Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling. https://doi.org/10.7554/eLife.05031.001 eLife digest Proteins are built based on instructions in template molecules called messenger RNAs (or mRNAs), which are copied from the DNA of genes. As they are made, proteins must fold into a specific three-dimensional shape and some proteins pass into a compartment in the cell, called the endoplasmic reticulum, in which they fold. So-called molecular chaperone proteins assist this folding process. From the endoplasmic reticulum, most proteins travel to other destinations within or outside of the cell. If the molecular chaperones in the endoplasmic reticulum are overwhelmed by their protein folding task, unfolded proteins accumulate; a situation that can be harmful to the cell. In eukaryotic cells including yeast, a sensor protein called Ire1 detects when unfolded proteins build up in the endoplasmic reticulum. As a result, the Ire1 sensor proteins join together to form clusters and an mRNA molecule called HAC1 is specifically recruited to the Ire1 clusters. The portions of the Ire1 protein that extend out from the endoplasmic reticulum into the cell proper then bind to HAC1 mRNA and cut a piece out of it. This edited mRNA encodes the instructions to build a protein that in turn boosts the expression of various components—including the appropriate molecular chaperones—that are needed to alleviate the stress caused by an excess of unfolded proteins. Within clusters, individual Ire1 proteins interact through the portions of the protein found on the inside of the endoplasmic reticulum. Now, van Anken et al. show that these interactions are sufficient for forming and maintaining clusters. The interactions between the portions of the Ire1 proteins outside of the endoplasmic reticulum are needed for editing the HAC1 mRNA but not for forming and maintaining the clusters or for recruiting the HAC1 mRNA molecule to bind to Ire1. Instead, van Anken et al. discovered an mRNA binding site on the Ire1 clusters, which is separate from the part of the Ire1 protein that cuts the mRNA molecules. The Ire1 protein needs to first bind the HAC1 mRNA molecule at this binding site before it can cut it; van Anken et al. suggest that this two-step process helps ensure accurate and efficient editing of the HAC1 mRNA by Ire1. This process could also help to minimize the chance of other mRNA molecules being edited by mistake. It will be of interest to investigate if similar safety measures are key for endoplasmic reticulum stress signaling mechanisms in humans, and whether these newly discovered steps can be targeted by drugs to treat disease. https://doi.org/10.7554/eLife.05031.002 Introduction Proteins that travel along the secretory pathway first enter the lumen of the endoplasmic reticulum (ER) as unfolded polypeptides. Assisted by ER-resident enzymes, they undergo oxidative folding, modification, and assembly reactions. When properly folded, they are packaged into ER exit vesicles and travel to their final destination in the endomembrane system, on the cell surface or, after secretion, outside of the cell. Proteins that do not reach maturity are degraded by the proteasome after retrotranslocation into the cytosol (via ER-associated degradation) or by autophagy (Ellgaard and Helenius, 2003; van Anken and Braakman, 2005; Bernales et al., 2006). Homeostasis in ER protein folding is achieved by fine-tuning the balance between the protein folding load and the protein folding capacity in the ER lumen (Mori, 2009; Kimata and Kohno, 2011; Walter and Ron, 2011). In yeast, Ire1 is the only known ER-stress sensor that responds to an accumulation of misfolded proteins in the ER lumen and transduces this information across the ER membrane. On the cytosolic side, Ire1 activation results in the non-conventional splicing of HAC1 mRNA, which is cleaved by Ire1's RNase domain at two splice sites, releasing a single intron (Sidrauski and Walter, 1997). Upon ligation of the severed exons, the spliced mRNA is translated to produce the Hac1 transcription activator that drives expression of UPR target genes to mitigate ER-stress (Travers et al., 2000; Walter and Ron, 2011). Ire1 is activated through higher-order oligomerization (Kimata et al., 2007; Aragón et al., 2009; Korennykh et al., 2009). Two ER-lumenal domain (LD) interfaces, IF1L and IF2L ('L' for lumenal), which were identified in the crystal structure of yeast Ire1 LD and validated by mutagenesis, mediate oligomeric assembly of the LD (Credle et al., 2005; Kimata et al., 2007; Aragón et al., 2009; Gardner and Walter, 2011). Dimerization via IF1L yields a composite groove extending across the LDs of two Ire1 molecules (Credle et al., 2005). Unfolded stretches of polypeptides bind within this groove of Ire1 LD, causing its oligomerization in vitro (Gardner and Walter, 2011). Proximal activation of the UPR in vivo coincides with the dissociation of Kar2 (the yeast homolog of the ER-lumenal hsp70 chaperone BiP) from Ire1 (Kimata et al., 2004; Pincus et al., 2010; Walter and Ron, 2011). Yet, Ire1 mutants with impaired Kar2 binding still respond to ER-stress, although the threshold for activation is lowered (Kimata et al., 2004; Pincus et al., 2010; Walter and Ron, 2011). Thus, misfolded proteins likely are direct ligands that activate Ire1, while Kar2 fine-tunes the signaling (Pincus et al., 2010; Gardner and Walter, 2011). On the cytosolic side of the ER membrane, Ire1 contains both a kinase and an RNase domain, which are tethered to the transmembrane domain via a linker (Mori, 2009; Kimata and Kohno, 2011; Walter and Ron, 2011). Three cytosolic assembly interfaces, IF1C, IF2C, and IF3C ('C' for cytosolic), were identified from the crystal structures of Ire1 kinase/RNase oligomers. IF1C creates back-to-back dimers of the kinase/RNase domains (Lee et al., 2008; Korennykh et al., 2009) that stack onto each other with an axial rotation via IF2C and IF3C to form filaments with a helical arrangement of seven Ire1 dimers per turn (Korennykh et al., 2009; Walter and Ron, 2011). The lumenal and cytosolic domain filaments predicted by the crystal structures have a different pitch and thus for steric reasons cannot be collinear. Instead, a two-dimensional arrangement of the two filaments, featuring ∼20–30 Ire1 molecules, provides a model for the higher-order assembly in vivo (Korennykh et al., 2009; Figure 1B). This model is compatible with the size of Ire1 foci observed by fluorescence microscopy (Aragón et al., 2009) and is sterically feasible despite the twists of the filaments on either side of the planar membrane, owing to the flexibility and length (>100 Å) of the linker domains on either side of the membrane, which can relieve the strain. Alternatively and not mutually exclusive, Ire1 clusters may be dynamic, such that constant rearrangements of the Ire1 molecules in clusters sustain transient intermittent oligomerization events on either side of the membrane. Figure 1 Download asset Open asset Oligomerization of Ire1's cytosolic domain is required for UPR signaling but not for Ire1 cluster formation or HAC1 mRNA recruitment. (A) Schematic of S. cerevisiae Ire1. The ER-lumenal portion of Ire1 is divided in an N-terminal domain (I, gray), a core lumenal—ER-stress-sensing—domain (cLD, light blue), and BiP binding domain (V, dark green), which is tethered via a transmembrane (TM, orange) stretch to Ire1's cytosolic portion that is composed of a linker (L, brown), a kinase (K, ochre), and an RNase (R, purple) domain (Walter and Ron, 2011). The activation loop (light green) and the αF–αEF (pink) loop protrude from the kinase domain (Lee et al., 2008; Korennykh et al., 2009). (B) A model architecture of a 24mer Ire1 cluster after oligomerization on either side of the ER membrane. Left: oligomerization via ER-lumenal interfaces IF1L (tan) and IF2L (steel blue) (top) and via cytosolic interfaces IF1C (indian red), IF2C (sea green), and IF3C (plum) (bottom). The 24 Ire1 molecules are labeled (A–H) A′–H′, and A′′–H′′. IF1C-mediated back-to-back dimers are between A & B and C & D, etc. IF2C, is formed between Ire1 molecules A and D, C and F, and so on. The third interface, IF3C, is stabilized by a phosphate in the activation loop resulting from Ire1 trans-autophosphorylation. Dimerization via IF3C is therefore directional from B → D → F and from E → C → A, etc. (Korennykh et al., 2009). Right: three-dimensional rendering of the same 24 Ire1 molecules colored as in (A). (C) Top: schematic of HAC1 mRNA. The HAC1 open reading frame (ORF) is divided into two exons (black). The intron (gray) base pairs with the 5′ UTR (gray), causing stalling of ribosomes. Ire1 cleaves the intron at the splice sites indicated by blue arrowheads. The 3′ UTR (gray) harbors a stem-loop structure with the 3′ BE (red) that facilitates recruitment of the HAC1 mRNA to Ire1 foci (Aragón et al., 2009). The 5′ m7G cap (•) and polyadenylation (polyA) signal are indicated. Middle: the green bar depicts the GFP ORF (green) that replaces part of the HAC1 sequence in the splicing reporter (SpR). Translation of GFP only occurs when the intron is spliced from the mRNA, because removal of the intron by Ire1's endonuclease activity lifts a translational block caused by base pairing between the intron and the 5′ UTR (Pincus et al., 2010). Bottom: 16 U1A binding sites (violet) were inserted into the 3′ UTR of the SpR mRNA, bearing the non-fluorescent GFP-R96A mutant (GFP*, gray), downstream of the 3′ BE containing stem-loop. Binding of GFP-tagged U1A protein allows visualization of the mRNA (Aragón et al., 2009). (D) Wild-type (WT) or ire1Δ cells, having a genomic copy of the SpR, were complemented with centromeric empty vector or bearing ire1 IF mutant alleles (Aragón et al., 2009; Korennykh et al., 2009) as indicated. Top: SpR assay of cells. GFP fluorescence for 10,000 cells was measured by FACS analysis before or after ER-stress induction with 2 mM DTT for 2 hr, as described (Pincus et al., 2010); mean and s.d. are shown (n = 2). Bar diagrams for IF mutants are color-coded as in (B) left. The signal of DTT treated WT was set at 100%, while the signal reached in DTT treated ire1Δ cells due to auto-fluorescence of 14% was set as background (light gray bar). Statistical significance in a Student's t-test of differences in splicing levels as compared with wild-type is indicated (*p ≤ 0.05; **p ≤ 0.01). Bottom: viability assay by 1:5 serial dilutions spotted onto solid media with 0.2 µg ml−1 of the ER-stress-inducer tunicamycin. Plates were photographed after 2–3 days at 30°C. (E) Localization of Ire1–GFP WT or IFL mutants before (left panel, control) and after (right panels, DTT) induction of ER-stress. (F) Schematic of Ire1–GFP and Ire1–mCherry with the fluorescent modules placed in the juxtamembrane region of the cytosolic linker. (G) Localization of Ire1–mCherry WT or IFC mutants, as well as SpRU1A mRNA decorated with U1A–GFP, after induction of ER-stress with DTT. (E, G) ER-stress was induced with 10 mM DTT for 45 min; imaging was performed in ire1Δ cells, complemented with Ire1 imaging constructs, as described (Aragón et al., 2009). Scale bars represent 5 µm. (H) Immunoblot of hemagglutinin (HA)-tagged Ire1 protein from lysates from strains as in panel (D) and (G). A sample from a strain that overexpressed HA-tagged Ire1 from a 2 µ plasmid served as a positive reference. Ire1 is denoted by an arrowhead. A background band, denoted by an asterisk (*), conveniently serves as a loading reference. (I) Bar diagrams depict the percentage of Ire1 signal in foci (red bars) and the co-localization index expressed in arbitrary units (yellow bars), as described (Aragón et al., 2009), for SpRU1A mRNA recruitment to foci of Ire1 variants shown in (G); mean and s.e.m. are shown, n = 5–8. There is no statistical significance in a Student's t-test of differences in foci formation and mRNA recruitments as compared with wild-type. https://doi.org/10.7554/eLife.05031.003 Figure 1—Source data 1 (A) Source data for Figure 1D and Figure 1H. https://doi.org/10.7554/eLife.05031.004 Download elife-05031-fig1-data1-v1.xlsx We previously have shown that Ire1 oligomerization allows selective recruitment of unspliced HAC1 mRNA to Ire1 clusters by virtue of a bipartite element in HAC1's 3′ untranslated region (UTR) (Aragón et al., 2009), which we named the 3′ BE. The 3′ BE is effective in targeting mRNA to Ire1 clusters as long as they are translationally repressed (Aragón et al., 2009). Stalling of HAC1 mRNA translation is afforded through base pairing between the intron and the 5′ UTR (Rüegsegger et al., 2001). Moreover, the in vitro endonuclease activity of Ire1 kinase/RNase domains is highly cooperative, indicating that oligomerization rather than dimerization leads to RNase activation (Korennykh et al., 2009). Intriguingly, the capacity of the kinase/RNase domains to oligomerize in vitro depends on a short stretch of the cytosolic linker that extends N-terminally from the kinase domain (Korennykh et al., 2009). In this study, we report that although oligomeric assembly of kinase/RNase domains is essential for activation of Ire1's in vivo mRNA processing capacity, it is not required for driving formation and maintenance of Ire1 clusters or for recruitment of its mRNA substrate. Instead, we discovered that a conserved positively charged element in Ire1's cytosolic linker mediates mRNA docking onto Ire1 clusters. Primary docking of the mRNA to this site is required for subsequent processing of the mRNA by Ire1's RNase domain. The staged way in which HAC1 mRNA is channeled to become subject to Ire1's endonuclease activity enhances efficiency and selectivity in the process and, thus, fidelity in UPR signaling. Results Oligomeric assembly of the kinase/RNase domains is essential for Ire1 function but not for its clustering or for HAC1 mRNA recruitment Efficiency of HAC1 mRNA splicing (Figure 1C) depends on clustering of ER-lumenal domains in vivo (Kimata et al., 2007; Aragón et al., 2009) and of cytosolic domains in vitro (Korennykh et al., 2009). To assess the contribution of cytosolic oligomerization events to Ire1 function in vivo, we analyzed mutations in the interfaces that govern Ire1 kinase/RNase oligomeric assembly by complementing ire1Δ yeast with centromeric plasmids. Driven by its autologous promoter, expression of (wild-type or mutant) Ire1 from these plasmids is at near-endogenous levels (Aragón et al., 2009). Disruption of IF1C abolished RNase function (Figure 1D) as monitored by the loss of expression of a fluorescent reporter protein (SpR; Figure 1C), whose levels report on Ire1 RNase activity (Pincus et al., 2010). Consequently, growth under ER-stress conditions was impaired (Figure 1D), as previously reported (Lee et al., 2008). Disruption of IF2C likewise disrupted RNase function and survival under ER-stress (Figure 1D), consistent with in vitro analyses (Korennykh et al., 2009). Mutations in IF3C led to a milder phenotype, sustaining intermediate levels of splicing and growth (Figure 1D), similar to the lumenal interface mutants that are shown for comparison (Aragón et al., 2009; Figure 1D). As expected, mutations in Ire1 did not affect growth under non-ER-stress conditions since growth of ire1Δ yeast is then also unaffected (Aragón et al., 2009). Under control conditions, Ire1 distributed diffusely throughout the ER but clustered into discrete foci upon ER-stress as visualized with fluorescently tagged Ire1–GFP (Aragón et al., 2009; Figure 1E,F). HAC1 mRNA is recruited to Ire1 foci under ER-stress via the 3′ BE targeting signal in the mRNA (Aragón et al., 2009; Figure 1C), which can be visualized in cells expressing HAC1 splicing reporter mRNA containing U1A binding sites (SpRU1A, Figure 1C) and GFP-tagged U1A RNA-binding protein (Brodsky and Silver, 2000; Aragón et al., 2009). Disruption of lumenal interfaces interfered with Ire1 clustering (Kimata et al., 2007; Aragón et al., 2009; Figure 1E) and, consequently, mRNA recruitment (Aragón et al., 2009). Disruption of cytosolic interfaces also compromised splicing activity of Ire1 and, hence, growth under ER-stress conditions (Figure 1D), but foci formation was unaffected (Figure 1G). Moreover, expression levels of the IFC mutants were comparable to wild-type (Figure 1H), and the percentage of Ire1 in foci as well as the extent of co-localization of mRNA at those foci, as determined by a customized MatLab script (Aragón et al., 2009), was similar for wild-type and IFC mutants (Figure 1G,I). Thus, all three cytosolic oligomeric interfaces are key for Ire1 function in vivo but not for Ire1 stability or its capacity to cluster and recruit HAC1 mRNA. The kinase and RNase domains are dispensable for Ire1 clustering and mRNA recruitment Tampering with the oligomeric assembly of the cytosolic domains of Ire1 gravely affected Ire1's endonuclease activity. To explore directly whether enzymatic activity of Ire1 is necessary for foci formation, we extended our assays using site-specific mutations that selectively disrupt Ire1's kinase and RNase activities ('KD' and 'RD' for kinase- and RNase-deficient, respectively). In line with previous results (Papa et al., 2003; Korennykh et al., 2011), splicing and survival under ER-stress were impaired in ire1(KD) and abolished in ire1(RD) mutant cells, while mutant Ire1 expression levels were similar to wild-type (Figure 2A). Yet, Ire1 foci formation and mRNA recruitment were unimpeded in either mutant (Figure 2B,F), indicating that neither of the enzymatic activity is required for Ire1 to recruit its mRNA substrate. Figure 2 Download asset Open asset The kinase and RNase domains of Ire1 are dispensable for foci formation and mRNA recruitment. (A) Splicing reporter assay before or after ER-stress induction with 2 mM DTT for 2 hr (top), Western blot of Ire1 (middle), and viability assay under ER-stress conditions (0.2 µg ml−1 tunicamycin; bottom) were performed in ire1Δ yeast containing a genomic copy of the SpR, complemented with wild-type (WT), kinase dead (KD), RNase dead (RD), and RNase truncation (ΔR) mutant alleles of ire1. Maximal (100%) and background level (14%, light gray bar) fluorescence are set as in Figure 1A. Mean and s.d. are shown (n = 2). Statistical significance in a Student's t-test of differences in splicing levels as compared with wild-type is indicated (**p ≤ 0.01). The arrowheads denote (mutant or truncated) Ire1 protein and the asterisk a background band on the immunoblot as in Figure 1H. (B, C) Top: schematic of the mCherry-tagged versions of the same ire1 mutants as in (A) as well as a kinase/RNase truncation (ΔKR) mutant, color-coded as in Figure 1A, except defective domains are black. (D) Schematic of a chimeric mRNA, SL-PGK1-3′ hac1U1A, which is PGK1U1A, bearing in its 3′ UTR the stem-loop structure with the 3′ BE of the HAC1 mRNA and in its 5′ UTR a small stem-loop (green) that confers translational repression (Aragón et al., 2009). (B, C, E) Localization of Ire1–mCherry and of U1A–GFP decorating either SpRU1A (B, C) or SL-PGK1-3′ hac1U1A (E) mRNA. ER-stress was induced with 10 mM DTT for 45 min; imaging was performed of ire1Δ cells, complemented with Ire1 imaging constructs, as depicted. Scale bars represent 5 µm. (F) Bar diagrams depict the percentage of Ire1 signal in foci (red bars) and the co-localization index for mRNA recruitment into foci of Ire1 variants shown in B, C, and E (mean and s.e.m., n = 5–10). There is no statistical significance in a Student's t-test of differences in foci formation and mRNA recruitments as compared with wild-type. https://doi.org/10.7554/eLife.05031.005 Figure 2—Source data 1 (A) Source data for Figure 2A and Figure 2F. https://doi.org/10.7554/eLife.05031.006 Download elife-05031-fig2-data1-v1.xlsx Strikingly, even removal of the entire RNase domain, either alone ('ΔR') or together with the kinase domain ('ΔKR'), left Ire1 foci formation and mRNA recruitment unaffected (Figure 2C,F, Figure 2—source data 1). These results show that oligomeric assembly of the kinase–RNase domain is dispensable for the Ire1 clustering. Moreover, these findings indicate that Ire1 must harbor an mRNA docking site within the linker that tethers the kinase/RNase to the transmembrane region because it is the only remaining cytosolic portion in the ire1(ΔKR) mutant. A heterologous mRNA, SL-PGK-3′ hac1U1A—which contains the 3′ BE of HAC1 mRNA, but lacks the HAC1 mRNA intron and splice sites, and a small stem-loop in its 5′ UTR to repress its translation (Aragón et al., 2009; and Figure 2D)—was also efficiently recruited to foci in ire1(ΔKR) mutant cells (Figure 2E,F, Figure 2—source data 1). These results were surprising, since mRNA recruitment to Ire1 serves to engage the splice sites in the HAC1 mRNA with Ire1's endonuclease domain for cleavage, yet neither the endonuclease domain nor the splice sites (or their context) are required for mRNA docking onto Ire1 clusters. Instead, our findings indicate that the core elements sufficient for the recruitment of HAC1 mRNA to and docking of the mRNA onto Ire1 clusters are contained in the 3′ BE of the mRNA and in Ire1's cytosolic linker domain. The cytosolic linker of Ire1 harbors a conserved positively charged motif that facilitates mRNA docking Ire1 is the only ER-stress sensor that is conserved in all eukaryotes. The kinase/RNase domains and the core lumenal ER-stress sensing domain are conserved, but other domains, including the cytosolic linker domain, show negligible sequence conservation (Figure 3A). The linker greatly varies in length between species but consistently harbors an unusually high number of basic and acidic residues. In fungal species, a short basic sequence stretch (henceforth referred to as '[+]-box') displays recognizable homology. In particular, sequence alignment reveals strict conservation of one lysine and two arginine residues as well as three glycine residues that intersperse the basic residues. The [+]-box is flanked on either one or both sides by acidic sequences. Figure 3 Download asset Open asset The cytosolic linker of Ire1 harbors a positively charged motif that is key for mRNA recruitment and splicing. (A) Conservation of Ire1. Top: mapped onto a schematic of Ire1 domains bordered by residues of which the number is denoted, bar diagrams display relative conservation of the Saccharomyces cerevisiae Ire1 protein sequence to homologs (lower, left) from other fungal species Kluyveromyces lactis, Candida glabrata, Aspergillus nidulans, Coccidioides posadasii, Gibberella zeae, Magnaporthe grisea, Neurospora crassa, and Schizosaccharomyces pombe, as well as from the animals Caenorhabditis elegans and Drosophila melanogaster, the two paralogues from the plant Arabidopsis thaliana (a and b) and from Homo sapiens (α and β). Domains are color-coded as in Figure 1A, except signal peptides (SP) are black; light green represents a loop inserted into the kinase domain of the A. thaliana Ire1s and crimson denotes C-terminal extensions in animal Ire1s. Expanded view (middle) of the linker domains that are aligned based on the stretch (gray box) for which the sequence alignment is shown on the right. Strictly conserved residues among fungal species except S. pombe are boxed. (A) lower right, (C) top, Basic (arginine and lysine) residues are shown in blue and acidic (aspartate and glutamate) residues in red. Glutamines replacing arginines and lysines in the Q-box mutant are black. (A) lower right, (B, C) Position of the [+]-box is indicated. (B, D–F) Localization of Ire1–GFP or Ire1–mCherry and of U1A–GFP decorated SpRU1A mRNA. Imaging was performed in ire1Δ yeast complemented with a genomic copy of C-terminally mCherry-tagged (ΔKR) or (ΔKR/Δ[+]-box) ire1 mutant alleles, as schematically shown (B top right), or with plasmids encoding IRE1 wild-type (C) or ire1 linker mutants (E, F), with the fluorescent modules GFP (E) and mCherry (C, F) placed in the αF–αEF loop, as schematically shown (D right), before (D upper panels, E, control) and after (B, D lower panels, F, DTT) induction of ER-stress with 10 mM DTT for 45 min. Scale bars represent 5 µm. Bar diagrams depict the percentage of Ire1 signal in foci (red bars) and the co-localization index for mRNA recruitment into foci of Ire1 variants (mean and s.e.m., n = 5–10) (B, bottom right, G). Statistical significance in a Student's t-test of differences in foci formation and mRNA recruitments as compared with wild-type is indicated (*p ≤ 0.05; **p ≤ 0.01). (C) Schematic of linker domains with mutations or truncations as in (A) (top). Splicing reporter assay before or after ER-stress induction with 2 mM DTT for 2 hr (left, middle), Western blot of Ire1 (left, bottom), and viability assay under ER-stress conditions (0.2 µg ml−1 tunicamycin; right, bottom). Assays were performed in ire1Δ yeast containing a genomic copy of the SpR, complemented with either IRE1 wild-type or ire1 linker mutants with mCherry in the αF–αEF loop. Mean and s.d. are shown (n = 2). Maximal (100%) and background level (14%, light gray bar) fluorescence are set as in Figure 1A. Statistical significance in a Student's t-test of differences in splicing levels as compared with wild-type is indicated (**p ≤ 0.01). The arrowheads denote (mutant or truncated) Ire1 protein and the asterisk a background band on the immunoblot as in Figure 1H. https://doi.org/10.7554/eLife.05031.007 Figure 3—Source data 1 (A) Source data for Figure 3B, Figure 3C and Figure 3G. https://doi.org/10.7554/eLife.05031.008 Download elife-05031-fig3-data1-v1.xlsx All fungal species with a [+]-box containing Ire1 linker have a conserved 3′ BE in their HAC1 mRNAs (Aragón et al., 2009). The only fungal species we found with a markedly divergent basic motif is Schizosaccharomyces pombe, which lacks a HAC1 gene altogether (Kimmig et al., 2012). In both Ire1 paralogs of Arabidopsis thaliana, the linkers harbor a basic motif that diverges from the fungal [+]-box (Figure 3A), but that motif is conserved among plants (not shown). Conversely, animal species lack any such recognizable motif (Figure 3A). Indicative of an important role for the [+]-box in UPR signaling is that it adorns the short linker extension, which facilitated oligomerization and markedly enhanced endonuclease activity of recombinant kinase/RNase domains in vitro (Korennykh et al., 2009). To analyze the role for the [+]-box in Ire1 function in vivo, we first truncated Ire1 further such that the [+]-box was deleted (Figure 3B). Since we could not obtain a centromeric plasmid of this construct, as it was toxic for Escherichia coli, we created ire1Δ strains with a genomic copy of the ire1ΔKR or ire1ΔKR/Δ[+]-box mCherry-tagged transgenes in the LEU2 locus. As expected, Ire1 clustering and mRNA recruitment were still at wild-type levels for ire1ΔKR when genomically integrated (Figure 3B), similar to what we observed when ire1ΔKR was expressed from a centromeric plasmid (Figure 2C,F). Further removal of the [+]-box did not affect Ire1 clustering but markedly reduced mRNA recruitment (Figure 3B). This finding suggests that the [+]-box is key for the docking of mRNA onto Ire1 clusters. Moreover, given that the extent of clustering of the ire1 (ΔKR/Δ[+]-box) mutant was similar to wild-type, we conclude that Ire1 clustering is driven by the lumenal domain alone: neither cytosolic oligomeric assembly (as facilitated by the kinase/RNase domains) nor mRNA docking (as facilitated by the [+]-box) is required for Ire1 foci formation. Next, we set out to explore the role of the [+]-box in the context of the full-length Ire1 using the mutants and truncations depicted in Figure 3C. The experiments presented so far employed Ire1 variants bearing GFP or mCherry modules in the linker (Aragón et al., 2009; Pincus et al., 2010; Rubio et al., 2011; Figure 1F; Figure 2B,C). To avoid interference with mutations in the linker, we relocated the fluorescent modules to the αF–αEF loop in the Ire1 kinase domain (Figure 3D). By contrast to the activation loop (Figure 1A), which becomes hyperphosphorylated due to Ire1's kinase activity, the αF–αEF loop is poorly conserved (Figure 3A) and dispensable for Ire1 activity in vit
Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly investigated, as in most studies the ER stress that is elicited cannot be overcome. Here we show that a proteostatic insult, provoked by persistent expression of the secretory heavy chain of immunoglobulin M (µs), is well-tolerated in HeLa cells. Upon µs expression, its levels temporarily eclipse those of the ER chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER stress in a ratiometric fashion. In this process, the ER expands about three-fold and becomes dominated by BiP. As the UPR is essential for successful ER homeostatic readjustment in the HeLa-µs model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER stress.
ABSTRACT Thioneins are cysteine-rich apoproteins that regulate divalent metal homeostasis by virtue of their metal-chelation properties resulting in the ligand-bound metallothionein state. Previous studies show transient upregulation of the metallothionein (MT) gene cluster as part of a complex transcriptional response to a class of histone demethylase tool compounds targeting human Fe 2+ dependent ketoglutarate oxygenases KDM6A (UTX) and KDM6B (JmjD3). The prototypic bioactive KDM6 inhibitor GSK-J4 induces apoptotic cell death in multiple myeloma cells and corresponding transcriptomic profiles are dominated by metal and metabolic stress response signatures, also observed in primary human myeloma cells. Here we investigate the hypothesis that metal-chelation by GSK-J4 provides the means for transport and intracellular release of Zn 2+ leading to a metallothionein transcriptomic response signature. Live cell imaging of myeloma cells shows transient increases in intracellular free Zn 2+ concentrations when exposed to GSK-J4, consistent with a model of inhibitor-mediated metal transport. Comparisons of GSK-J4 and ZnSO 4 treatments in the presence or absence of metal chelators show that both treatment conditions induce different transcription factor repertoires with an overlapping MTF1 transcriptional regulation responsible for metallothionein and metal ion transport regulation. The data provide a possible explanation for the observed metal response upon GSK-J4 inhibition however the relationship with the pro-apoptotic mechanism in myeloma cells requires further investigation.
Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.
Abstract Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 different stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.
Endoplasmic-reticulum-associated protein degradation
How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible bulk expression of secretory immunoglobulin M heavy chain (μs) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µs is restored lead to µs-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (CH1) from µs. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem.
Endoplasmic-reticulum-associated protein degradation
Article29 January 2018Open Access Source DataTransparent process ERAD-dependent control of the Wnt secretory factor Evi Kathrin Glaeser Kathrin Glaeser Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Manuela Urban Manuela Urban Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Emma Fenech Emma Fenech Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Oksana Voloshanenko Oksana Voloshanenko Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Dominique Kranz Dominique Kranz Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Federica Lari Federica Lari Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author John C Christianson John C Christianson orcid.org/0000-0002-0474-1207 Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Michael Boutros Corresponding Author Michael Boutros [email protected] orcid.org/0000-0002-9458-817X Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Kathrin Glaeser Kathrin Glaeser Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Manuela Urban Manuela Urban Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Emma Fenech Emma Fenech Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Oksana Voloshanenko Oksana Voloshanenko Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Dominique Kranz Dominique Kranz Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Federica Lari Federica Lari Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author John C Christianson John C Christianson orcid.org/0000-0002-0474-1207 Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Michael Boutros Corresponding Author Michael Boutros [email protected] orcid.org/0000-0002-9458-817X Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany Search for more papers by this author Author Information Kathrin Glaeser1, Manuela Urban1, Emma Fenech2, Oksana Voloshanenko1, Dominique Kranz1, Federica Lari2, John C Christianson2 and Michael Boutros *,1 1Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany 2Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK *Corresponding author. Tel: +49 6221 421950; E-mail [email protected] The EMBO Journal (2018)37:e97311https://doi.org/10.15252/embj.201797311 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β-catenin by the ubiquitin-proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)-associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP-dependent manner. Ubiquitination of Evi involves the E2-conjugating enzyme UBE2J2 and the E3-ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD-dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions. Synopsis Wnt palmitoylation by O-acetyltransferase Porcupine (PORCN) along the secretory pathway regulates the stability of the Wnt secretory factor Evi/Wls by protecting Evi/Wls from ubiquitination and degradation via the ERAD pathway in the endoplasmic reticulum. An increase in palmitoylated Wnt levels leads to Evi/Wls protein stabilization. In the absence of Wnt proteins, Evi/Wls is ubiquitinated by the E2-conjugating enzyme UBE2J2 and the ubiquitin E3 ligase CGRRF1 and proteasomally degraded. An ERAD triaging complex consisting of PORCN and VCP/p97 regulates the levels of the Wnt cargo receptor Evi/Wls. In turn, higher Evi/Wls levels in the endoplasmic reticulum promote Wnt secretion. Introduction Post-translational protein regulation is an important mechanism that provides cells with rapid responses to changes in intra-and extracellular conditions (Lee & Yaffe, 2016). Steady-state protein levels are controlled by changes in transcription and translation as well as by their intrinsic stability, which is affected by folding state, localization, and co-assembly with partner proteins. For proteins of the secretory pathway, endoplasmic reticulum (ER)-associated degradation (ERAD) is a key cellular process to ensure protein quality and quantity control (Hegde & Ploegh, 2010). ERAD is a specialized ubiquitin-proteasome system (UPS)-dependent process that prevents the secretion and aggregation of proteins that failed to fold or assemble appropriately (Vembar & Brodsky, 2008). Following coordinated recognition and delivery to membrane-embedded ubiquitination machineries, ERAD substrates are retrotranslocated across the ER membrane into the cytosol through the AAA-ATPase VCP (also known as p97) and degraded by 26S proteasomes (Ye et al, 2001; Vembar & Brodsky, 2008; Smith et al, 2011; Olzmann et al, 2015). Since ERAD-dependent quality control has been linked to a range of cellular processes and human diseases (Zettl et al, 2011; Guerriero & Brodsky, 2012; Perrody et al, 2016), understanding its molecular mechanisms is an important step to develop potential treatment strategies (Tsai & Weissman, 2010; Hetz et al, 2013). In addition to its role in protein quality control of nascent polypeptides, ERAD has been implicated in regulating the abundance of mature proteins in response to changes in physiological conditions (Wiertz et al, 1996a,b; Sever et al, 2003; Brodsky & Fisher, 2008; Adle et al, 2009; Foresti et al, 2013; Avci et al, 2014; van den Boomen et al, 2014). However, only a few regulatory ERAD substrates have been described so far. Given the number of ubiquitin ligases present in the ER (Neutzner et al, 2011), the physiological role of regulatory ERAD is likely underestimated at present. Wnt signaling is crucial during development and adult tissue homeostasis (Logan & Nusse, 2004), and its aberrant regulation has been linked to many diseases, including cancer (Clevers & Nusse, 2012; Zhan et al, 2017). Nineteen Wnt ligands are produced and subsequently processed in the endoplasmic reticulum (ER) by the attachment of palmitoleic acid to a conserved serine residue by the acyltransferase Porcupine (Porcn) (Kadowaki et al, 1996; Willert et al, 2003; Takada et al, 2006). In the Wnt secretory pathway, this lipid modification is important for the interaction between Wnt proteins and the conserved transmembrane protein Evi (Wls, GPR177), which shuttles lipid-modified Wnt proteins to the plasma membrane and onto exovesicles (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006; Gross et al, 2012; Herr & Basler, 2012). Evi is then recycled via the retromer complex back to the Golgi and ER (Coudreuse, 2006; Belenkaya et al, 2008; Franch-Marro et al, 2008; Pan et al, 2008; Port et al, 2008; Yang et al, 2008; Yu et al, 2014). The levels of both Wnt and Evi proteins are often aberrantly regulated in cancer (Augustin et al, 2012; Voloshanenko et al, 2013; Stewart et al, 2015), which contributes to high Wnt activity during tumorigenesis. Here, we identified a regulatory ERAD mechanism that continuously removes the Wnt shuttling receptor Evi from the ER in the absence of lipid-modified Wnt proteins. Poly-ubiquitination and degradation of Evi involve the E2 ubiquitin-conjugating enzyme UBE2J2, the ubiquitin ligase CGRRF1, and the AAA-ATPase VCP. Responding to Wnt protein availability in the ER, a triaging complex containing Porcn and VCP directs Evi either toward ERAD or the secretory pathway. Since expression of different Wnt proteins prevents ERAD-dependent degradation of Evi, our data demonstrate that Wnt-induced Evi stabilization automatically adjusts the amount of Evi to accommodate secretion of Wnt ligands. Control of Evi abundance through ERAD represents a cellular feedback mechanism to control Wnt protein secretion. Results Wnt proteins stabilize the cargo receptor Evi Increased Evi protein abundance coincides with elevated Wnt3 expression in colorectal cancer (Fig EV1A; TCGA, 2012; Voloshanenko et al, 2013). However, transcriptome profiles of 456 colon adenocarcinoma samples showed no increase in Evi mRNA levels (Fig EV1B; TCGA, 2012). Similarly, immunohistochemistry and in situ RNA hybridization carried out on sequential tissue sections found cases of colon carcinoma where increased Evi protein was not matched by a concomitant increase in Evi mRNA (Fig 1A; Appendix Fig S1A and B). These observations raised the question how Evi protein levels might be regulated under physiological conditions. Click here to expand this figure. Figure EV1. Evi is not transcriptionally regulated by Wnt A, B. FPKM-normalized RNA-seq. data of the TCGA Research Network (TCGA-COAD; http://cancergenome.nih.gov/; 09/25/2017) were log-transformed to illustrate the relative expression of (A) Wnt3 and (B) Evi in healthy colon (41) versus colon adenocarcinoma (456). The distribution into tumor and healthy samples was based on their barcodes as described in TCGA Wiki. Statistical significance of gene expression differences was determined using a Student's t-test under the alternative hypothesis H1 that gene expression is higher in tumors compared to healthy tissue. The boxplot diagram shows the median as line within the box, the 25th and 75th percentiles as the upper and lower part of the box, the 10th and 90th percentiles as error bars and outliers as circles. C. HEK293T cells were transfected with the indicated expression constructs, treated with 100 ng/ml recombinant mouse Wnt3A (rec. W3A) or with 10 μM GSK3 inhibitor SB216763 for the indicated hours (h). AXIN2 and Evi mRNA levels were analyzed by qRT–PCR and normalized to GAPDH expression. Results are shown as mean ± s.d. from three independent experiments. D, D′. Twenty-four hours after reverse transfection with Ctrl or CTNNB1 siRNA, HEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression plasmids and analyzed (D) for the indicated proteins via immunoblotting or (D′) for canonical Wnt activity using the TCF-Luciferase Wnt reporter assay. Immunoblotting is representative of three independent experiments, and Wnt reporter activity was calculated as mean from three independent experiments ± s.d. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Wnt ligand production increases Evi protein levels In situ RNA hybridization (red dots) and immunohistochemistry (brown staining) of Evi were performed on sequential FFPE tissue slides of healthy colon and matched colon cancer tissue from five patients. The illustrated example is representative for three patients. Scale bar: 40 μm. Specificity of Evi probes was confirmed in Appendix Fig S1B. Wild-type (wt) or EviKO HEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression plasmids and subjected to Western blot analysis. Specific Evi bands are indicated by arrows and unspecific bands by asterisks. Endogenous Evi is not only detectable as a monomeric form (46 kDa) but also as SDS-resistant dimers (80 kDa). Clonal EviKO HEK293T cells were generated via CRISPR/Cas9 using Evi sgRNA #2 (EviKO2.9) or Evi sgRNA #1 (EviKO1.1; Appendix Fig S2). HEK293T cells were transfected with Wnt expression plasmids and analyzed for endogenous Evi levels by immunoblotting with a mouse monoclonal Evi antibody (Biolegend, clone YJ5). HEK293T cells were transfected with the indicated overexpression constructs, treated with 100 ng/ml recombinant mouse Wnt3A (rec. W3A) or with 10 μM GSK3β inhibitor SB216763 for the indicated hours (h). The obtained cell lysates were used for Western blot analysis. Representative Western blots of three independent experiments are shown. β-Actin or N-cadherin were used as a loading control, and LRP6 served as a reference membrane protein. Scheme showing that Evi is regulated through Wnt proteins within the Wnt-producing cell. Canonical Wnt signaling can be activated by Wnt ligands, Dishevelled (Dvl) overexpression or by the GSK3β inhibitor SB216763. Source data are available online for this figure. Source Data for Figure 1 [embj201797311-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Since several Wnt proteins are upregulated in colorectal cancer (TCGA, 2012; Voloshanenko et al, 2013), we assumed that an increase in Wnt proteins might affect the levels of the cargo receptor Evi, allowing the cells to adjust to the molecular requirements for Wnt secretion. To test this hypothesis, we transiently increased Wnt levels in HEK293T cells and observed an elevation in Evi protein levels upon Wnt3A expression, but not upon expression of a secreted control protein, IGFBP5-V5 (Fig 1B). The Wnt-regulated protein bands were specific for Evi since they were absent in Evi knockout (EviKO) HEK293T cells (Fig 1B; Appendix Fig S2). Wnt-induced increase in protein abundance appeared to be selective for Evi since levels of the Wnt co-receptor LRP6 were not affected by Wnt3A expression (Fig 1B). The human genome encodes 19 different Wnt proteins (van Amerongen & Nusse, 2009) and so we asked next whether also other Wnts are capable to increase Evi protein levels. Expression of different canonical and non-canonical Wnt ligands strongly increased Evi protein levels, which was not observed upon expression of secreted luciferase (sLuc) or IGFBP5 (Fig 1C). Overexpression of Wnt11 led to only a modest increase in Evi levels. To address whether Wnt ligands elevated Evi levels by inducing the intracellular Wnt signaling cascade, we monitored Evi not only upon Wnt3A expression, but also upon activation of the Wnt cascade below the receptor level by expressing the Wnt activator Dishevelled2 (Dvl2) or by blocking GSK3β activity with the small molecule SB216763 (Fig 1E). While the Wnt target gene AXIN2 was upregulated under all conditions (Fig EV1C), Evi protein levels increased only upon Wnt3A expression (Fig 1D). These experiments suggest that the increase in Evi protein levels is specifically mediated by Wnt ligands and not by Wnt pathway activation (Fig 1E). Notably, exogenously provided recombinant Wnt3A was not sufficient to increase Evi protein levels (Fig 1D, lanes 5–8), indicating that cell-autonomous Wnt protein production is required. The Wnt-dependent increase in Evi protein abundance could be attributed to upregulated transcription of Evi. However, we did not observe changes in Evi mRNA levels upon Wnt3A expression (Fig EV1C), which is consistent with a lack of increased Evi expression in colorectal cancer (Figs 1A and EV1B). To confirm that the observed increase in Evi protein levels was not due to transcriptional changes upon canonical Wnt pathway activation, we monitored Evi levels following knockdown of β-catenin. Upon Wnt3A expression, β-catenin knockdown reduced canonical Wnt reporter activity to basal levels (Fig EV1D′) but did not affect the Wnt-induced increase in Evi protein (Fig EV1D). Taken together, these results support a model whereby Evi protein levels are post-transcriptionally regulated by the concomitant secretion of Wnt proteins (Fig 1E). Evi regulation depends on Wnt palmitoylation Wnt proteins are palmitoylated at a conserved serine residue by the ER-resident O-acyltransferase Porcn (Kadowaki et al, 1996; Willert et al, 2003; Takada et al, 2006), which is required for the Wnt proteins to interact with Evi (Herr & Basler, 2012) (Fig 2A). To determine whether Wnt palmitoylation and consequently Evi-Wnt interactions are required for the Wnt-mediated increase in Evi protein, we treated wild-type, Wnt3, or Wnt5B stable transfected HEK293T cells with the Porcn inhibitor LGK974 to block Wnt palmitoylation. Strikingly, the Wnt-induced increase in Evi protein levels was blocked upon Porcn inhibition, indicating that Porcn activity is required for Wnt-mediated Evi regulation (Fig 2B). In contrast to the Wnt target gene AXIN2, mRNA levels of Evi were not affected by LGK974 treatment indicating a post-transcriptional regulation of Evi (Fig EV2A). Figure 2. Evi stabilization is dependent on Wnt palmitoylation Schematic illustration of the Porcn-mediated Wnt palmitoylation, which is important for Evi-Wnt interaction and which is blocked upon Porcn inhibition (LGK974), in PorcnKO cells and by using a palmitoylation-deficient S209A Wnt3A mutant. Wild-type or stable Wnt3-and Wnt5B-expressing HEK293T cells were treated with 5 μM LGK974 for 48 h and subjected to Western blot analysis. Western blot analysis of endogenous Evi in wt, PorcnKO, or EviKO HEK293T cells upon overexpression of Wnt3A or IGFBP5-V5. PorcnKO1.2 and PorcnKO1.4 indicate clone #2 and clone #4 of PorcnKO HEK293T cells generated with Porcn sgRNA1 (Appendix Fig S3). Clonal EviKO HEK293T cells were generated with Evi sgRNA2 (EviKO2.9; clone #9) or Evi sgRNA1 (EviKO1.1; clone #1; Appendix Fig S2). Increase in total β-catenin protein served as control for Wnt pathway activation. Western blot analysis of endogenous Evi in HEK293T cells transfected with the indicated overexpression plasmids. When indicated, the cells were additionally treated with 5 μM LGK974 for 48 h. Western blot analysis of endogenous Evi in HCT116 or A375 cells treated with 5 μM LGK974 or DMSO for the indicated hours (h). All Western blots are representative of three independent experiments. β-Actin was used as a loading control, LRP6 as a reference membrane protein and EGF-Myc and IGFBP5-V5 as controls for secreted proteins. Specific Evi bands are indicated by arrows, and unspecific bands are marked by asterisks. Scheme: Wnt-induced Evi stabilization is blocked in the absence of Wnt palmitoylation (PorcnKO, LGK974, Wnt3A S209A). Source data are available online for this figure. Source Data for Figure 2 [embj201797311-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Porcn inhibition does not affect Evi transcription A. Wild-type or stable Wnt3- and Wnt5B-expressing HEK293T cells were treated with 5 μM LGK974 for 48 h, and AXIN2 and Evi mRNA levels were quantified by qRT–PCR. Results are shown as mean ± s.d. from three independent experiments. B. Wild-type (wt), PorcnKO1.2, and PorcnKO1.4 HEK293T cells were transfected with the indicated overexpression constructs and analyzed in Wnt Luciferase activity assays. Results are representative of three independent experiments and shown as mean of seven technical replicates ± s.d. C, D. HCT116 (C) or A375 cells (D) were treated with 5 μM LGK974 for the indicated hours (h). Evi mRNA was quantified by qRT–PCR and normalized to GAPDH. Results are shown as mean ± s.d. from three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint Porcn knockout (PorcnKO) HEK293T cells were generated (Appendix Fig S3) to confirm the importance of Porcn for Wnt-mediated Evi regulation. Unlike wild-type HEK293T cells, PorcnKO cells were not responsive to Wnt3A expression despite retaining intact Wnt signaling capability as confirmed by Wnt pathway activation upon Dvl3 overexpression (Fig EV2B). Using these cell lines, we found that Wnt3A expression led to an increase in Evi protein levels in wild-type but not in PorcnKO HEK293T cells (Fig 2C). To investigate whether the reduced steady-state levels of Evi upon Porcn inhibition are caused by the lack of Wnt palmitoylation, HEK293T cells were transfected with the palmitoylation mutant Wnt3A S209A and several wild-type Wnt3A constructs. All tested Wnt3A variants stabilized Evi with the exception of Wnt3A S209A, which produced a phenotype similar to that of Porcn inhibition (Fig 2D). To confirm the role of Porcn function in Evi regulation without manipulating Wnt expression, we monitored Evi in the colon cancer cell line HCT116, that depends on Wnt secretion (Voloshanenko et al, 2013) and in the melanoma cell line A375, that expresses Wnt5A and Wnt10B endogenously (Yang et al, 2012). LGK974 treatment of HCT116 and A375 cells reduced Evi protein levels in a time-dependent manner and depleted Evi below the detection limit after 24–48 h (Fig 2E) without reducing Evi mRNA levels (Fig EV2C and D). These results demonstrate that the palmitoylation of Wnts by Porcn is required for the Wnt-induced accumulation of Evi (Fig 2F). Evi is poly-ubiquitinated and degraded by the proteasome We next asked whether palmitoylated Wnt proteins increase Evi protein levels by altering its rate of degradation. For this purpose, Evi levels were monitored following impairment of the lysosomal and proteasomal degradation pathways. Blocking lysosomal degradation by treatment with the V-ATPase inhibitor Bafilomycin A increased Evi protein levels independent of Wnt3A expression (Fig EV3A). In contrast, inhibition of proteasomal degradation by treatment with the proteasome inhibitors MG132 (Fig 3A) or bortezomib (Fig EV3B) increased Evi protein levels in the absence of Wnt3A, while no additional increase was observed when Wnt3A was expressed. The presence of cell-autonomous Wnt proteins thus seems to be sufficient to prevent proteasome-dependent degradation of Evi, promoting its stability. Click here to expand this figure. Figure EV3. Evi is ubiquitinated and degraded from the ER by the proteasome HEK293T cells were transfected with the indicated expression constructs and treated with Bafilomycin A (Bafilo) in the indicated concentrations for 24 h. Lysosomal inhibition additionally increased Evi protein on top of Wnt3A expression indicating that Evi is degraded by the lysosome also in the presence of Wnt proteins. HEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression plasmids and treated for 24 h with DMSO, MG132, or bortezomib in the indicated concentrations. Following MG132 treatment (1 μM for 24 h), poly-ubiquitinated proteins were precipitated using TUBE2 agarose and incubated with DUB buffer, supplemented with the catalytic domain of the DUB enzyme USP2, if indicated. The precipitates were assayed for endogenous Evi or K48 poly-ubiquitin. Ctrl agarose beads were used to confirm specificity of the TUBE2 assay for ubiquitinated proteins. HEK293T cells were transfected with the indicated overexpression constructs and treated with 5 μM LGK974 for 48 h, if indicated. Secreted Wnt3A proteins were precipitated from conditioned medium and analyzed via immunoblotting. Compared to wild-type Wnt3A, Wnt3A-KDEL is not secreted into the medium affirming cellular retention. HSC70 served as loading control. All Western blots are representative of three independent experiments. Upon VCP knockdown in HEK293T cells using single or pooled siRNAs, Evi and VCP mRNA were analyzed via qRT–PCR. mRNA levels are shown as mean ± s.d. relative to GAPDH mRNA levels from three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint Figure 3. Evi poly-ubiquitination is regulated by the presence of Wnt proteins HEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression constructs and treated with MG132 at the indicated concentrations for 24 h. Cell lysates were analyzed for endogenous Evi by immunoblotting. Total β-catenin protein was used to assess MG132 efficiency. Wild-type (wt), stable Wnt3-expressing, or EviKO2.9 HEK293T cells were treated with 1 μM MG132 for 24 h. TUBE2 immunoprecipitates were assayed for endogenous Evi or K48 poly-ubiquitin by immunoblotting. To confirm specificity of the TUBE2 assay, Ctrl agarose beads were used as control. The asterisk at the β-actin blot indicates Wnt3A proteins blotted before membrane stripping. Scheme illustrating ubiquitination and proteasomal degradation of Evi, which is blocked in the presence of Wnt ligands. HEK293T cells were transfected with the indicated plasmids and additionally treated with 5 μM LGK974 for 48 h when indicated. In case of Wnt3A-KDEL, the ER-retaining sequence KDEL was C-terminally fused to Wnt3A. Dvl2-HA, Dvl3, and β-catenin-YFP overexpression was used as negative control to verify that Evi stabilization was not due to downstream activation of Wnt signaling. All Western blots are representative of three independent experiments. β-Actin was used as loading control. Specific Evi bands are marked by arrows and unspecific bands by asterisks. Source data are available online for this figure. Source Data for Figure 3 [embj201797311-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint Proteins targeted for proteasomal degradation are conventionally marked by covalent poly-ubiquitin chains. Using ubiquitin-affinity precipitation from lysates of MG132-treated cells, we were able to detect poly-ubiquitination of endogenous Evi (Fig 3B). Treatment with the deubiquitinating enzyme (DUB) Usp2 reduced the signal of poly-ubiquitinated Evi (Fig EV3C), confirming direct ubiquitin-modification of Evi. Notably, the amount of poly-ubiquitinated Evi was reduced upon Wnt3 expression (Fig 3B, lane 4), supporting the ability of Wnts to diminish the ubiquitination and thus proteasomal degradation of Evi (Fig 3C). Proteasomal degradation of integral membrane proteins like Evi in the early secretory pathway could be mediated through ERAD (Vembar & Brodsky, 2008). To ascertain whether Wnts stabilize Evi within the ER, we fused a KDEL retrieval sequence to Wnt3A (Wnt3A-KDEL) to restrict its trafficking (Fig EV3D) and monitored changes in Evi abundance. We found that the expression of ER-retained Wnt3A-KDEL stabilized Evi protein levels, which was blocked by LGK974 treatment (Fig 3D). These results confirm that palmitoylated Wnt proteins stabilize Evi within the ER. The AAA-ATPase VCP is required for Evi degradation Proteasome-dependent degradation from the ER in the absence of Wnt proteins suggests that Evi might be constitutively targeted for ERAD. To determine whether Evi is an endogenous ERAD substrate, we silenced the expression of several known ERAD components and monitored changes in Evi abundance. The AAA-ATPase VCP is an essential component of ERAD processing by promoting the dislocation of ERAD substrates across the ER membrane into the cytosol to allow proteasomal degradation (Ye et al, 2001; Fig 4A). Knockdown of VCP by both pooled and single siRNAs stabilized endogenous Evi protein without affecting its mRNA levels (Figs 4B and EV3E). Similarly, treating HEK293T cells with the specific allosteric VCP inhibitor NMS-873 (Magnaghi et al, 2013) stabilized Evi in a concentration-dependent manner (Fig 4C). Multiple Evi-immunoreactive bands at higher molecular weights in the absence of VCP activity are consistent with the accumulation of poly-ubiquitinated species and impaired delivery to the proteasome. Figure 4. The ERAD-protein VCP is required for Evi destabilization ERAD-dependent degradation requires recognition, ubiquitination, and subsequent dislocation of the selected substrates through the ER membra
Endoplasmic-reticulum-associated protein degradation
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