The endoplasmic reticulum associated degradation (ERAD) pathway ubiquitylates and removes misfolded proteins from the ER. Reduced ERAD function leads to accumulation of misfolded protein and activation of the ER Unfolded Protein Response (UPRER). We investigated the C. elegans ERAD E2 conjugase genes ubc-6 and ubc-7. Δubc-6 animals do not accumulate the misfolded protein CPL-Y32AW35A::YFP, but they upregulate the UPRER reporter gene, hsp-4 under basal and ER stress conditions. Δubc-7 animals accumulate significantly more CPL-Y32AW35A::YFP compared to wildtype, but do not hyper-activate the hsp-4 reporter. Our findings suggest that ubc-6 and ubc-7 contributed differently to ER and protein homeostasis in C. elegans.
","acknowledgements":"This work was initiated as part of a Course-based Undergraduate Research Experience (CUREs) in Biology 487 and Biology 324 (AYs 2019-2024). We thank the students from Biology 487 for help with designing and testing primers and initiating crosses, and Dr. Jackie Rose and Dr. Adrienne Wang for insightful comments during the writing process. The Δubc-6 and Δubc-7 animals were a kind gift from Dr. Don Moerman’s laboratory and we thank Vinci Au, Erika Li-Leger, and Mark Edgely for their generosity in creating the deletion mutants. hhsIs113 was generously provided by Dr. Thorsten Hoppe. This manuscript is dedicated to the memory of Don Moerman.
","authors":[{"affiliations":["Western Washington University, Bellingham, WA, United States","University of Washington, Seattle, WA, United States"],"departments":["Biology",""],"credit":["formalAnalysis","investigation","methodology","conceptualization"],"email":"breernelson@gmail.com","firstName":"Bree","lastName":"Nelson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["conceptualization","investigation","formalAnalysis","writing_originalDraft","validation"],"email":"summerkatherine2003@gmail.com","firstName":"Summer","lastName":"Simpson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["dataCuration","methodology","visualization","writing_originalDraft","writing_reviewEditing","formalAnalysis"],"email":"avawilkinson7264@gmail.com","firstName":"Ava","lastName":"Wilkinson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["formalAnalysis","writing_originalDraft","writing_reviewEditing","visualization","investigation"],"email":"wirthe@wwu.edu","firstName":"Eva","lastName":"Wirth","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["validation","visualization","writing_reviewEditing"],"email":"peachc2@wwu.edu","firstName":"Connor","lastName":"Peach","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["investigation","supervision","validation"],"email":"sutera3@wwu.edu","firstName":"Casper ","lastName":"Suter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, Washington, United States"],"departments":["Biology"],"credit":["conceptualization","project","resources","writing_originalDraft","writing_reviewEditing","supervision"],"email":"dahlbec@wwu.edu","firstName":"Caroline","lastName":"Dahlberg","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-6940-4888"}],"awards":[{"awardId":"GM157704-01","funderName":"National Institutes of Health (United States)","awardRecipient":"Dahlberg"},{"awardId":"IUSE # 1612252","funderName":"National Science Foundation (United States)","awardRecipient":"Dahlberg"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Western Washington University Office of Research and Sponsored Programs through an Undergraduate Research and Creative Opportunities Grant (B. Nelson)
","image":{"url":"https://portal.micropublication.org/uploads/126585ecb3b7d4178bb805dcae891657.png"},"imageCaption":"A. Simplified model of the ERAD pathway that UBC-6 and UBC-7 participate in. A misfolded protein (CPL-Y32AW35A::YFP) is translocated to the cytosol where it is ubiquitylated and degraded by the proteasome. The UPRER reporter, Phsp-4::GFP, is active at low levels in basal conditions and is upregulated in response to ER stress. Model created using BioRender.
B. Schematic diagram of the ubc-6 and ubc-7 genes and the deletion alleles, gk3799 (Δubc-6) and gk5037 (Δubc-7).
C. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing CPL-Y32AW35A::YFP under the control of the nhx-2 promoter.
D. Quantification of the size, in pixels2 , of puncta caused by CPL-Y32AW35A::YFP accumulation in wildtype, Δubc-6, Δubc-7 animals.
E. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter.
F. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
G. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter in control (DMSO) and stress (2.5 ng/mL tunicamycin) conditions. Images for DMSO and tunicamycin treatments were processed separately to preserve visible differences.
H. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, and Δubc-7 animals. All images were quantified at the same time, with identical processing..
*, p<0.05; **, p<0.001; ***, p<0.0001; ns, not significant. Tukey post-hoc test
","imageTitle":"Model of ERAD and fluorescence imaging of C. elegans lacking ubc-6 or ubc-7
","methods":"Animal maintenance and crossing
Animals were reared at 20°C on NGM. Genetic crosses were performed using established methods and genotypes were confirmed as outlined below.
Genotyping
Worms were lysed in a buffer of 50mM KCl, 10mM Tris, 2.5mM MgCl2, 0.45% Tween 20, and 0.45% NP-40; proteinase K was added in 200μg/mL final concentration. Lysate was used in PCRs with Apex 2.0X Taq RED Master Mix (1.5mM) from Genesee Scientific according to manufacturer directions (annealing: 55 C, 30 seconds; 90 second extension) with the following primers.
Primer | Sequence |
ubc-6 FWD | 5'-ATGAGTGAGCAGT-3' |
ubc-6 REV | 5'-ACTCGGAACCGTC-3' |
ubc-7 FWD | 5' gacaggtaccaaaaaaTGGAGCAATCCTC -3'* |
ubc-7 REV | 5'-gacggaattcTCATTCTTCTTGACTTCTGCG -3'* |
* lowercase sequence indicates exogenous sequence used for unrelated subcloning experiments.
Tunicamycin treatment
L4 stage C. elegans were incubated in 2.5 ng/ul tunicamycin or DMSO as vehicle control (both Fisher Scientific) for 4 hours with microfuge lids open at 20 degrees C. After incubation, animals were settled by microcentrifugation at 2000 rpm for 30 seconds and liquid was removed from the worm pellet.
Fluorescence widefield microscopy
Animals from all conditions for each experiment were imaged on the same day over several imaging sessions. Slides were prepared using 1% levamisole in 1% agarose in H2O. L4 worms were collected from plates and placed into 1% levamisole in M9 onto agarose slides. Using an eyebrow pick, worms were moved into stacks (arranged side by side in a row) on the slides. Images were collected on the Leica iDM6000 widefield microscope at 10X, 1 gain, and 200 ms exposure.
Quantification/Statistical analysis
Microscope images were processed in ImageJ (FIJI). To quantify GFP expression under the control of the hsp-4 promoter (Phsp-4::GFP), the perimeter of each animal was traced, and mean fluorescence brightness was recorded and subtracted from corresponding background. To quantify CPL-1::YFP puncta, images of animals with the Pnhx-2::CPL-Y32AW35A::YFP transgene were enhanced in FIJI using the difference of Gaussians algorithm. Images were then thresholded using the IsoData algorithm with a lower intensity limit of 10 and upper intensity limit of 225. A watershed was applied to isolate overlapping puncta and the area of each punctum was calculated. Stacks of worms were traced to exclude surrounding noise. Significance (p < 0.05) was determined using an ANOVA followed by a Tukey post-hoc test. Statistical tests and graphs were done in Rstudio version 4.5.3.
","reagents":"C. elegans strains
Name | Genotype | Purpose | Source |
Wildtype | Control for no YFP expression | CGC | |
Control for baseline level of CPL-Y32AW35A::YFP accumulation | CGC, T. Hoppe lab | ||
Control for expression of hsp-4 | CGC | ||
Experimental strain for CPL-Y32AW35A::YFP accumulation with ubc-7 deletion | This manuscript | ||
Experimental strain for CPL-Y32AW35A::YFP accumulation with ubc-6 deletion | This manuscript |
The endoplasmic reticulum (ER) regulates post-translational protein folding and maturation to maintain protein homeostasis and limit the accumulation of misfolded proteins. Accumulation of misfolded protein at the ER activates the ER Unfolded Protein Response (UPRER) and the downstream ER-associated protein degradation (ERAD) pathway, which alleviates the build-up of unfolded and/or unprocessed proteins (Christianson and Carvalho 2022). ERAD is facilitated by a protein complex that includes ubiquitin conjugating enzymes that target substrates on the cytosolic side of the ER membrane (Christianson and Ye 2014; Christianson et al. 2023). Ubiquitylated ERAD targets are retrotranslocated from the ER to the cytosol and degraded by the proteasome (Vembar and Brodsky 2008; Neal et al. 2018; Lopata et al. 2020; Christianson and Carvalho 2022). In S. cerevisiae, two ERAD E2 ligases, Ubc6 and Ubc7 have complementary roles in which they sequentially ubiquitylate misfolded proteins translocated from the ER lumen to the cytosol (Weber et al. 2016; Christianson and Carvalho 2022). Ubc6 adds the primary ubiquitin to lysines or hydroxylated amino acid residues (serine or threonine) on target proteins, while Ubc7 elongates the ubiquitin chain. Ubc7 has a more expansive role in ERAD than Ubc6 as it is also able to prime target proteins with ubiquitin on lysine residues prior to elongation of the ubiquitin chain (Weber et al. 2016). The mammalian orthologs to Ubc6 and Ubc7, Ube2j1/Ube2j2 and Ube2g1/Ube2g2, respectively, work with E3 conjugases to maintain ER protein homeostasis in a similar manner (Wang et al. 2009; Burr et al. 2013; Christianson and Ye 2014; Christianson and Carvalho 2022; Abdul Rehman et al. 2024).
In C. elegans, there are two predicted orthologs of ER-associated E2 conjugating enzymes, UBC-6 and UBC-7 (Figure 1A). We investigated the roles of the ubc-6 and ubc-7 genes in C. elegans by characterizing the effect of deletion alleles on the UPRER and ERAD. We used genetic reporters to measure accumulation of misfolded proteins and activation of the UPRER in strains with CRISPR-Cas9-targeted deletion alleles of either ubc-6 (gk3799) (hereafter Δubc-6) or ubc-7 (gk5037) (Δubc-7) (Figure 1B), which we crossed into two different reporter lines. To measure the accumulation of misfolded proteins, we used Pnhx-2::CPL-Y32AW35A::YFP (hhIs113), which serves as a reporter for ERAD function because CPL-Y32AW35A::YFP is engineered to misfold, but is normally cleared from the ER lumen via ERAD (Miedel et al. 2012; Efstathiou et al. 2022). In order to observe the activation of the UPRER, we measured the expression of GFP under the control of the hsp-4 promoter Phsp-4::GFP (zcIs4), whose activity increases in response to heat shock and other forms of ER stress (Ye et al. 2004; Walter and Ron 2011; Bar-Ziv et al. 2020). Using the Δubc-6 and Δubc-7 alleles, we observed how misfolded protein accumulation and regulation of a UPRER reporter depend on each gene in C. elegans.
We used fluorescence microscopy to determine how the loss of the E2 conjugases ubc-6 and ubc-7 affect the accumulation of CPL-Y32AW35A and the levels of hsp-4 expression. We measured CPL-Y32AW35A::YFP accumulation by quantifying the size of CPL-**::YFP puncta in Δubc-6 and Δubc-7 animals. Compared to wildtype, the average puncta area of CPL-Y32AW35A::YFP that accumulated was statistically unchanged in Δubc-6 animals (21.7 pixels2 compared to 14.5 pixels2 in wildtype), whereas Δubc-7 animals had larger average puncta size (53.8 pixels2 compared to 14.5 pixels2 in wildtype and 21.7 pixels2 in Δubc-6 animals) (Figure 1C, D). Immunoblot analysis (not shown) confirmed these observations. This result aligns with previous results from yeast, in which Ubc6 mutants accumulate less misfolded protein in the ER lumen compared to Ubc7 mutants (Weber et al. 2016). Because Δubc-7 animals have high accumulation of CPL-Y32AW35A::YFP, we expected that they would have a similarly high expression of the Phsp-4::GFP reporter, compared to wildtype and Δubc-6 animals. However, animals lacking ubc-6 exhibited 1.75 times higher GFP expression compared to the wildtype, whereas animals lacking ubc-7 had similar GFP levels as wildtype (Figure 1 E,F). These results were also confirmed by immunoblotting (not shown). To determine if either mutant was more sensitive to an acute ER stressor, we treated animals with tunicamycin, a compound that prevents protein glycosylation in the ER. All animals were affected by tunicamycin as measured by Phsp-4::GFP activation , which shows that Δubc-7 animals are able to respond to ER stress. We also found that, similar to basal conditions, animals lacking ubc-6, had an elevated response, 1.75 times stronger compared with wild-type animals (Figure 1, G,H) .
Our results show that loss of ubc-7 has a detrimental effect on the ability of cells to clear the misfolded protein CPL-Y32AW35A::YFP, which suggests that it is particularly important for maintaining ER protein homeostasis. Despite the increase in protein CPL-Y32AW35A::YFP accumulation, loss of ubc-7 does not lead to an upregulation of the Phsp-4::GFP reporter over wildtype levels, which suggests that either Δubc-7 animals are unable to mount a higher UPRER response via hsp-4, or that a redundant pathway is activated but not detected in our assays. Our quantification of Phsp-4::GFP expression also shows that loss of ubc-6 leads to the activation of the UPRER reporter above the wildtype levels in control and stress (tunicamycin) conditions. Therefore, although Δubc-6 animals do not accumulate significantly more CPL-Y32AW35A::YFP than wildtype animals, the loss of ubc-6 induces an elevated UPRER response. In conclusion, while ubc-6 and ubc-7 are necessary for ERAD and activation of the UPR, they seem to have independent, though related, functions. These experiments were carried out in single mutants, but it will be interesting to determine the epistatic relationship of ubc-6 and ubc-7, especially because Ubc6 and Ubc7 deletions in yeast can have additive effects on stress responses (Owutey et al. 2024). In addition, future work will help determine if UBC-6 and UBC-7 share the biochemical features of their yeast and mammalian orthologs, such as preferences for different amino acids and addition of primary ubiquitin versus chain elongation.
","references":[{"reference":"Abdul Rehman SA, Cazzaniga C, Di Nisio E, Antico O, Knebel A, Johnson C, et al., De Cesare. 2024. Discovery and characterization of noncanonical E2-conjugating enzymes. Science Advances 10: 10.1126/sciadv.adh0123.
","pubmedId":"","doi":"10.1126/sciadv.adh0123"},{"reference":"Bar Ziv R, Frakes AE, Higuchi Sanabria R, Bolas T, Frankino PA, Gildea HK, Metcalf MG, Dillin A. 2020. Measurements of Physiological Stress Responses in C. Elegans. Journal of Visualized Experiments: JoVE 5137.","pubmedId":"","doi":"10.3791/61001"},{"reference":"Burr ML, Boomen DJH, Bye H, Antrobus R, Wiertz EJ, Lehner PJ. 2013. MHC class I molecules are preferentially ubiquitinated on endoplasmic. Proceedings of the National Academy of Sciences of the United States o. 110: 14290-14295. 4816.","pubmedId":"","doi":"10.1073/pnas.1303380110"},{"reference":"Christianson JC, Carvalho P. 2022. Order through destruction: how ER‐associated protein degradation contr. The EMBO Journal. 41: e109845. 4515.","pubmedId":"","doi":"10.15252/embj.2021109845"},{"reference":"Christianson JC, Jarosch E, Sommer T. 2023. Mechanisms of substrate processing during ER-associated protein degrad. Nature Reviews. Molecular Cell Biology. 24: 777-796. 4812.","pubmedId":"","doi":"10.1038/s41580-023-00633-8"},{"reference":"Christianson JC, Ye Y. 2014. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nature Structural & Molecular Biology. 21: 325-335. 5141.","pubmedId":"","doi":"10.1038/nsmb.2793"},{"reference":"Efstathiou S, Ottens F, Schutter LS, Ravanelli S, Charmpilas N, Gutschmidt A, et al., Hoppe T. 2022. ER-associated RNA silencing promotes ER quality control. Nature Cell Biology. 24: 1714-1725. 4541.","pubmedId":"","doi":"10.1038/s41556-022-01025-4"},{"reference":"Lopata A, Kniss A, Lohr F, Rogov VV, Dotsch V. 2020. Ubiquitination in the ERAD Process. International Journal of Molecular Sciences. 21: 5369. 5135.","pubmedId":"","doi":"10.3390/ijms21155369"},{"reference":"Miedel MT, Graf NJ, Stephen KE, Long OS, Pak SC, Perlmutter DH, Silverman GA, Luke CJ. 2012. A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticu. PloS One. 7: e40145. 4576.","pubmedId":"","doi":"10.1371/journal.pone.0040145"},{"reference":"Neal S, Jaeger PA, Duttke SH, Benner C, K Glass C, Ideker T, Hampton RY. 2018. The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Me. Molecular Cell. 69: 306-320.e4. 4564.","pubmedId":"","doi":"10.1016/j.molcel.2017.12.012"},{"reference":"Owutey SL, Procuniar KA, Akoto E, Davis JC, Vachon RM, O Malley LF, et al., Rubenstein EM. 2024. Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating. microPublication Biology. 2024 5139.","pubmedId":"","doi":"10.17912/micropub.biology.001276"},{"reference":"Vembar SS, Brodsky JL. 2008. One step at a time: endoplasmic reticulum-associated degradation. Nature Reviews. Molecular Cell Biology. 9: 944-957. 715.","pubmedId":"","doi":"10.1038/nrm2546"},{"reference":"Walter P, Ron D. 2011. The Unfolded Protein Response: From Stress Pathway to Homeostatic Regu. Science. 334: 1081-1086. 4533.","pubmedId":"","doi":""},{"reference":"Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJHJ, Hansen TH. 2009. Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degrada. Journal of Cell Biology. 187: 655-668. 4928.","pubmedId":"","doi":"10.1083/jcb.200908036"},{"reference":"Weber A, Cohen I, Popp O, Dittmar G, Reiss Y, Sommer T, Ravid T, Jarosch E. 2016. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expa. Molecular Cell. 63: 827-839. 1012.","pubmedId":"","doi":"10.1016/j.molcel.2016.07.020"},{"reference":"Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA. 2004. A membrane protein complex mediates retro-translocation from the ER lu. Nature. 429: 841-847. 4535.","pubmedId":"","doi":"10.1038/nature02656"}],"title":"Effects of loss of the E2 conjugating enzymes ubc-6 and ubc-7 on the unfolded protein response and protein homeostasis in C. elegans
","reviews":[{"reviewer":{"displayName":"Eric Rubenstein"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"609726b2-3d44-4053-b12d-c6cd6604a43b","decision":"accept","abstract":"The endoplasmic reticulum associated degradation (ERAD) pathway ubiquitylates and removes misfolded proteins from the ER to prevent the accumulation of misfolded protein and activation of the ER Unfolded Protein Response (UPRER). We investigated the C. elegans ERAD E2 conjugase genes ubc-6 and ubc-7. Δubc-6 animals do not accumulate the misfolded protein CPL-Y32AW35A::YFP, but they upregulate the UPRER reporter gene, Phsp-4::GFP. Δubc-7 animals accumulate significantly more CPL-Y32AW35A::YFP compared to wildtype, but do not hyper-activate the hsp-4 reporter. Our findings suggest that ubc-6 and ubc-7 contribute differently to ER and protein homeostasis in C. elegans.
","acknowledgements":"This work was initiated as part of a Course-based Undergraduate Research Experience (CUREs) in Biology 487 and Biology 324 (AYs 2019-2024). We thank the students from Biology 487 for help with designing and testing primers and initiating crosses, and Dr. Jackie Rose and Dr. Adrienne Wang for insightful comments during the writing process. The Δubc-6 and Δubc-7 animals were a kind gift from Dr. Don Moerman’s laboratory and we thank Vinci Au, Erika Li-Leger, and Mark Edgely for their generosity in creating the deletion mutants. hhsIs113 was generously provided by Dr. Thorsten Hoppe. This manuscript is dedicated to the memory of Don Moerman.
","authors":[{"affiliations":["Western Washington University, Bellingham, WA, United States","University of Washington, Seattle, WA, United States"],"departments":["Biology",""],"credit":["formalAnalysis","investigation","methodology","conceptualization"],"email":"breernelson@gmail.com","firstName":"Bree","lastName":"Nelson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["conceptualization","investigation","formalAnalysis","writing_originalDraft","validation"],"email":"summerkatherine2003@gmail.com","firstName":"Summer","lastName":"Simpson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["dataCuration","methodology","visualization","writing_originalDraft","writing_reviewEditing","formalAnalysis"],"email":"avawilkinson7264@gmail.com","firstName":"Ava","lastName":"Wilkinson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["formalAnalysis","writing_originalDraft","writing_reviewEditing","visualization","investigation"],"email":"wirthe@wwu.edu","firstName":"Eva","lastName":"Wirth","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["validation","visualization","writing_reviewEditing"],"email":"peachc2@wwu.edu","firstName":"Connor","lastName":"Peach","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["investigation","supervision","validation"],"email":"sutera3@wwu.edu","firstName":"Casper ","lastName":"Suter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, Washington, United States"],"departments":["Biology"],"credit":["conceptualization","project","resources","writing_originalDraft","writing_reviewEditing","supervision"],"email":"dahlbec@wwu.edu","firstName":"Caroline","lastName":"Dahlberg","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-6940-4888"}],"awards":[{"awardId":"GM157704-01","funderName":"National Institutes of Health (United States)","awardRecipient":"Dahlberg"},{"awardId":"IUSE # 1612252","funderName":"National Science Foundation (United States)","awardRecipient":"Dahlberg"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Western Washington University Office of Research and Sponsored Programs through an Undergraduate Research and Creative Opportunities Grant (B. Nelson)
","image":{"url":"https://portal.micropublication.org/uploads/1030e49396c129e4acd09f7409710979.png"},"imageCaption":"A. Simplified model of the ERAD pathway that UBC-6 and UBC-7 participate in. A misfolded protein (CPL-Y32AW35A::YFP) is translocated to the cytosol where it is ubiquitylated and degraded by the proteasome. The UPRER reporter, Phsp-4::GFP, is active at low levels in basal conditions and is upregulated in response to ER stress. Model created using BioRender.
B. Schematic diagram of the coding regions of the ubc-6 and ubc-7 genes and their deletion alleles, gk3799 (Δubc-6, teal arrow) and gk5037 (Δubc-7, magenta arrow). Black boxes indicate exons, separated by spliced introns, which are designated by black linkers. White boxes indicate untranslated sequences.
C. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing CPL-Y32AW35A::YFP under the control of the nhx-2 promoter.
D. Quantification of the size, in pixels2, of puncta caused by CPL-Y32AW35A::YFP accumulation in wildtype, Δubc-6, Δubc-7 animals.
E. Quantification of the total CPL-Y32AW35A::YFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
F. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter. All images were identical processed using the same parameters.
G. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
H. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter in control (DMSO) and stress (2.5 ng/mL tunicamycin) conditions. Images for DMSO and tunicamycin treatments were processed separately to preserve visible differences.
I. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, and Δubc-7 animals treated with DMSO or tunicamycin.
*, p<0.05; **, p<0.001; ***, p<0.0001; ns, not significant. Tukey post-hoc test
","imageTitle":"Model of ERAD and fluorescence imaging of C. elegans lacking ubc-6 or ubc-7
","methods":"Animal maintenance and crossing
Animals were reared at 20°C on NGM. Genetic crosses were performed using established methods and genotypes were confirmed as outlined below.
Genotyping
Worms were lysed in a buffer of 50mM KCl, 10mM Tris, 2.5mM MgCl2, 0.45% Tween 20, and 0.45% NP-40; proteinase K was added in 200μg/mL final concentration. Lysate was used in PCRs with Apex 2.0X Taq RED Master Mix (1.5mM) from Genesee Scientific according to manufacturer directions (annealing: 55 C, 30 seconds; 90 second extension) with the following primers.
Primer | Sequence |
ubc-6 FWD | 5'-ATGAGTGAGCAGT-3' |
ubc-6 REV | 5'-ACTCGGAACCGTC-3' |
ubc-7 FWD | 5' gacaggtaccaaaaaaTGGAGCAATCCTC -3'* |
ubc-7 REV | 5'-gacggaattcTCATTCTTCTTGACTTCTGCG -3'* |
* lowercase sequence indicates exogenous sequence used for unrelated subcloning experiments.
Tunicamycin treatment
L4 stage C. elegans were incubated in 2.5 ng/ul tunicamycin or DMSO as vehicle control (both Fisher Scientific) for 4 hours with microfuge lids open at 20 degrees C. After incubation, animals were settled by microcentrifugation at 2000 rpm for 30 seconds and liquid was removed from the worm pellet.
Fluorescence widefield microscopy
Animals from all conditions for each experiment were imaged on the same day over several imaging sessions. Slides were prepared using 1% levamisole in 1% agarose in H2O. L4 worms were collected from plates and placed into 1% levamisole in M9 onto agarose slides. Using an eyebrow pick, worms were moved into stacks (arranged side by side in a row) on the slides. Images were collected on the Leica iDM6000 widefield microscope at 10X, 1 gain, and 200 ms exposure.
Quantification/Statistical analysis
Microscope images were processed in ImageJ (FIJI). To quantify GFP expression under the control of the hsp-4 promoter (Phsp-4::GFP), the perimeter of each animal was traced, and mean fluorescence brightness was recorded and subtracted from corresponding background. To quantify CPL-1::YFP puncta, images of animals with the Pnhx-2::CPL-Y32AW35A::YFP transgene were enhanced in FIJI using the difference of Gaussians algorithm. Images were then thresholded using the IsoData algorithm with a lower intensity limit of 10 and upper intensity limit of 225. A watershed was applied to isolate overlapping puncta and the area of each punctum was calculated. Stacks of worms were traced to exclude surrounding noise. Significance (p < 0.05) was determined using an ANOVA followed by a Tukey post-hoc test. Statistical tests and graphs were done in Rstudio version 4.5.3.
","reagents":"C. elegans strains
Name | Genotype | Purpose | Source |
Wildtype | Control for no YFP expression | CGC | |
Control for baseline level of CPL-Y32AW35A::YFP accumulation | CGC, T. Hoppe lab | ||
Control for expression of hsp-4 | CGC | ||
Experimental strain for CPL-Y32AW35A::YFP accumulation with ubc-7 deletion | This manuscript | ||
Experimental strain for CPL-Y32AW35A::YFP accumulation with ubc-6 deletion | This manuscript |
The endoplasmic reticulum (ER) regulates post-translational protein folding and maturation to maintain protein homeostasis and limit the accumulation of misfolded proteins. Accumulation of misfolded protein at the ER activates the ER Unfolded Protein Response (UPRER) and the downstream ER-associated protein degradation (ERAD) pathway, which alleviates the build-up of unfolded and/or unprocessed proteins (Christianson and Carvalho 2022). ERAD is facilitated by a protein complex that includes ubiquitin conjugating enzymes that target substrates on the cytosolic side of the ER membrane (Christianson and Ye 2014; Christianson et al. 2023). Ubiquitylated ERAD targets are retrotranslocated from the ER to the cytosol and degraded by the proteasome (Vembar and Brodsky 2008; Neal et al. 2018; Lopata et al. 2020; Christianson and Carvalho 2022). In S. cerevisiae, two ERAD E2 ligases, Ubc6 and Ubc7 have complementary roles in which they sequentially ubiquitylate misfolded proteins translocated from the ER lumen to the cytosol (Weber et al. 2016; Christianson and Carvalho 2022). Ubc6 adds the primary ubiquitin to lysines or hydroxylated amino acid residues (serine or threonine) on target proteins, while Ubc7 elongates the ubiquitin chain. Ubc7 has a more expansive role in ERAD than Ubc6 as it is also able to prime target proteins with ubiquitin on lysine residues prior to elongation of the ubiquitin chain (Weber et al. 2016). The mammalian orthologs to Ubc6 and Ubc7, Ube2j1/Ube2j2 and Ube2g1/Ube2g2, respectively, work with E3 conjugases to maintain ER protein homeostasis in a similar manner (Wang et al. 2009; Burr et al. 2013; Christianson and Ye 2014; Christianson and Carvalho 2022; Abdul Rehman et al. 2024).
In C. elegans, there are two predicted orthologs of ER-associated E2 conjugating enzymes, UBC-6 and UBC-7 (Figure 1A). We investigated the roles of the ubc-6 and ubc-7 genes in C. elegans by characterizing the effect of deletion alleles on the UPRER and ERAD. We used genetic reporters to measure accumulation of misfolded proteins and activation of the UPRER in strains with CRISPR-Cas9-targeted deletion alleles of either ubc-6 (gk3799) (hereafter Δubc-6) or ubc-7 (gk5037) (Δubc-7) (Figure 1B), which we crossed into two different reporter lines. To measure the accumulation of misfolded proteins, we used Pnhx-2::CPL-Y32AW35A::YFP (hhIs113), which serves as a reporter for ERAD function because CPL-Y32AW35A::YFP is engineered to misfold, but is normally cleared from the ER lumen via ERAD (Miedel et al. 2012; Efstathiou et al. 2022). In order to observe the activation of the UPRER, we measured the expression of GFP under the control of the hsp-4 promoter Phsp-4::GFP (zcIs4), whose activity increases in response to heat shock and other forms of ER stress (Ye et al. 2004; Walter and Ron 2011; Bar-Ziv et al. 2020). Using the Δubc-6 and Δubc-7 alleles, we observed how misfolded protein accumulation and regulation of a UPRER reporter depend on each gene in C. elegans.
We used fluorescence microscopy to determine how the loss of the E2 conjugases ubc-6 and ubc-7 affect the accumulation of CPL-Y32AW35A and the levels of hsp-4 expression. We measured CPL-Y32AW35A::YFP accumulation by quantifying the size of CPL-**::YFP puncta in Δubc-6 and Δubc-7 animals. Compared to wildtype, the average puncta area of CPL-Y32AW35A::YFP that accumulated was statistically unchanged in Δubc-6 animals (21.7 pixels2 compared to 14.5 pixels2 in wildtype), whereas Δubc-7 animals had larger average puncta size (53.8 pixels2 compared to 14.5 pixels2 in wildtype and 21.7 pixels2 in Δubc-6 animals) (Figure 1C, D). The overall signal intensity correlated with the puncta area since wildtype and Δubc-6 animals had essentially the same overall fluorescence, while Δubc-7 animals were, on average 1.45 times brighter (Figure 1E). Immunoblot analysis (not shown) confirmed these observations. This result aligns with previous results from yeast, in which Ubc6 mutants accumulate less misfolded protein in the ER lumen compared to Ubc7 mutants (Weber et al. 2016). Because Δubc-7 animals have high accumulation of CPL-Y32AW35A::YFP, we expected that they would have a similarly high expression of the Phsp-4::GFP reporter, compared to wildtype and Δubc-6 animals. However, animals lacking ubc-6 exhibited 1.75 times higher GFP expression compared to the wildtype, whereas animals lacking ubc-7 had similar GFP levels as wildtype (Figure 1 F,G). These results were also confirmed by immunoblotting (not shown). To determine if either mutant was more sensitive to an acute ER stressor, we treated animals with tunicamycin, a compound that prevents protein glycosylation in the ER, or DMSO as a vehicle control. All animals were affected by tunicamycin as measured by Phsp-4::GFP activation, which shows that Δubc-7 animals are able to respond to ER stress. We also found that animals lacking ubc-6 had significantly higher Phsp-4::GFP activation in response to tunicamycin compared with both wild-type and Δubc-7 animals (Figure 1, H, I).
Our results show that loss of ubc-7 has a detrimental effect on the ability of cells to clear the misfolded protein CPL-Y32AW35A::YFP, which suggests that it is particularly important for maintaining ER protein homeostasis. Despite the increase in protein CPL-Y32AW35A::YFP accumulation, loss of ubc-7 does not lead to an upregulation of the Phsp-4::GFP reporter over wildtype levels, which suggests that either Δubc-7 animals are unable to mount a higher UPRER response via hsp-4, or that a redundant pathway is activated but not detected in our assays. Our quantification of Phsp-4::GFP expression also shows that loss of ubc-6 leads to the activation of the UPRER reporter above the wildtype levels in control and stress (tunicamycin) conditions. Therefore, although Δubc-6 animals do not accumulate significantly more CPL-Y32AW35A::YFP than wildtype animals, the loss of ubc-6 induces an elevated UPRER response. In conclusion, while ubc-6 and ubc-7 are necessary for ERAD and activation of the UPR, they seem to have independent, though related, functions. These experiments were carried out in single mutants, but it will be interesting to determine the epistatic relationship of ubc-6 and ubc-7, especially because Ubc6 and Ubc7 deletions in yeast can have additive effects on stress responses (Owutey et al. 2024). In addition, future work will help determine if UBC-6 and UBC-7 share the biochemical features of their yeast and mammalian orthologs, such as preferences for different amino acids and addition of primary ubiquitin versus chain elongation.
","references":[{"reference":"Abdul Rehman SA, Cazzaniga C, Di Nisio E, Antico O, Knebel A, Johnson C, et al., De Cesare. 2024. Discovery and characterization of noncanonical E2-conjugating enzymes. Science Advances 10: 10.1126/sciadv.adh0123.
","pubmedId":"","doi":"10.1126/sciadv.adh0123"},{"reference":"Bar Ziv R, Frakes AE, Higuchi Sanabria R, Bolas T, Frankino PA, Gildea HK, Metcalf MG, Dillin A. 2020. Measurements of Physiological Stress Responses in C. Elegans. Journal of Visualized Experiments: JoVE 5137.","pubmedId":"","doi":"10.3791/61001"},{"reference":"Burr ML, Boomen DJH, Bye H, Antrobus R, Wiertz EJ, Lehner PJ. 2013. MHC class I molecules are preferentially ubiquitinated on endoplasmic. Proceedings of the National Academy of Sciences of the United States o. 110: 14290-14295. 4816.","pubmedId":"","doi":"10.1073/pnas.1303380110"},{"reference":"Christianson JC, Carvalho P. 2022. Order through destruction: how ER‐associated protein degradation contr. The EMBO Journal. 41: e109845. 4515.","pubmedId":"","doi":"10.15252/embj.2021109845"},{"reference":"Christianson JC, Jarosch E, Sommer T. 2023. Mechanisms of substrate processing during ER-associated protein degrad. Nature Reviews. Molecular Cell Biology. 24: 777-796. 4812.","pubmedId":"","doi":"10.1038/s41580-023-00633-8"},{"reference":"Christianson JC, Ye Y. 2014. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nature Structural & Molecular Biology. 21: 325-335. 5141.","pubmedId":"","doi":"10.1038/nsmb.2793"},{"reference":"Efstathiou S, Ottens F, Schutter LS, Ravanelli S, Charmpilas N, Gutschmidt A, et al., Hoppe T. 2022. ER-associated RNA silencing promotes ER quality control. Nature Cell Biology. 24: 1714-1725. 4541.","pubmedId":"","doi":"10.1038/s41556-022-01025-4"},{"reference":"Lopata A, Kniss A, Lohr F, Rogov VV, Dotsch V. 2020. Ubiquitination in the ERAD Process. International Journal of Molecular Sciences. 21: 5369. 5135.","pubmedId":"","doi":"10.3390/ijms21155369"},{"reference":"Miedel MT, Graf NJ, Stephen KE, Long OS, Pak SC, Perlmutter DH, Silverman GA, Luke CJ. 2012. A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticu. PloS One. 7: e40145. 4576.","pubmedId":"","doi":"10.1371/journal.pone.0040145"},{"reference":"Neal S, Jaeger PA, Duttke SH, Benner C, K Glass C, Ideker T, Hampton RY. 2018. The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Me. Molecular Cell. 69: 306-320.e4. 4564.","pubmedId":"","doi":"10.1016/j.molcel.2017.12.012"},{"reference":"Owutey SL, Procuniar KA, Akoto E, Davis JC, Vachon RM, O Malley LF, et al., Rubenstein EM. 2024. Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating. microPublication Biology. 2024 5139.","pubmedId":"","doi":"10.17912/micropub.biology.001276"},{"reference":"Vembar SS, Brodsky JL. 2008. One step at a time: endoplasmic reticulum-associated degradation. Nature Reviews. Molecular Cell Biology. 9: 944-957. 715.","pubmedId":"","doi":"10.1038/nrm2546"},{"reference":"Walter P, Ron D. 2011. The Unfolded Protein Response: From Stress Pathway to Homeostatic Regu. Science. 334: 1081-1086. 4533.","pubmedId":"","doi":""},{"reference":"Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJHJ, Hansen TH. 2009. Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degrada. Journal of Cell Biology. 187: 655-668. 4928.","pubmedId":"","doi":"10.1083/jcb.200908036"},{"reference":"Weber A, Cohen I, Popp O, Dittmar G, Reiss Y, Sommer T, Ravid T, Jarosch E. 2016. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expa. Molecular Cell. 63: 827-839. 1012.","pubmedId":"","doi":"10.1016/j.molcel.2016.07.020"},{"reference":"Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA. 2004. A membrane protein complex mediates retro-translocation from the ER lu. Nature. 429: 841-847. 4535.","pubmedId":"","doi":"10.1038/nature02656"}],"title":"Effects of loss of the E2 conjugating enzymes ubc-6 and ubc-7 on the unfolded protein response and protein homeostasis in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":"1793322403930"}]},{"id":"470ee093-40b1-47e2-ade4-1b9441d29db7","decision":"revise","abstract":"The endoplasmic reticulum associated degradation (ERAD) pathway ubiquitylates and removes misfolded proteins from the ER to prevent the accumulation of misfolded protein and activation of the ER Unfolded Protein Response (UPRER). We investigated the C. elegans ERAD E2 conjugase genes ubc-6 and ubc-7. Δubc-6 animals do not accumulate the misfolded protein CPL-1Y32AW35A::YFP, but they upregulate the UPRER reporter gene, Phsp-4::GFP. Δubc-7 animals accumulate significantly more CPL-1Y32AW35A::YFP compared to wildtype, but do not hyper-activate the hsp-4 reporter. Our findings suggest that ubc-6 and ubc-7 contribute differently to ER and protein homeostasis in C. elegans.
","acknowledgements":"This work was initiated as part of a Course-based Undergraduate Research Experience (CUREs) in Biology 487 and Biology 324 (AYs 2019-2024). We thank the students from Biology 487 for help with designing and testing primers and initiating crosses, and Dr. Jackie Rose and Dr. Adrienne Wang for insightful comments during the writing process. The Δubc-6 and Δubc-7 animals were a kind gift from Dr. Don Moerman’s laboratory and we thank Vinci Au, Erika Li-Leger, and Mark Edgely for their generosity in creating the deletion mutants. hhsIs113 was generously provided by Dr. Thorsten Hoppe. This manuscript is dedicated to the memory of Don Moerman.
","authors":[{"affiliations":["Western Washington University, Bellingham, WA, United States","University of Washington, Seattle, WA, United States"],"departments":["Biology",""],"credit":["formalAnalysis","investigation","methodology","conceptualization"],"email":"breernelson@gmail.com","firstName":"Bree","lastName":"Nelson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["conceptualization","investigation","formalAnalysis","writing_originalDraft","validation"],"email":"summerkatherine2003@gmail.com","firstName":"Summer","lastName":"Simpson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["dataCuration","methodology","visualization","writing_originalDraft","writing_reviewEditing","formalAnalysis"],"email":"avawilkinson7264@gmail.com","firstName":"Ava","lastName":"Wilkinson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["formalAnalysis","writing_originalDraft","writing_reviewEditing","visualization","investigation"],"email":"wirthe@wwu.edu","firstName":"Eva","lastName":"Wirth","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["validation","visualization","writing_reviewEditing"],"email":"peachc2@wwu.edu","firstName":"Connor","lastName":"Peach","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["investigation","supervision","validation"],"email":"sutera3@wwu.edu","firstName":"Casper ","lastName":"Suter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["conceptualization","project","resources","writing_originalDraft","writing_reviewEditing","supervision"],"email":"dahlbec@wwu.edu","firstName":"Caroline","lastName":"Dahlberg","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-6940-4888"}],"awards":[{"awardId":"GM157704-01","funderName":"National Institutes of Health (United States)","awardRecipient":"Dahlberg"},{"awardId":"IUSE # 1612252","funderName":"National Science Foundation (United States)","awardRecipient":"Dahlberg"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Western Washington University Office of Research and Sponsored Programs through an Undergraduate Research and Creative Opportunities Grant (B. Nelson)
","image":{"url":"https://portal.micropublication.org/uploads/1030e49396c129e4acd09f7409710979.png"},"imageCaption":"A. Simplified model of the ERAD pathway that UBC-6 and UBC-7 participate in. A misfolded protein (CPL-1Y32AW35A::YFP) is translocated to the cytosol where it is ubiquitylated and degraded by the proteasome. The UPRER reporter, Phsp-4::GFP, is active at low levels in basal conditions and is upregulated in response to ER stress. Model created using BioRender.
B. Schematic diagram of the coding regions of the ubc-6 and ubc-7 genes and their deletion alleles, gk3799 (Δubc-6, teal arrow) and gk5037 (Δubc-7, magenta arrow). Black boxes indicate exons, separated by spliced introns, which are designated by black linkers. White boxes indicate untranslated sequences.
C. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing CPL-1Y32AW35A::YFP under the control of the nhx-2 promoter (CPL-1**::YFP).
D. Quantification of the size, in pixels2, of puncta caused by CPL-1Y32AW35A::YFP (CPL-1**::YFP) accumulation in wildtype, Δubc-6, Δubc-7 animals.
E. Quantification of the total CPL-1Y32AW35A::YFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
F. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter. All images were identical processed using the same parameters.
G. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
H. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter in control (DMSO) and stress (2.5 ng/mL tunicamycin) conditions. Images for DMSO and tunicamycin treatments were processed separately to preserve visible differences.
I. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, and Δubc-7 animals treated with DMSO or tunicamycin.
*, p<0.05; **, p<0.001; ***, p<0.0001; ns, not significant. Tukey post-hoc test
","imageTitle":"Model of ERAD and fluorescence imaging of C. elegans lacking ubc-6 or ubc-7
","methods":"Animal maintenance and crossing
Animals were reared at 20°C on NGM. Genetic crosses were performed using established methods and genotypes were confirmed as outlined below.
Genotyping
Worms were lysed in a buffer of 50mM KCl, 10mM Tris, 2.5mM MgCl2, 0.45% Tween 20, and 0.45% NP-40; proteinase K was added in 200μg/mL final concentration. Lysate was used in PCRs with Apex 2.0X Taq RED Master Mix (1.5mM) from Genesee Scientific according to manufacturer directions (annealing: 55 C, 30 seconds; 90 second extension) with the following primers.
Primer | Sequence |
ubc-6 FWD | 5'-ATGAGTGAGCAGT-3' |
ubc-6 REV | 5'-ACTCGGAACCGTC-3' |
ubc-7 FWD | 5' gacaggtaccaaaaaaTGGAGCAATCCTC -3'* |
ubc-7 REV | 5'-gacggaattcTCATTCTTCTTGACTTCTGCG -3'* |
* lowercase sequence indicates exogenous sequence used for unrelated subcloning experiments.
Tunicamycin treatment
L4 stage C. elegans were incubated in 2.5 ng/ul tunicamycin or DMSO as vehicle control (both Fisher Scientific) for 4 hours with microfuge lids open at 20 degrees C. After incubation, animals were settled by microcentrifugation at 2000 rpm for 30 seconds and liquid was removed from the worm pellet.
Fluorescence widefield microscopy
Animals from all conditions for each experiment were imaged on the same day over several imaging sessions. Slides were prepared using 1% levamisole in 1% agarose in H2O. L4 worms were collected from plates and placed into 1% levamisole in M9 onto agarose slides. Using an eyebrow pick, worms were moved into stacks (arranged side by side in a row) on the slides. Images were collected on the Leica iDM6000 widefield microscope at 10X, 1 gain, and 200 ms exposure.
Quantification/Statistical analysis
Microscope images were processed in ImageJ (FIJI). To quantify GFP expression under the control of the hsp-4 promoter (Phsp-4::GFP), the perimeter of each animal was traced, and mean fluorescence brightness was recorded and subtracted from corresponding background. To quantify CPL-1Y32AW35A::YFP puncta, images of animals with the Pnhx-2::CPL-1Y32AW35A::YFP transgene were enhanced in FIJI using the difference of Gaussians algorithm. Images were then thresholded using the IsoData algorithm with a lower intensity limit of 10 and upper intensity limit of 225. A watershed was applied to isolate overlapping puncta and the area of each punctum was calculated. Stacks of worms were traced to exclude surrounding noise. Significance (p < 0.05) was determined using an ANOVA followed by a Tukey post-hoc test. Statistical tests and graphs were done in Rstudio version 4.5.3.
","reagents":"C. elegans strains
Name | Genotype | Purpose | Source |
Wildtype | Control for no YFP expression | CGC | |
Control for baseline level of CPL-1Y32AW35A::YFP accumulation | CGC, T. Hoppe lab | ||
Control for expression of hsp-4 | CGC | ||
Experimental strain for CPL-1Y32AW35A::YFP accumulation with ubc-7 deletion | This manuscript | ||
Experimental strain for CPL-1Y32AW35A::YFP accumulation with ubc-6 deletion | This manuscript |
The endoplasmic reticulum (ER) regulates post-translational protein folding and maturation to maintain protein homeostasis and limit the accumulation of misfolded proteins. Accumulation of misfolded protein at the ER activates the ER Unfolded Protein Response (UPRER) and the downstream ER-associated protein degradation (ERAD) pathway, which alleviates the build-up of unfolded and/or unprocessed proteins (Christianson and Carvalho 2022). ERAD is facilitated by a protein complex that includes ubiquitin conjugating enzymes that target substrates on the cytosolic side of the ER membrane (Christianson and Ye 2014; Christianson et al. 2023). Ubiquitylated ERAD targets are retrotranslocated from the ER to the cytosol and degraded by the proteasome (Vembar and Brodsky 2008; Neal et al. 2018; Lopata et al. 2020; Christianson and Carvalho 2022). In S. cerevisiae, two ERAD E2 ligases, Ubc6 and Ubc7 have complementary roles in which they sequentially ubiquitylate misfolded proteins translocated from the ER lumen to the cytosol (Weber et al. 2016; Christianson and Carvalho 2022). Ubc6 adds the primary ubiquitin to lysines or hydroxylated amino acid residues (serine or threonine) on target proteins, while Ubc7 elongates the ubiquitin chain. Ubc7 has a more expansive role in ERAD than Ubc6 as it is also able to prime target proteins with ubiquitin on lysine residues prior to elongation of the ubiquitin chain (Weber et al. 2016). The mammalian orthologs to Ubc6 and Ubc7, Ube2j1/Ube2j2 and Ube2g1/Ube2g2, respectively, work with E3 conjugases to maintain ER protein homeostasis in a similar manner (Wang et al. 2009; Burr et al. 2013; Christianson and Ye 2014; Christianson and Carvalho 2022; Abdul Rehman et al. 2024).
In C. elegans, there are two predicted orthologs of ER-associated E2 conjugating enzymes, UBC-6 and UBC-7 (Figure 1A). We investigated the roles of the ubc-6 and ubc-7 genes in C. elegans by characterizing the effect of deletion alleles on the UPRER and ERAD. We used genetic reporters to measure accumulation of misfolded proteins and activation of the UPRER in strains with CRISPR-Cas9-targeted deletion alleles of either ubc-6 (gk3799) (hereafter Δubc-6) or ubc-7 (gk5037) (Δubc-7) (Figure 1B), which we crossed into two different reporter lines. To measure the accumulation of misfolded proteins, we used Pnhx-2::CPL-1Y32AW35A::YFP (hhIs113), which serves as a reporter for ERAD function because CPL-1Y32AW35A::YFP is engineered to misfold, but is normally cleared from the ER lumen via ERAD (Miedel et al. 2012; Efstathiou et al. 2022). In order to observe the activation of the UPRER, we measured the expression of GFP under the control of the hsp-4 promoter Phsp-4::GFP (zcIs4), whose activity increases in response to heat shock and other forms of ER stress (Ye et al. 2004; Walter and Ron 2011; Bar-Ziv et al. 2020). Using the Δubc-6 and Δubc-7 alleles, we observed how misfolded protein accumulation and regulation of a UPRER reporter depend on each gene in C. elegans.
We used fluorescence microscopy to determine how the loss of the E2 conjugases ubc-6 and ubc-7 affect the accumulation of CPL-1Y32AW35A and the levels of hsp-4 expression. We measured CPL-1Y32AW35A::YFP accumulation by quantifying the size of CPL-1Y32AW35A::YFP puncta in Δubc-6 and Δubc-7 animals. Compared to wildtype, the average puncta area of CPL-1Y32AW35A::YFP that accumulated was statistically unchanged in Δubc-6 animals (21.7 pixels2 compared to 14.5 pixels2 in wildtype), whereas Δubc-7 animals had larger average puncta size (53.8 pixels2 compared to 14.5 pixels2 in wildtype and 21.7 pixels2 in Δubc-6 animals) (Figure 1C, D). The overall signal intensity correlated with the puncta area since wildtype and Δubc-6 animals had essentially the same overall fluorescence, while Δubc-7 animals were, on average 1.45 times brighter (Figure 1E). Immunoblot analysis (not shown) confirmed these observations. This result aligns with previous results from yeast, in which Ubc6 mutants accumulate less misfolded protein in the ER lumen compared to Ubc7 mutants (Weber et al. 2016). Because Δubc-7 animals have high accumulation of CPL-1Y32AW35A::YFP, we expected that they would have a similarly high expression of the Phsp-4::GFP reporter, compared to wildtype and Δubc-6 animals. However, animals lacking ubc-6 exhibited 1.75 times higher GFP expression compared to the wildtype, whereas animals lacking ubc-7 had similar GFP levels as wildtype (Figure 1 F,G). These results were also confirmed by immunoblotting (not shown). To determine if either mutant was more sensitive to an acute ER stressor, we treated animals with tunicamycin, a compound that prevents protein glycosylation in the ER, or DMSO as a vehicle control. All animals were affected by tunicamycin as measured by Phsp-4::GFP activation, which shows that Δubc-7 animals are able to respond to ER stress. We also found that animals lacking ubc-6 had significantly higher Phsp-4::GFP activation in response to tunicamycin compared with both wild-type and Δubc-7 animals (Figure 1, H, I).
Our results show that loss of ubc-7 has a detrimental effect on the ability of cells to clear the misfolded protein CPL-1Y32AW35A::YFP, which suggests that it is particularly important for maintaining ER protein homeostasis. Despite the increase in protein CPL-1Y32AW35A::YFP accumulation, loss of ubc-7 does not lead to an upregulation of the Phsp-4::GFP reporter over wildtype levels, which suggests that either Δubc-7 animals are unable to mount a higher UPRER response via hsp-4, or that a redundant pathway is activated but not detected in our assays. Our quantification of Phsp-4::GFP expression also shows that loss of ubc-6 leads to the activation of the UPRER reporter above the wildtype levels in control and stress (tunicamycin) conditions. Therefore, although Δubc-6 animals do not accumulate significantly more CPL-1Y32AW35A::YFP than wildtype animals, the loss of ubc-6 induces an elevated UPRER response. In conclusion, while ubc-6 and ubc-7 are necessary for ERAD and activation of the UPR, they seem to have independent, though related, functions. These experiments were carried out in single mutants, but it will be interesting to determine the epistatic relationship of ubc-6 and ubc-7, especially because Ubc6 and Ubc7 deletions in yeast can have additive effects on stress responses (Owutey et al. 2024). In addition, future work will help determine if UBC-6 and UBC-7 share the biochemical features of their yeast and mammalian orthologs, such as preferences for different amino acids and addition of primary ubiquitin versus chain elongation.
","references":[{"reference":"Abdul Rehman SA, Cazzaniga C, Di Nisio E, Antico O, Knebel A, Johnson C, et al., De Cesare. 2024. Discovery and characterization of noncanonical E2-conjugating enzymes. Science Advances 10: 10.1126/sciadv.adh0123.
","pubmedId":"","doi":"10.1126/sciadv.adh0123"},{"reference":"Bar Ziv R, Frakes AE, Higuchi Sanabria R, Bolas T, Frankino PA, Gildea HK, Metcalf MG, Dillin A. 2020. Measurements of Physiological Stress Responses in C. Elegans. Journal of Visualized Experiments: JoVE 5137.","pubmedId":"","doi":"10.3791/61001"},{"reference":"Burr ML, Boomen DJH, Bye H, Antrobus R, Wiertz EJ, Lehner PJ. 2013. MHC class I molecules are preferentially ubiquitinated on endoplasmic. Proceedings of the National Academy of Sciences of the United States o. 110: 14290-14295. 4816.","pubmedId":"","doi":"10.1073/pnas.1303380110"},{"reference":"Christianson JC, Carvalho P. 2022. Order through destruction: how ER‐associated protein degradation contr. The EMBO Journal. 41: e109845. 4515.","pubmedId":"","doi":"10.15252/embj.2021109845"},{"reference":"Christianson JC, Jarosch E, Sommer T. 2023. Mechanisms of substrate processing during ER-associated protein degrad. Nature Reviews. Molecular Cell Biology. 24: 777-796. 4812.","pubmedId":"","doi":"10.1038/s41580-023-00633-8"},{"reference":"Christianson JC, Ye Y. 2014. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nature Structural & Molecular Biology. 21: 325-335. 5141.","pubmedId":"","doi":"10.1038/nsmb.2793"},{"reference":"Efstathiou S, Ottens F, Schutter LS, Ravanelli S, Charmpilas N, Gutschmidt A, et al., Hoppe T. 2022. ER-associated RNA silencing promotes ER quality control. Nature Cell Biology. 24: 1714-1725. 4541.","pubmedId":"","doi":"10.1038/s41556-022-01025-4"},{"reference":"Lopata A, Kniss A, Lohr F, Rogov VV, Dotsch V. 2020. Ubiquitination in the ERAD Process. International Journal of Molecular Sciences. 21: 5369. 5135.","pubmedId":"","doi":"10.3390/ijms21155369"},{"reference":"Miedel MT, Graf NJ, Stephen KE, Long OS, Pak SC, Perlmutter DH, Silverman GA, Luke CJ. 2012. A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticu. PloS One. 7: e40145. 4576.","pubmedId":"","doi":"10.1371/journal.pone.0040145"},{"reference":"Neal S, Jaeger PA, Duttke SH, Benner C, K Glass C, Ideker T, Hampton RY. 2018. The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Me. Molecular Cell. 69: 306-320.e4. 4564.","pubmedId":"","doi":"10.1016/j.molcel.2017.12.012"},{"reference":"Owutey SL, Procuniar KA, Akoto E, Davis JC, Vachon RM, O Malley LF, et al., Rubenstein EM. 2024. Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating. microPublication Biology. 2024 5139.","pubmedId":"","doi":"10.17912/micropub.biology.001276"},{"reference":"Vembar SS, Brodsky JL. 2008. One step at a time: endoplasmic reticulum-associated degradation. Nature Reviews. Molecular Cell Biology. 9: 944-957. 715.","pubmedId":"","doi":"10.1038/nrm2546"},{"reference":"Walter P, Ron D. 2011. The Unfolded Protein Response: From Stress Pathway to Homeostatic Regu. Science. 334: 1081-1086. 4533.","pubmedId":"","doi":""},{"reference":"Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJHJ, Hansen TH. 2009. Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degrada. Journal of Cell Biology. 187: 655-668. 4928.","pubmedId":"","doi":"10.1083/jcb.200908036"},{"reference":"Weber A, Cohen I, Popp O, Dittmar G, Reiss Y, Sommer T, Ravid T, Jarosch E. 2016. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expa. Molecular Cell. 63: 827-839. 1012.","pubmedId":"","doi":"10.1016/j.molcel.2016.07.020"},{"reference":"Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA. 2004. A membrane protein complex mediates retro-translocation from the ER lu. Nature. 429: 841-847. 4535.","pubmedId":"","doi":"10.1038/nature02656"}],"title":"Effects of loss of the E2 conjugating enzymes ubc-6 and ubc-7 on the unfolded protein response and protein homeostasis in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"069f73cc-c775-4a1f-b19b-48cea6ef6d60","decision":"publish","abstract":"The endoplasmic reticulum associated degradation (ERAD) pathway ubiquitylates and removes misfolded proteins from the ER to prevent the accumulation of misfolded protein and activation of the ER Unfolded Protein Response (UPRER). We investigated the C. elegans ERAD E2 conjugase genes ubc-6 and ubc-7. Δubc-6 animals do not accumulate the misfolded protein CPL-1Y32AW35A::YFP, but they upregulate the UPRER reporter gene, Phsp-4::GFP. Δubc-7 animals accumulate significantly more CPL-1Y32AW35A::YFP compared to wildtype, but do not hyper-activate the hsp-4 reporter. Our findings suggest that ubc-6 and ubc-7 contribute differently to ER and protein homeostasis in C. elegans.
","acknowledgements":"This work was initiated as part of a Course-based Undergraduate Research Experience (CUREs) in Biology 487 and Biology 324 (AYs 2019-2024). We thank the students from Biology 487 for help with designing and testing primers and initiating crosses, and Dr. Jackie Rose and Dr. Adrienne Wang for insightful comments during the writing process. The Δubc-6 and Δubc-7 animals were a kind gift from Dr. Don Moerman’s laboratory and we thank Vinci Au, Erika Li-Leger, and Mark Edgely for their generosity in creating the deletion mutants. hhsIs113 was generously provided by Dr. Thorsten Hoppe. This manuscript is dedicated to the memory of Don Moerman.
","authors":[{"affiliations":["Western Washington University, Bellingham, WA, United States","University of Washington, Seattle, WA, United States"],"departments":["Biology",""],"credit":["formalAnalysis","investigation","methodology","conceptualization"],"email":"breernelson@gmail.com","firstName":"Bree","lastName":"Nelson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["conceptualization","investigation","formalAnalysis","writing_originalDraft","validation"],"email":"summerkatherine2003@gmail.com","firstName":"Summer","lastName":"Simpson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["dataCuration","methodology","visualization","writing_originalDraft","writing_reviewEditing","formalAnalysis"],"email":"avawilkinson7264@gmail.com","firstName":"Ava","lastName":"Wilkinson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["formalAnalysis","writing_originalDraft","writing_reviewEditing","visualization","investigation"],"email":"wirthe@wwu.edu","firstName":"Eva","lastName":"Wirth","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["validation","visualization","writing_reviewEditing"],"email":"peachc2@wwu.edu","firstName":"Connor","lastName":"Peach","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["investigation","supervision","validation"],"email":"sutera3@wwu.edu","firstName":"Casper ","lastName":"Suter","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Western Washington University, Bellingham, WA, United States"],"departments":["Biology"],"credit":["conceptualization","project","resources","writing_originalDraft","writing_reviewEditing","supervision"],"email":"dahlbec@wwu.edu","firstName":"Caroline","lastName":"Dahlberg","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-6940-4888"}],"awards":[{"awardId":"GM157704-01","funderName":"National Institutes of Health (United States)","awardRecipient":"Dahlberg"},{"awardId":"IUSE # 1612252","funderName":"National Science Foundation (United States)","awardRecipient":"Dahlberg"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Western Washington University Office of Research and Sponsored Programs through an Undergraduate Research and Creative Opportunities Grant (B. Nelson)
","image":{"url":"https://portal.micropublication.org/uploads/afaff04335d1eb0d4e3f810503e3c2f9.png"},"imageCaption":"A. Simplified model of the ERAD pathway that UBC-6 and UBC-7 participate in. A misfolded protein (CPL-1Y32AW35A::YFP) is translocated to the cytosol where it is ubiquitylated and degraded by the proteasome. The UPRER reporter, Phsp-4::GFP, is active at low levels in basal conditions and is upregulated in response to ER stress. Model created using BioRender.
B. Schematic diagram of the coding regions of the ubc-6 and ubc-7 genes and their deletion alleles, gk3799 (Δubc-6, teal arrow) and gk5037 (Δubc-7, magenta arrow). Black boxes indicate exons, separated by spliced introns, which are designated by black linkers. White boxes indicate untranslated sequences.
C. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing CPL-1Y32AW35A::YFP under the control of the nhx-2 promoter (CPL-1**::YFP).
D. Quantification of the size, in pixels2, of puncta caused by CPL-1Y32AW35A::YFP (CPL-1**::YFP) accumulation in wildtype, Δubc-6, Δubc-7 animals.
E. Quantification of the total CPL-1Y32AW35A::YFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
F. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter. All images were identical processed using the same parameters.
G. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, Δubc-7 animals.
H. Fluorescence images of wildtype, Δubc-6, and Δubc-7 animals expressing GFP under the control of the hsp-4 promoter in control (DMSO) and stress (2.5 ng/mL tunicamycin) conditions. Images for DMSO and tunicamycin treatments were processed separately to preserve visible differences.
I. Quantification of the total GFP fluorescence per animal in wildtype, Δubc-6, and Δubc-7 animals treated with DMSO or tunicamycin.
*, p<0.05; **, p<0.001; ***, p<0.0001; ns, not significant. Tukey post-hoc test
","imageTitle":"Model of ERAD and fluorescence imaging of C. elegans lacking ubc-6 or ubc-7
","methods":"Animal maintenance and crossing
Animals were reared at 20°C on NGM. Genetic crosses were performed using established methods and genotypes were confirmed as outlined below.
Genotyping
Worms were lysed in a buffer of 50mM KCl, 10mM Tris, 2.5mM MgCl2, 0.45% Tween 20, and 0.45% NP-40; proteinase K was added in 200μg/mL final concentration. Lysate was used in PCRs with Apex 2.0X Taq RED Master Mix (1.5mM) from Genesee Scientific according to manufacturer directions (annealing: 55 C, 30 seconds; 90 second extension) with the following primers.
Primer | Sequence |
ubc-6 FWD | 5'-ATGAGTGAGCAGT-3' |
ubc-6 REV | 5'-ACTCGGAACCGTC-3' |
ubc-7 FWD | 5' gacaggtaccaaaaaaTGGAGCAATCCTC -3'* |
ubc-7 REV | 5'-gacggaattcTCATTCTTCTTGACTTCTGCG -3'* |
* lowercase sequence indicates exogenous sequence used for unrelated subcloning experiments.
Tunicamycin treatment
L4 stage C. elegans were incubated in 2.5 ng/ul tunicamycin or DMSO as vehicle control (both Fisher Scientific) for 4 hours with microfuge lids open at 20 degrees C. After incubation, animals were settled by microcentrifugation at 2000 rpm for 30 seconds and liquid was removed from the worm pellet.
Fluorescence widefield microscopy
Animals from all conditions for each experiment were imaged on the same day over several imaging sessions. Slides were prepared using 1% levamisole in 1% agarose in H2O. L4 worms were collected from plates and placed into 1% levamisole in M9 onto agarose slides. Using an eyebrow pick, worms were moved into stacks (arranged side by side in a row) on the slides. Images were collected on the Leica iDM6000 widefield microscope at 10X, 1 gain, and 200 ms exposure.
Quantification/Statistical analysis
Microscope images were processed in ImageJ (FIJI). To quantify GFP expression under the control of the hsp-4 promoter (Phsp-4::GFP), the perimeter of each animal was traced, and mean fluorescence brightness was recorded and subtracted from corresponding background. To quantify CPL-1Y32AW35A::YFP puncta, images of animals with the Pnhx-2::CPL-1Y32AW35A::YFP transgene were enhanced in FIJI using the difference of Gaussians algorithm. Images were then thresholded using the IsoData algorithm with a lower intensity limit of 10 and upper intensity limit of 225. A watershed was applied to isolate overlapping puncta and the area of each punctum was calculated. Stacks of worms were traced to exclude surrounding noise. Significance (p < 0.05) was determined using an ANOVA followed by a Tukey post-hoc test. Statistical tests and graphs were done in Rstudio version 4.5.3.
","reagents":"C. elegans strains
Name | Genotype | Purpose | Source |
Wildtype | Control for no YFP expression | CGC | |
Control for baseline level of CPL-1Y32AW35A::YFP accumulation | CGC, T. Hoppe lab | ||
Control for expression of hsp-4 | CGC | ||
Experimental strain for CPL-1Y32AW35A::YFP accumulation with ubc-7 deletion | This manuscript | ||
Experimental strain for CPL-1Y32AW35A::YFP accumulation with ubc-6 deletion | This manuscript |
The endoplasmic reticulum (ER) regulates post-translational protein folding and maturation to maintain protein homeostasis and limit the accumulation of misfolded proteins. Accumulation of misfolded protein at the ER activates the ER Unfolded Protein Response (UPRER) and the downstream ER-associated protein degradation (ERAD) pathway, which alleviates the build-up of unfolded and/or unprocessed proteins (Christianson and Carvalho 2022). ERAD is facilitated by a protein complex that includes ubiquitin conjugating enzymes that target substrates on the cytosolic side of the ER membrane (Christianson and Ye 2014; Christianson et al. 2023). Ubiquitylated ERAD targets are retrotranslocated from the ER to the cytosol and degraded by the proteasome (Vembar and Brodsky 2008; Neal et al. 2018; Lopata et al. 2020; Christianson and Carvalho 2022). In S. cerevisiae, two ERAD E2 ligases, Ubc6 and Ubc7 have complementary roles in which they sequentially ubiquitylate misfolded proteins translocated from the ER lumen to the cytosol (Weber et al. 2016; Christianson and Carvalho 2022). Ubc6 adds the primary ubiquitin to lysines or hydroxylated amino acid residues (serine or threonine) on target proteins, while Ubc7 elongates the ubiquitin chain. Ubc7 has a more expansive role in ERAD than Ubc6 as it is also able to prime target proteins with ubiquitin on lysine residues prior to elongation of the ubiquitin chain (Weber et al. 2016). The mammalian orthologs to Ubc6 and Ubc7, Ube2j1/Ube2j2 and Ube2g1/Ube2g2, respectively, work with E3 conjugases to maintain ER protein homeostasis in a similar manner (Wang et al. 2009; Burr et al. 2013; Christianson and Ye 2014; Christianson and Carvalho 2022; Abdul Rehman et al. 2024).
In C. elegans, there are two predicted orthologs of ER-associated E2 conjugating enzymes, UBC-6 and UBC-7 (Figure 1A). We investigated the roles of the ubc-6 and ubc-7 genes in C. elegans by characterizing the effect of deletion alleles on the UPRER and ERAD. We used genetic reporters to measure accumulation of misfolded proteins and activation of the UPRER in strains with CRISPR-Cas9-targeted deletion alleles of either ubc-6 (gk3799) (hereafter Δubc-6) or ubc-7 (gk5037) (Δubc-7) (Figure 1B), which we crossed into two different reporter lines. To measure the accumulation of misfolded proteins, we used Pnhx-2::CPL-1Y32AW35A::YFP (hhIs113), which serves as a reporter for ERAD function because CPL-1Y32AW35A::YFP is engineered to misfold, but is normally cleared from the ER lumen via ERAD (Miedel et al. 2012; Efstathiou et al. 2022). In order to observe the activation of the UPRER, we measured the expression of GFP under the control of the hsp-4 promoter Phsp-4::GFP (zcIs4), whose activity increases in response to heat shock and other forms of ER stress (Ye et al. 2004; Walter and Ron 2011; Bar-Ziv et al. 2020). Using the Δubc-6 and Δubc-7 alleles, we observed how misfolded protein accumulation and regulation of a UPRER reporter depend on each gene in C. elegans.
We used fluorescence microscopy to determine how the loss of the E2 conjugases ubc-6 and ubc-7 affect the accumulation of CPL-1Y32AW35A and the levels of hsp-4 expression. We measured CPL-1Y32AW35A::YFP accumulation by quantifying the size of CPL-1Y32AW35A::YFP puncta in Δubc-6 and Δubc-7 animals. Compared to wildtype, the average puncta area of CPL-1Y32AW35A::YFP that accumulated was statistically unchanged in Δubc-6 animals (21.7 pixels2 compared to 14.5 pixels2 in wildtype), whereas Δubc-7 animals had larger average puncta size (53.8 pixels2 compared to 14.5 pixels2 in wildtype and 21.7 pixels2 in Δubc-6 animals) (Figure 1C, D). The overall signal intensity correlated with the puncta area since wildtype and Δubc-6 animals had essentially the same overall fluorescence, while Δubc-7 animals were, on average 1.45 times brighter (Figure 1E). Immunoblot analysis (not shown) confirmed these observations. This result aligns with previous results from yeast, in which Ubc6 mutants accumulate less misfolded protein in the ER lumen compared to Ubc7 mutants (Weber et al. 2016). Because Δubc-7 animals have high accumulation of CPL-1Y32AW35A::YFP, we expected that they would have a similarly high expression of the Phsp-4::GFP reporter, compared to wildtype and Δubc-6 animals. However, animals lacking ubc-6 exhibited 1.75 times higher GFP expression compared to the wildtype, whereas animals lacking ubc-7 had similar GFP levels as wildtype (Figure 1 F,G). These results were also confirmed by immunoblotting (not shown). To determine if either mutant was more sensitive to an acute ER stressor, we treated animals with tunicamycin, a compound that prevents protein glycosylation in the ER, or DMSO as a vehicle control. All animals were affected by tunicamycin as measured by Phsp-4::GFP activation, which shows that Δubc-7 animals are able to respond to ER stress. We also found that animals lacking ubc-6 had significantly higher Phsp-4::GFP activation in response to tunicamycin compared with both wild-type and Δubc-7 animals (Figure 1, H, I).
Our results show that loss of ubc-7 has a detrimental effect on the ability of cells to clear the misfolded protein CPL-1Y32AW35A::YFP, which suggests that it is particularly important for maintaining ER protein homeostasis. Despite the increase in protein CPL-1Y32AW35A::YFP accumulation, loss of ubc-7 does not lead to an upregulation of the Phsp-4::GFP reporter over wildtype levels, which suggests that either Δubc-7 animals are unable to mount a higher UPRER response via hsp-4, or that a redundant pathway is activated but not detected in our assays. Our quantification of Phsp-4::GFP expression also shows that loss of ubc-6 leads to the activation of the UPRER reporter above the wildtype levels in control and stress (tunicamycin) conditions. Therefore, although Δubc-6 animals do not accumulate significantly more CPL-1Y32AW35A::YFP than wildtype animals, the loss of ubc-6 induces an elevated UPRER response. In conclusion, while ubc-6 and ubc-7 are necessary for ERAD and activation of the UPR, they seem to have independent, though related, functions. These experiments were carried out in single mutants, but it will be interesting to determine the epistatic relationship of ubc-6 and ubc-7, especially because Ubc6 and Ubc7 deletions in yeast can have additive effects on stress responses (Owutey et al. 2024). In addition, future work will help determine if UBC-6 and UBC-7 share the biochemical features of their yeast and mammalian orthologs, such as preferences for different amino acids and addition of primary ubiquitin versus chain elongation.
","references":[{"reference":"Abdul Rehman SA, Cazzaniga C, Di Nisio E, Antico O, Knebel A, Johnson C, et al., De Cesare. 2024. Discovery and characterization of noncanonical E2-conjugating enzymes. Science Advances 10: 10.1126/sciadv.adh0123.
","pubmedId":"","doi":"10.1126/sciadv.adh0123"},{"reference":"Bar Ziv R, Frakes AE, Higuchi Sanabria R, Bolas T, Frankino PA, Gildea HK, Metcalf MG, Dillin A. 2020. Measurements of Physiological Stress Responses in C. Elegans. Journal of Visualized Experiments: JoVE 5137.","pubmedId":"","doi":"10.3791/61001"},{"reference":"Burr ML, Boomen DJH, Bye H, Antrobus R, Wiertz EJ, Lehner PJ. 2013. MHC class I molecules are preferentially ubiquitinated on endoplasmic. Proceedings of the National Academy of Sciences of the United States o. 110: 14290-14295. 4816.","pubmedId":"","doi":"10.1073/pnas.1303380110"},{"reference":"Christianson JC, Carvalho P. 2022. Order through destruction: how ER‐associated protein degradation contr. The EMBO Journal. 41: e109845. 4515.","pubmedId":"","doi":"10.15252/embj.2021109845"},{"reference":"Christianson JC, Jarosch E, Sommer T. 2023. Mechanisms of substrate processing during ER-associated protein degrad. Nature Reviews. Molecular Cell Biology. 24: 777-796. 4812.","pubmedId":"","doi":"10.1038/s41580-023-00633-8"},{"reference":"Christianson JC, Ye Y. 2014. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nature Structural & Molecular Biology. 21: 325-335. 5141.","pubmedId":"","doi":"10.1038/nsmb.2793"},{"reference":"Efstathiou S, Ottens F, Schutter LS, Ravanelli S, Charmpilas N, Gutschmidt A, et al., Hoppe T. 2022. ER-associated RNA silencing promotes ER quality control. Nature Cell Biology. 24: 1714-1725. 4541.","pubmedId":"","doi":"10.1038/s41556-022-01025-4"},{"reference":"Lopata A, Kniss A, Lohr F, Rogov VV, Dotsch V. 2020. Ubiquitination in the ERAD Process. International Journal of Molecular Sciences. 21: 5369. 5135.","pubmedId":"","doi":"10.3390/ijms21155369"},{"reference":"Miedel MT, Graf NJ, Stephen KE, Long OS, Pak SC, Perlmutter DH, Silverman GA, Luke CJ. 2012. A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticu. PloS One. 7: e40145. 4576.","pubmedId":"","doi":"10.1371/journal.pone.0040145"},{"reference":"Neal S, Jaeger PA, Duttke SH, Benner C, K Glass C, Ideker T, Hampton RY. 2018. The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Me. Molecular Cell. 69: 306-320.e4. 4564.","pubmedId":"","doi":"10.1016/j.molcel.2017.12.012"},{"reference":"Owutey SL, Procuniar KA, Akoto E, Davis JC, Vachon RM, O Malley LF, et al., Rubenstein EM. 2024. Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating. microPublication Biology. 2024 5139.","pubmedId":"","doi":"10.17912/micropub.biology.001276"},{"reference":"Vembar SS, Brodsky JL. 2008. One step at a time: endoplasmic reticulum-associated degradation. Nature Reviews. Molecular Cell Biology. 9: 944-957. 715.","pubmedId":"","doi":"10.1038/nrm2546"},{"reference":"Walter P, Ron D. 2011. The Unfolded Protein Response: From Stress Pathway to Homeostatic Regu. Science. 334: 1081-1086. 4533.","pubmedId":"","doi":""},{"reference":"Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJHJ, Hansen TH. 2009. Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degrada. Journal of Cell Biology. 187: 655-668. 4928.","pubmedId":"","doi":"10.1083/jcb.200908036"},{"reference":"Weber A, Cohen I, Popp O, Dittmar G, Reiss Y, Sommer T, Ravid T, Jarosch E. 2016. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expa. Molecular Cell. 63: 827-839. 1012.","pubmedId":"","doi":"10.1016/j.molcel.2016.07.020"},{"reference":"Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA. 2004. A membrane protein complex mediates retro-translocation from the ER lu. Nature. 429: 841-847. 4535.","pubmedId":"","doi":"10.1038/nature02656"}],"title":"Effects of loss of the E2 conjugating enzymes ubc-6 and ubc-7 on the unfolded protein response and protein homeostasis in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes 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