady state in wild-type cells, and many more bands are present than those observed with the corresponding HA construct (D. J. Krysan, unpublished results). Therefore, we suspect that the intrinsic instability of Crh2-GFP may result in a more rapid clearance of the protein from the ER and, therefore, less activation of the UPR. The more stable Crh2-HA protein, on the other hand, may require a more extensive activation of the UPR to efficiently remove it from the ER.
Although overexpressed Crh2-HA/GFP certainly leads to ER stress, it is important to consider whether these constructs represent an appropriate model substrate for the study of mechanisms of GPI-linked protein quality control. Prior to this study, only one other putatively misfolded GPI-linked protein has been studied: a point mutant of Gas1p (13
). Gas1p is a GPI-linked protein that is mainly plasma membrane localized and, like Crh2p, is thought to be involved in the remodeling of cell wall polysaccharides. Fujita et al. previously constructed a point mutant of Gas1p that shows an accumulation of the ER form at steady state and undergoes proteasome-mediated degradation (13
). Their studies of the processing of misfolded Gas1p revealed that its proteasome-mediated degradation was independent of the key ERAD proteins Hrd1p and Doa10p and dependent on the inositol deacetylase Bst1p.
If overexpressed Crh2-HA/GFP is an appropriate model substrate for studies of the processing of aberrant or excess GPI proteins, then its processing should share features with those observed for misfolded Gas1p. Similarly to misfolded Gas1p (13
), overexpressed Crh2-HA leads to high levels of its ER form, and the deletion of Bst1p significantly delays the decay of the ER form of Crh2-HA in cycloheximide chase experiments. Taken together, the observations that overexpressed Crh2-HA/GFP induces ER stress and is processed in part by pathways previously shown to be involved in the processing of misfolded GPI proteins strongly suggest that this represents an informative system for the study of GPI protein quality control.
In addition to being processed through the Bst1p-dependent proteasome-mediated degradative pathway, we observed that significant amounts of both Crh2-HA and Crh2-GFP are secreted to the extracellular space. The secretion of Crh2-HA/GFP is dependent on the UPR and is increased in strains lacking Bst1p, an inositol deacetylase involved in the proteasome-mediated degradation of GPI proteins (13
). Based on these and other observations described above, we propose that the secretion of Crh2-HA/GFP represents a previously unrecognized pathway by which excess or aberrant secretory proteins are removed from the cell when other degradative pathways are saturated or inoperative. It is important that the overexpression of the extensively studied ERAD and UPR substrate CPY* does not lead to secretion (D. Ng, personal communication), suggesting that only some ER-stress-inducing secretory proteins are processed through this process.
To our knowledge, the ER-stress-induced release of secretory pathway proteins to the extracellular space has been described only once previously. Specifically, Belden and Barlowe showed that ER stress induced either by the mutation of proteins in the p24 family of ER transport proteins or by the treatment of cells with the reducing agent β-mercaptoethanol causes the release of the ER-resident chaperone Kar2p/BiP to the extracellular medium (3
). This release is completely blocked when the UPR is disabled through the deletion of IRE1
, leading those authors to propose that Kar2p secretion is a “hallmark of UPR” (3
). Our results indicate that the high-level expression of a GPI-linked cargo protein that induces UPR activation leads to both Kar2p secretion and secretion of the cargo protein, suggesting that this UPR-mediated secretory pathway may not represent simply a nonspecific release of secretory pathway proteins to the extracellular space.
At this point, we cannot completely exclude the possibility that the release of Kar2p/Crh2p is due to stress-induced ER and secretory pathway dysfunction. However, a number of observations from this work and from the work of Belden and Barlowe (3
) support the notion that this secretory pathway is not an indirect consequence of ER dysfunction. First, we have shown that the secretion of Crh2-HA is dependent on ER-to-Golgi transport via Sec18p, indicating that components of the trafficking machinery are required for the process. Second, Belden and Barlowe found previously that under conditions in which Kar2p is released, there is very little change in the pattern or quantity of proteins secreted into the growth medium compared to wild-type cells (3
). This finding suggests that Kar2p secretion is not due to generalized defects in secretory pathway function leading to a nonspecific release of proteins into the extracellular space. Third, the UPR is an important mechanism by which ER and secretory pathway function is maintained (23
). Therefore, if the release of Crh2-HA was the result of ER-stress-mediated secretory pathway dysfunction, then the additional ER stress generated by the loss of the UPR through the deletion of Ire1p would be expected to increase secretion. Hence, the decrease in Crh2-HA and Kar2p secretion observed for ire1
Δ mutants is inconsistent with a nonspecific breakdown in secretory pathway function but is consistent with the idea that secretion represents a UPR-mediated pathway by which aberrant proteins are removed by secretion to the extracellular space.
Although more work will be required to fully characterize the secretory pathway proteins involved in the delivery of Crh2-HA/GFP to the extracellular space, our initial results suggest that this pathway is distinct from the pathway by which normal GPI proteins are delivered to the cell surface. First, ire1
Δ mutants, which do not secrete Crh2-HA/GFP, still localize a significant portion of the protein to the cell wall, indicating that secretion is not required for appropriate localization and supporting the idea that secretion is distinct from normal GPI protein trafficking. Second, the deletion of two proteins involved in GPI protein trafficking, Emp24p and Erv25p, does not block secretion; indeed, the deletion of either protein leads to Kar2p secretion (3
), suggesting that the loss of these proteins may actually trigger the pathway. Third, Western blots of strains with decreased secretion show amounts of fully mature Crh2-HA that are similar to those in wild-type cells (). The pathway does, however, require Sec18p, a protein required for ER-to-Golgi transport. Thus, the pathway by which Crh2-GFP/HA is secreted to the extracellular space must utilize at least some components of the machinery involved in the trafficking of normal proteins. Since mutants that affect the proteasome-mediated processing of aberrant GPI proteins increase Crh2-HA secretion, we suspect that this pathway represents a mechanism that is operative mainly when the standard, proteasome-mediated pathway is saturated by severe ER stress.
An important question regarding the yapsins is, where do they cleave their substrates? Recently, Gagnon-Arsenault et al. reported an elegant study that strongly suggested that yapsin proteolysis occurs at the cell surface and is regulated by the pH of the compartment (14
). This finding is consistent with the results of Komano et al. showing that the yapsin-specific cleavage of fluorescent model substrates occurs at the surface of intact cells (19
). Yps1p and Yps2p have both been shown to have optimal activity at a low pH, which is present in the extracellular/periplasmic space in yeast. Since the early compartments of the secretory pathway are more basic than those that lead to efficient yapsin proteolysis, cleavage is unlikely to occur within early portions of the pathway, such as the ER (14
); indeed, our results show that the trapping of Crh2-HA in the ER blocks proteolysis (). It is, however, possible that some portion of the proteolysis occurs within the Golgi apparatus. Sievi et al. showed previously the Yps1p-mediated degradation of a mammalian sialyltransferase ectodomain fusion protein (STE6Ne) in the Golgi apparatus of a strain with a temperature-sensitive block in Golgi transport; Yps2p had no effect on the processing of this substrate (33
Seivi et al. also showed that Yps1p is mislocalized from the plasma membrane to the vacuole in erg6
Δ mutants and processes STE6Ne in that acidic compartment (33
). Although the cleavage of Crh2-HA could occur in this compartment, the fact that we did not observed an accumulation of uncleaved Crh2-GFP in the vacuole of yapsin mutants () argues against this possibility. However, since Crh2-GFP processing and Crh2-HA processing are not identical, we cannot rule out the vacuole as a site of Crh2-HA processing. This model, however, requires that the cleavage products then be transported from the vacuole to the cell surface, a process that is unprecedented to our knowledge.
In addition to the vacuolar targeting model, our observations are consistent with at least three other scenarios for the events leading to the yapsin-mediated release of misfolded/excess proteins such as Crh2-HA. First, the substrates and proteases could traffic in the same vesicles as normal proteins to the cell surface and then undergo proteolysis. In this model, selection between folded and misfolded proteins could result from differential rates between further processing and proteolysis. For example, a cell wall-targeted GPI protein must be cleaved from its anchor and incorporated into the cell wall. If a protein is misfolded or excess protein has saturated the capacity of this process, then its delay may then allow proteolysis to compete, leading to the release of the misfolded, or excess, GPI protein. Second, a similar series of events may occur but within specific vesicles that contain misfolded proteins. An indirect precedence for the packaging of misfolded cargo proteins into distinct vesicles is based on the fact that excess misfolded protein can be trafficked directly to the vacuole when ERAD is saturated, a process that is likely to involve vesicles distinct from those destined for the plasma membrane (35
). Last, yapsin cleavage could occur in the Golgi apparatus, as suggested by the results described previously by Seivi et al. (33
). Recent work has also revealed that protein quality control surveillance is operative within the Golgi apparatus (40
), and thus, it is possible that discrimination between abnormal and normal proteins is made at the Golgi apparatus. After proteolysis, the fragments could then be transported to the surface via either normal vesicles or, alternatively, specific vesicles containing misfolded proteins. The small punctate structures to which Crh2-GFP localizes in the yps2
Δ mutant could represent the Golgi apparatus () and therefore would provide support for this model. It must be stressed that these models are rather speculative at this point and that additional work will be required to identify the pathways that are involved in the UPR/yapsin-dependent secretion of excess/misfolded proteins.
Although the yapsin family of GPI-linked aspartyl proteases was identified over 15 years ago (15
), an understanding of their physiological function is only recently beginning to emerge (1
). To date, S. cerevisiae
yapsins have been shown to play a role in yeast cell wall integrity (20
), the processing of the signaling mucin Msb2p during the transition to filamentous growth (38
), and the release of both Gas1p and Pir4p from the cell surface (14
). In this work, we have found evidence supporting a role for both Yps1p and Yps2p in protein quality control. YPS1
expression is increased by ER stress in an Ire1p-dependent fashion, a finding that is consistent with data from previously reported transcriptional profiling experiments (36
) and is further supported by the epistatic relationship between ire1
Δ and yps1
Δ mutants during ER stress (). Yps2p also appears to play a role in protein quality control based on the fact that yps2
Δ and ire1
Δ mutants display a synthetic genetic interaction during ER stress.
expression does not appear to vary significantly during cellular stress (15
), and its genetic interaction with Ire1p suggests that its function is independent of the UPR. Furthermore, the surface yapsin activity is dependent almost exclusively on Yps2p under nonstress conditions, while Yps1p is undetectable at the surface unless the cells are exposed to stresses known to increase YPS1
expression levels (15
). We therefore propose that Yps2p is constitutively active during unstressed growth and is sufficient to process the basal levels of misfolded or excess protein within the secretory pathway. During ER stress, the UPR is activated and induces YPS1
expression to increase the processing capacity. This model provides a possible explanation for the observation that Yps2p is the only yapsin required for Crh2-GFP processing while both Yps1p and Yps2p process Crh2-HA. Crh2-GFP does not activate the UPR as intensely, as Crh2-HA presumably leads to relatively low levels of Yps1p, and thus, Yps2p carries out the bulk of the processing. Crh2-HA induces the UPR more strongly, and consequently, Yps1p is available to process Crh2-HA. Taken together with previous studies of yapsin function (14
) in S. cerevisiae
as well as in the closely related pathogenic yeast C. glabrata
), the yapsins appear to mediate the release of proteins from the surface of yeast cells during a variety of cellular process, including cell wall remodeling, cell surface-initiated signaling, and protein quality control.
A number of groups have found that the presence of yapsins and their homologues adversely affects the production of heterologously expressed, recombinant proteins in S. cerevisiae
and Pichia pastoris
) by causing a large amount of degraded protein. Our data implicating yapsins in secretory pathway quality control provide a compelling physiological explanation for these observations. Yps1p has been found to be particularly deleterious to recombinant protein production, and its presence leads to the degradation of a variety of recombinant proteins (5
). The regulation of YPS1
expression by the UPR suggests that heterologous or high-level expression could activate the UPR, leading to increased expression levels of YPS1
and, ultimately, to an increased degradation of the secreted protein. Recent results indicating that Yps2p also contributes to the degradation of some recombinant proteins (10
) are also consistent with our findings that Yps1p and Yps2p both have a role in secretory pathway homeostasis.
In summary, we have provided evidence for a previously unrecognized, UPR-mediated pathway by which an ER-stress-inducing GPI-linked protein is secreted from the cell. We propose that this pathway represents a mechanism by which the cell removes excess or aberrant proteins from the cell when other pathways for degradation are saturated or inoperative. In addition, we have identified a new substrate for the yapsins and provided evidence supporting a role for this family of proteins in protein quality control. Studies designed to further characterize the types of proteins secreted to the extracellular space by this pathway as well as the components of the secretory pathway that mediate this secretion are in progress and will be reported in due course.