Aberrant and excess proteins are destroyed by compartment-specific protein quality control mechanisms. In Saccharomyces cerevisiae, endoplasmic reticulum (ER)-associated degradation (ERAD) and inner nuclear membrane (INM)-associated degradation (INMAD) requires the Ubc6 and Ubc7 ubiquitin-conjugating enzymes. Ubc6 is an integral membrane protein. By contrast, Ubc7 is a soluble protein tethered to the ER and INM membranes by the transmembrane protein Cue1. Here, we assessed the requirement of Cue1 in resisting proteotoxic stress. CUE1 loss sensitized cells to hygromycin B to a similar extent as UBC7 deletion, consistent with a shared role for Cue1 and Ubc7 in ER and INM protein quality control.
","acknowledgements":"Experiments to determine sensitivity of cue1Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Kalie Adams, Reva Allam, Alexis Baynes, Dilynn Blair, Olivia Coltharp, Piper Conway, La’Naviya Farries, Morgan Hensley, Sydney Hrehor, Madyson Jones, Zachary Mann, Abigail McLaughlin, Dailey Morris, Skyler Petro, Ken Reed, Mercedes Sanders, Alonna Williams) and high school students in the Spring 2025 Genetics course at the Indiana Academy for Science, Mathematics, and Humanities (Sylvester Acherekoh, Sebastian Bilbo, Jackson Booth, Sophia Bortolotti, Natalie Garringer, MaKaela Gist, Ella Johnson, Victoria Johnson, Emma-Lee Kennedy, Elizabeth Long, Cole McDermott, Jayden McDole, Kha Nguyen, Mallory Saldana). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model, as described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Christopher Hickey for generously sharing yeast strains. We thank the Saccharomyces Genome Database for serving as an invaluable repository for yeast genetic information (Wong et al., 2023). We thank the Ball State University Division of Online and Strategic Learning for supporting an initiative to transform undergraduate laboratory courses into authentic research-based learning experiences. This manuscript is dedicated to the memory of our friend, Ian Tesch, whose generosity of spirit was unmatched. Thank you for being a part of our team.
","authors":[{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","investigation","validation","writing_reviewEditing"],"email":"james.avaala@bsu.edu","firstName":"James A","lastName":"Avaala","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0008-4575-7329"},{"affiliations":["Ball State University","The Indiana Academy for Science, Mathematics, and Humanities"],"departments":["Department of Biology",""],"credit":["investigation","writing_reviewEditing","validation"],"email":"melissa.palackel@bsu.edu","firstName":"Melissa","lastName":"Palackel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0007-2695-8603"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["resources","investigation"],"email":"IanTesch90@gmail.com","firstName":"Ian M","lastName":"Tesch","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Ball State University","Midwestern University"],"departments":["Department of Biology","Chicago College of Osteopathic Medicine"],"credit":["supervision","writing_reviewEditing"],"email":"joseph.gumina@midwestern.edu","firstName":"Joseph","lastName":"Gumina","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"chance.creviston@bsu.edu","firstName":"Chance S","lastName":"Creviston","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0005-8785-1581"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"affiliations":["The Indiana Academy for Science, Mathematics, and Humanities","Ball State University"],"departments":["Division of Natural, Physical, and Computer Sciences","Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"justin.crowder@bsu.edu","firstName":"Justin J","lastName":"Crowder","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-6327-6888"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["fundingAcquisition","supervision","writing_originalDraft"],"email":"emrubenstein@bsu.edu","firstName":"Eric M","lastName":"Rubenstein","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0003-4983-1430"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This work was funded by NIH grant R15 GM111713 (EMR). JAA was supported by a Ball State University Aspire student research grant. Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology and the Indiana Academy for Science, Mathematics, and Humanities. This project was conceived while EMR was supported in part by a Ball State University Excellence in Teaching award (sponsored by the Ball State University Division of Online and Strategic Learning and the Office of the Provost).
","image":{"url":"https://portal.micropublication.org/uploads/2ccf6d16e98491ffa45ecb3e07fba2cb.png"},"imageCaption":"(A) By anchoring the ubiquitin-conjugating enzyme Ubc7 to the ER and inner nuclear membranes, Cue1 contributes to protein degradation mediated by the Hrd1, Doa10, and Asi ubiquitin ligases. See text for details. (B) Sixfold serial dilutions of yeast of the indicated genotypes were spotted on medium lacking (No Drug) or containing hygromycin B at the indicated concentrations. cue1Δ #1 and cue1Δ #2 are two independent clones resulting from homologous recombination-mediated gene replacement. Plates were incubated at 30°C and imaged at the indicated times. (C) As in (B), but using strains from a distinct genetic background. Experiments were performed three times.
","imageTitle":"CUE1 confers resistance to hygromycin B
","methods":"CUE1 gene replacement
To generate yeast strains VJY336 and VJY337, CUE1 was replaced by natMX4 through homologous recombination. A 1464-bp nat4MX4 cassette with termini possessing sequences flanking the CUE1 gene was PCR-amplified from pAG25 (Goldstein & McCusker, 1999) using primers VJR253 (5’ CGCCATAAAGCATTACAATCTACGATCGCGCAAACTTTTTTCTTTTGGCCCACATACGATTTAGGTGACAC) and VJR254 (5’ TTATGCGCATTATGGGCACACTTGCGTGTTCCCGACAAGCACTTAAGCGTAATACGACTCACTATAGGGAG 3’). The natMX4 cassette was introduced into VJY6 yeast by lithium acetate transformation (Guthrie & Fink, 2004), followed by selection on medium containing nourseothricin. Successful integration was verified by PCR at 5’ and 3’ recombination junctions.
Growth assays
Sixfold serial dilutions of indicated yeast strains were spotted onto yeast extract-peptone-dextrose medium lacking or containing hygromycin B (Gibco) at the indicated concentrations (Guthrie & Fink, 2004) followed by incubation at 30°C for the indicated time, as described in (Watts et al., 2015).
","reagents":"Name | Genotype | Figure | Reference |
VJY6 (alias MHY500) | MATa his3-ΔD200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 | 1B | Chen et al., 1993 |
VJY50 (alias MHY551) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc7Δ::LEU2 | 1B | Chen et al., 1993 |
VJY324 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 cue1Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY336 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 1 | 1B | This study |
VJY337 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 2 | 1B | This study |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1C | Tong et al., 2001 |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY1098 (MHY11132, ABM297) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 asi1Δ::kanMX6 | 1B | Mehrtash & Hochstrasser, 2023 |
Cellular and organismal health depends on protein quality control (Badawi et al., 2023; Guerriero & Brodsky, 2012; Higuchi-Sanabria et al., 2018). Aberrant and excess endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed by ER-associated degradation (ERAD) and INM-associated degradation (INMAD), respectively (reviewed in (Mehrtash & Hochstrasser, 2019)). In Saccharomyces cerevisiae, the Hrd1 and Doa10 ubiquitin ligases (E3s) mediate ERAD of soluble, transmembrane, and translocon-clogging proteins at the ER membrane (Carvalho et al., 2006; Huyer et al., 2004; Metzger et al., 2008; Rubenstein et al., 2012; Runnebohm, Richards, et al., 2020; Sato et al., 2009; Swanson et al., 2001) (Figure 1A). Doa10 also localizes to the INM, where it and the heterotrimeric E3 Asi (comprised of Asi1, Asi2, and Asi3) catalyze INMAD of transmembrane INM and soluble nucleoplasmic proteins (Deng & Hochstrasser, 2006; Foresti et al., 2014; Khmelinskii et al., 2014; Swanson et al., 2001). Additional E3s, including Ubr1, Ltn1, and the anaphase-promoting complex, contribute to ER and INM protein quality control (Arakawa et al., 2016; Crowder et al., 2015; Koch et al., 2019; Ruggiano et al., 2016; Stolz et al., 2013).
Ubiquitin-conjugating enzymes (E2s) deliver ubiquitin molecules to E3s for substrate ligation. Hrd1 functions with the E2 Ubc7, while Doa10 and Asi work with two E2s, Ubc6 and Ubc7 (Bays et al., 2001; Foresti et al., 2014; Khmelinskii et al., 2014; Lips et al., 2020; Plemper et al., 1999; Swanson et al., 2001). Ubc7 is anchored at the ER membrane, stabilized, and activated by the transmembrane protein Cue1 (Biederer et al., 1997; Kostova et al., 2009; Ravid & Hochstrasser, 2007). While a direct Cue1 homolog is not present in mammals, E3s and E3-accessory proteins anchor, stabilize, and activate the Ubc7 homolog UBE2G2 to ensure functional ERAD in mammalian cells (Chen et al., 2006; Das et al., 2009; Smith et al., 2021).
The aminoglycoside hygromycin B distorts ribosome A sites (Brodersen et al., 2000; Ganoza & Kiel, 2001), likely increasing synthesis of the types of aberrant proteins that contribute to age-related neurodegenerative and other diseases (Akbergenov et al., 2025). We and others have demonstrated that several genes required for efficient protein quality control enable cells to resist stress caused by hygromycin B (Akoto et al., 2025; Bengtson & Joazeiro, 2010; Chuang & Madura, 2005; Daraghmi et al., 2023; Flagg et al., 2023; Jaeger et al., 2018; Niekamp et al., 2019; Verma et al., 2013). Deletion of genes encoding ERAD and INMAD E2s and E3s profoundly sensitizes yeast to hygromycin B (Crowder et al., 2015; Doss et al., 2023; Runnebohm, Evans, et al., 2020; Turk et al., 2023; Woodruff et al., 2021). Simultaneous loss of E3s Hrd1, Doa10, and Asi1 causes greater hygromycin B sensitivity than loss of E2s Ubc6 and Ubc7 (Owutey et al., 2024), consistent with compensatory contributions by other E2s to ERAD and INMAD (Bays et al., 2001). Large-scale genetic analyses indicated CUE1 deletion reduces hygromycin B tolerance (Brown et al., 2006; Dudley et al., 2005), but this result has not been validated in targeted, small-scale studies.
We tested the hypothesis that Cue1 is required for proteotoxic stress resistance. We compared cellular fitness of wild type yeast, two independent CUE1 knockouts, yeast lacking the E2 Ubc7, and yeast lacking the three major ERAD and INMAD E3s (hrd1Δ doa10Δ asi1Δ) (Figure 1B) on media lacking or containing hygromycin B. Deletion of CUE1 and UBC7 sensitized yeast to hygromycin B to a similar extent, consistent with a shared role in protein quality control. As previously observed (Owutey et al., 2024), deletion of genes encoding ERAD and INMAD E3s caused a more profound growth defect in the presence of drug. Hygromycin B sensitivity of cue1Δ yeast from a distinct genetic background validates this result (Figure 1C).
Hypersensitivity of cue1Δ and ubc7Δ yeast to proteotoxic stress is consistent with function of a Cue1-Ubc7 subcomplex in ERAD and INMAD (Biederer et al., 1997; Buchanan et al., 2016; Khmelinskii et al., 2014; Pantazopoulou et al., 2016). Large-scale studies have demonstrated that CUE1 promotes resistance to a range of stressors, including transition metals, which oxidatively damage proteins (Ruotolo et al., 2008; Zhao et al., 2020), genotoxic agents (Alamgir et al., 2010; Gaytan et al., 2013; Kapitzky et al., 2010; Ogbede et al., 2021), and sterol biosynthesis disruption (Kapitzky et al., 2010). Our results support a crucial function for Cue1 in maintaining proteostasis in yeast.
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Inner Nuclear Membrane Asi Ubiquitin Ligase Catalytic Subunits Asi1p and Asi3p, but not Asi2p, confer resistance to aminoglycoside hygromycin B in Saccharomyces cerevisiae. MicroPubl Biol. 2021 43.","pubmedId":"34095778","doi":"10.17912/micropub.biology.000403"},{"reference":"Zhao YY, Cao CL, Liu YL, Wang J, Li SY, Li J, Deng Y. 2020. Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast. Biochim Biophys Acta Gen Subj. 1864: 129516. 50.","pubmedId":"31904504","doi":"10.1016/j.bbagen.2020.129516"}],"title":"Ubiquitin-conjugating enzyme membrane anchor Cue1 confers resistance to hygromycin B in Saccharomyces cerevisiae
","reviews":[],"curatorReviews":[]},{"id":"26bbf173-c3a4-44f7-8847-b44b380c3413","decision":"revise","abstract":"Aberrant and excess proteins are destroyed by compartment-specific protein quality control mechanisms. In Saccharomyces cerevisiae, endoplasmic reticulum (ER)-associated degradation (ERAD) and inner nuclear membrane (INM)-associated degradation (INMAD) requires the Ubc6 and Ubc7 ubiquitin-conjugating enzymes. Ubc6 is an integral membrane protein. By contrast, Ubc7 is a soluble protein tethered to the ER and INM membranes by the transmembrane protein Cue1. Here, we assessed the requirement of Cue1 in resisting proteotoxic stress. CUE1 loss sensitized cells to hygromycin B to a similar extent as UBC7 deletion, consistent with a shared role for Cue1 and Ubc7 in ER and INM protein quality control.
","acknowledgements":"Experiments to determine sensitivity of cue1Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Kalie Adams, Reva Allam, Alexis Baynes, Dilynn Blair, Olivia Coltharp, Piper Conway, La’Naviya Farries, Morgan Hensley, Sydney Hrehor, Madyson Jones, Zachary Mann, Abigail McLaughlin, Dailey Morris, Skyler Petro, Ken Reed, Mercedes Sanders, Alonna Williams) and high school students in the Spring 2025 Genetics course at the Indiana Academy for Science, Mathematics, and Humanities (Sylvester Acherekoh, Sebastian Bilbo, Jackson Booth, Sophia Bortolotti, Natalie Garringer, MaKaela Gist, Ella Johnson, Victoria Johnson, Emma-Lee Kennedy, Elizabeth Long, Cole McDermott, Jayden McDole, Kha Nguyen, Mallory Saldana). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model, as described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Christopher Hickey for generously sharing yeast strains. We thank the Saccharomyces Genome Database for serving as an invaluable repository for yeast genetic information (Wong et al., 2023). We thank the Ball State University Division of Online and Strategic Learning for supporting an initiative to transform undergraduate laboratory courses into authentic research-based learning experiences. This manuscript is dedicated to the memory of our friend, Ian Tesch, whose generosity of spirit was unmatched. Thank you for being a part of our team.
","authors":[{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","investigation","validation","writing_reviewEditing"],"email":"james.avaala@bsu.edu","firstName":"James A","lastName":"Avaala","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0008-4575-7329"},{"affiliations":["Ball State University","The Indiana Academy for Science, Mathematics, and Humanities"],"departments":["Department of Biology",""],"credit":["investigation","writing_reviewEditing","validation"],"email":"melissa.palackel@bsu.edu","firstName":"Melissa","lastName":"Palackel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0007-2695-8603"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["resources","investigation"],"email":"IanTesch90@gmail.com","firstName":"Ian M","lastName":"Tesch","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Ball State University","Midwestern University"],"departments":["Department of Biology","Chicago College of Osteopathic Medicine"],"credit":["supervision","writing_reviewEditing"],"email":"joseph.gumina@midwestern.edu","firstName":"Joseph","lastName":"Gumina","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"chance.creviston@bsu.edu","firstName":"Chance S","lastName":"Creviston","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0005-8785-1581"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"affiliations":["The Indiana Academy for Science, Mathematics, and Humanities","Ball State University"],"departments":["Division of Natural, Physical, and Computer Sciences","Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"justin.crowder@bsu.edu","firstName":"Justin J","lastName":"Crowder","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-6327-6888"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["fundingAcquisition","supervision","writing_originalDraft"],"email":"emrubenstein@bsu.edu","firstName":"Eric M","lastName":"Rubenstein","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0003-4983-1430"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This work was funded by NIH grant R15 GM111713 (EMR). JAA was supported by a Ball State University Aspire student research grant. Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology and the Indiana Academy for Science, Mathematics, and Humanities. This project was conceived while EMR was supported in part by a Ball State University Excellence in Teaching award (sponsored by the Ball State University Division of Online and Strategic Learning and the Office of the Provost).
","image":{"url":"https://portal.micropublication.org/uploads/2ccf6d16e98491ffa45ecb3e07fba2cb.png"},"imageCaption":"(A) By anchoring the ubiquitin-conjugating enzyme Ubc7 to the ER and inner nuclear membranes, Cue1 contributes to protein degradation mediated by the Hrd1, Doa10, and Asi ubiquitin ligases. See text for details. (B) Sixfold serial dilutions of yeast of the indicated genotypes were spotted on medium lacking (No Drug) or containing hygromycin B at the indicated concentrations. cue1Δ #1 and cue1Δ #2 are two independent clones resulting from homologous recombination-mediated gene replacement. Plates were incubated at 30°C and imaged at the indicated times. (C) As in (B), but using strains from a distinct genetic background. Experiments were performed three times.
","imageTitle":"CUE1 confers resistance to hygromycin B
","methods":"CUE1 gene replacement
To generate yeast strains VJY336 and VJY337, CUE1 was replaced by natMX4 through homologous recombination. A 1464-bp nat4MX4 cassette with termini possessing sequences flanking the CUE1 gene was PCR-amplified from pAG25 (Goldstein & McCusker, 1999) using primers VJR253 (5’ CGCCATAAAGCATTACAATCTACGATCGCGCAAACTTTTTTCTTTTGGCCCACATACGATTTAGGTGACAC) and VJR254 (5’ TTATGCGCATTATGGGCACACTTGCGTGTTCCCGACAAGCACTTAAGCGTAATACGACTCACTATAGGGAG 3’). The natMX4 cassette was introduced into VJY6 yeast by lithium acetate transformation (Guthrie & Fink, 2004), followed by selection on medium containing nourseothricin. Successful integration was verified by PCR at 5’ and 3’ recombination junctions.
Growth assays
Sixfold serial dilutions of indicated yeast strains were spotted onto yeast extract-peptone-dextrose medium lacking or containing hygromycin B (Gibco) at the indicated concentrations (Guthrie & Fink, 2004) followed by incubation at 30°C for the indicated time, as described in (Watts et al., 2015).
","reagents":"Name | Genotype | Figure | Reference |
VJY6 (alias MHY500) | MATa his3-ΔD200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 | 1B | Chen et al., 1993 |
VJY50 (alias MHY551) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc7Δ::LEU2 | 1B | Chen et al., 1993 |
VJY324 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 cue1Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY336 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 1 | 1B | This study |
VJY337 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 2 | 1B | This study |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1C | Tong et al., 2001 |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY1098 (MHY11132, ABM297) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 asi1Δ::kanMX6 | 1B | Mehrtash & Hochstrasser, 2023 |
Cellular and organismal health depends on protein quality control (Badawi et al., 2023; Guerriero & Brodsky, 2012; Higuchi-Sanabria et al., 2018). Aberrant and excess endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed by ER-associated degradation (ERAD) and INM-associated degradation (INMAD), respectively (reviewed in (Mehrtash & Hochstrasser, 2019)). In Saccharomyces cerevisiae, the Hrd1 and Doa10 ubiquitin ligases (E3s) mediate ERAD of soluble, transmembrane, and translocon-clogging proteins at the ER membrane (Carvalho et al., 2006; Huyer et al., 2004; Metzger et al., 2008; Rubenstein et al., 2012; Runnebohm, Richards, et al., 2020; Sato et al., 2009; Swanson et al., 2001) (Figure 1A). Doa10 also localizes to the INM, where it and the heterotrimeric E3 Asi (comprised of Asi1, Asi2, and Asi3) catalyze INMAD of transmembrane INM and soluble nucleoplasmic proteins (Deng & Hochstrasser, 2006; Foresti et al., 2014; Khmelinskii et al., 2014; Swanson et al., 2001). Additional E3s, including Ubr1, Ltn1, and the anaphase-promoting complex, contribute to ER and INM protein quality control (Arakawa et al., 2016; Crowder et al., 2015; Koch et al., 2019; Ruggiano et al., 2016; Stolz et al., 2013).
Ubiquitin-conjugating enzymes (E2s) deliver ubiquitin molecules to E3s for substrate ligation. Hrd1 functions with the E2 Ubc7, while Doa10 and Asi work with two E2s, Ubc6 and Ubc7 (Bays et al., 2001; Foresti et al., 2014; Khmelinskii et al., 2014; Lips et al., 2020; Plemper et al., 1999; Swanson et al., 2001). Ubc7 is anchored at the ER membrane, stabilized, and activated by the transmembrane protein Cue1 (Biederer et al., 1997; Kostova et al., 2009; Ravid & Hochstrasser, 2007). While a direct Cue1 homolog is not present in mammals, E3s and E3-accessory proteins anchor, stabilize, and activate the Ubc7 homolog UBE2G2 to ensure functional ERAD in mammalian cells (Chen et al., 2006; Das et al., 2009; Smith et al., 2021).
The aminoglycoside hygromycin B distorts ribosome A sites (Brodersen et al., 2000; Ganoza & Kiel, 2001), likely increasing synthesis of the types of aberrant proteins that contribute to age-related neurodegenerative and other diseases (Akbergenov et al., 2025). We and others have demonstrated that several genes required for efficient protein quality control enable cells to resist stress caused by hygromycin B (Akoto et al., 2025; Bengtson & Joazeiro, 2010; Chuang & Madura, 2005; Daraghmi et al., 2023; Flagg et al., 2023; Jaeger et al., 2018; Niekamp et al., 2019; Verma et al., 2013). Deletion of genes encoding ERAD and INMAD E2s and E3s profoundly sensitizes yeast to hygromycin B (Crowder et al., 2015; Doss et al., 2023; Runnebohm, Evans, et al., 2020; Turk et al., 2023; Woodruff et al., 2021). Simultaneous loss of E3s Hrd1, Doa10, and Asi1 causes greater hygromycin B sensitivity than loss of E2s Ubc6 and Ubc7 (Owutey et al., 2024), consistent with compensatory contributions by other E2s to ERAD and INMAD (Bays et al., 2001). Large-scale genetic analyses indicated CUE1 deletion reduces hygromycin B tolerance (Brown et al., 2006; Dudley et al., 2005), but this result has not been validated in targeted, small-scale studies.
We tested the hypothesis that Cue1 is required for proteotoxic stress resistance. We compared cellular fitness of wild type yeast, two independent CUE1 knockouts, yeast lacking the E2 Ubc7, and yeast lacking the three major ERAD and INMAD E3s (hrd1Δ doa10Δ asi1Δ) (Figure 1B) on media lacking or containing hygromycin B. Deletion of CUE1 and UBC7 sensitized yeast to hygromycin B to a similar extent, consistent with a shared role in protein quality control. As previously observed (Owutey et al., 2024), deletion of genes encoding ERAD and INMAD E3s caused a more profound growth defect in the presence of drug. Hygromycin B sensitivity of cue1Δ yeast from a distinct genetic background validates this result (Figure 1C).
Hypersensitivity of cue1Δ and ubc7Δ yeast to proteotoxic stress is consistent with function of a Cue1-Ubc7 subcomplex in ERAD and INMAD (Biederer et al., 1997; Buchanan et al., 2016; Khmelinskii et al., 2014; Pantazopoulou et al., 2016). Large-scale studies have demonstrated that CUE1 promotes resistance to a range of stressors, including transition metals, which oxidatively damage proteins (Ruotolo et al., 2008; Zhao et al., 2020), genotoxic agents (Alamgir et al., 2010; Gaytan et al., 2013; Kapitzky et al., 2010; Ogbede et al., 2021), and sterol biosynthesis disruption (Kapitzky et al., 2010). Our results support a crucial function for Cue1 in maintaining proteostasis in yeast.
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Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae. J Vis Exp: e52428. 57.","pubmedId":"25742191","doi":"10.3791/52428"},{"reference":"Wong ED, Miyasato SR, Aleksander S, Karra K, Nash RS, Skrzypek MS, et al., Cherry JM. 2023. Saccharomyces genome database update: server architecture, pan-genome nomenclature, and external resources. Genetics. 224 59.","pubmedId":"36607068","doi":"10.1093/genetics/iyac191"},{"reference":"Woodruff KA, Richards KA, Evans MD, Scott AR, Voas BM, Irelan CB, et al., Rubenstein EM. 2021. Inner Nuclear Membrane Asi Ubiquitin Ligase Catalytic Subunits Asi1p and Asi3p, but not Asi2p, confer resistance to aminoglycoside hygromycin B in Saccharomyces cerevisiae. MicroPubl Biol. 2021 43.","pubmedId":"34095778","doi":"10.17912/micropub.biology.000403"},{"reference":"Zhao YY, Cao CL, Liu YL, Wang J, Li SY, Li J, Deng Y. 2020. Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast. Biochim Biophys Acta Gen Subj. 1864: 129516. 50.","pubmedId":"31904504","doi":"10.1016/j.bbagen.2020.129516"}],"title":"Ubiquitin-conjugating enzyme membrane anchor Cue1 confers resistance to hygromycin B in Saccharomyces cerevisiae
","reviews":[{"reviewer":{"displayName":"Oliver Kerscher"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Stacia Engel"},"openAcknowledgement":false,"submitted":"1771972776439"}]},{"id":"e622ceb0-12f5-4200-bbb3-9766e228be25","decision":"accept","abstract":"Aberrant and excess proteins are destroyed by compartment-specific protein quality control mechanisms. In Saccharomyces cerevisiae, endoplasmic reticulum (ER)-associated degradation (ERAD) and inner nuclear membrane (INM)-associated degradation (INMAD) requires the Ubc6 and Ubc7 ubiquitin-conjugating enzymes. Ubc6 is an integral membrane protein. By contrast, Ubc7 is a soluble protein tethered to the ER and INM membranes by the transmembrane protein Cue1. Here, we assessed the requirement of Cue1 in resisting proteotoxic stress. CUE1 loss sensitized cells to hygromycin B to a similar extent as UBC7 deletion, consistent with a shared role for Cue1 and Ubc7 in ER and INM protein quality control.
","acknowledgements":"Experiments to determine sensitivity of cue1Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Kalie Adams, Reva Allam, Alexis Baynes, Dilynn Blair, Olivia Coltharp, Piper Conway, La’Naviya Farries, Morgan Hensley, Sydney Hrehor, Madyson Jones, Zachary Mann, Abigail McLaughlin, Dailey Morris, Skyler Petro, Ken Reed, Mercedes Sanders, Alonna Williams) and high school students in the Spring 2025 Genetics course at the Indiana Academy for Science, Mathematics, and Humanities (Sylvester Acherekoh, Sebastian Bilbo, Jackson Booth, Sophia Bortolotti, Natalie Garringer, MaKaela Gist, Ella Johnson, Victoria Johnson, Emma-Lee Kennedy, Elizabeth Long, Cole McDermott, Jayden McDole, Kha Nguyen, Mallory Saldana). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model, as described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Christopher Hickey for generously sharing yeast strains and plasmids. We thank the Saccharomyces Genome Database for serving as an invaluable repository for yeast genetic information (Wong et al., 2023). We thank the Ball State University Division of Online and Strategic Learning for supporting an initiative to transform undergraduate laboratory courses into authentic research-based learning experiences.
This manuscript is dedicated to the memory of our friend, Ian Tesch, whose generosity of spirit was unmatched. Thank you for being a part of our team.
","authors":[{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","investigation","validation","writing_reviewEditing"],"email":"james.avaala@bsu.edu","firstName":"James A","lastName":"Avaala","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0008-4575-7329"},{"affiliations":["Ball State University","The Indiana Academy for Science, Mathematics, and Humanities"],"departments":["Department of Biology",""],"credit":["investigation","writing_reviewEditing","validation"],"email":"melissa.palackel@bsu.edu","firstName":"Melissa","lastName":"Palackel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0007-2695-8603"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["resources","investigation"],"email":"IanTesch90@gmail.com","firstName":"Ian M","lastName":"Tesch","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Ball State University","Midwestern University"],"departments":["Department of Biology","Chicago College of Osteopathic Medicine"],"credit":["supervision","writing_reviewEditing"],"email":"joseph.gumina@midwestern.edu","firstName":"Joseph","lastName":"Gumina","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"chance.creviston@bsu.edu","firstName":"Chance S","lastName":"Creviston","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0005-8785-1581"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"affiliations":["The Indiana Academy for Science, Mathematics, and Humanities","Ball State University"],"departments":["Division of Natural, Physical, and Computer Sciences","Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"justin.crowder@bsu.edu","firstName":"Justin J","lastName":"Crowder","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-6327-6888"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["fundingAcquisition","supervision","writing_originalDraft"],"email":"emrubenstein@bsu.edu","firstName":"Eric M","lastName":"Rubenstein","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0003-4983-1430"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was funded by NIH grant R15 GM111713 (EMR). JAA was supported by a Ball State University Aspire student research grant. Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology and the Indiana Academy for Science, Mathematics, and Humanities. This project was conceived while EMR was supported in part by a Ball State University Excellence in Teaching award (sponsored by the Ball State University Division of Online and Strategic Learning and the Office of the Provost).
","image":{"url":"https://portal.micropublication.org/uploads/7f9217398ef1e2d02047c056d638ecd1.png"},"imageCaption":"(A) By anchoring the ubiquitin-conjugating enzyme Ubc7 to the endoplasmic reticulum and inner nuclear membranes, Cue1 contributes to protein degradation mediated by the Hrd1, Doa10, and Asi ubiquitin ligases. See text for details. (B-D) Sixfold serial dilutions of yeast of the indicated genotypes were spotted on medium lacking (No Drug) or containing hygromycin B at the indicated concentrations. Strains in (D) were transformed with an empty vector or a plasmid encoding Cue1. Strains used in the experiments in (B and D) are derived from the MHY500 genetic background, whereas strains in (C) are derived from BY4741. cue1Δ #1 and cue1Δ #2 are independent clones resulting from homologous recombination-mediated gene replacement. Plates were incubated at 30°C and imaged at the indicated times. Experiments in (B and C) were performed three times. The experiment in (D) was performed twice.
","imageTitle":"CUE1 confers resistance to hygromycin B
","methods":"CUE1 gene replacement
To generate yeast strains VJY336 and VJY337, CUE1 was replaced by natMX4 through homologous recombination. A 1464-bp nat4MX4 cassette with termini possessing sequences flanking the CUE1 gene was PCR-amplified from pAG25 (Goldstein & McCusker, 1999) using primers VJR253 (5’ CGCCATAAAGCATTACAATCTACGATCGCGCAAACTTTTTTCTTTTGGCCCACATACGATTTAGGTGACAC) and VJR254 (5’ TTATGCGCATTATGGGCACACTTGCGTGTTCCCGACAAGCACTTAAGCGTAATACGACTCACTATAGGGAG 3’). The natMX4 cassette was introduced into VJY6 yeast by lithium acetate transformation (Guthrie & Fink, 2004), followed by selection on medium containing nourseothricin. Successful integration was verified by PCR at 5’ and 3’ recombination junctions.
Growth assays
Sixfold serial dilutions of indicated yeast strains were spotted onto yeast extract-peptone-dextrose medium lacking or containing hygromycin B (Gibco) at the indicated concentrations (Guthrie & Fink, 2004) followed by incubation at 30°C for the indicated time, as described in (Watts et al., 2015).
","reagents":"Yeast strains used in this study.
Name | Genotype | Figure | Reference |
VJY6 (alias MHY500) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 | 1B, 1D | Chen et al., 1993 |
VJY50 (alias MHY551) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc7Δ::LEU2 | 1B | Chen et al., 1993 |
VJY324 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 cue1Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY336 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 1 | 1B, 1D | This study |
VJY337 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 2 | 1B | This study |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1C | Tong et al., 2001 |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY1098 (MHY11132, ABM297) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 asi1Δ::kanMX6 | 1B | Mehrtash & Hochstrasser, 2023 |
Plasmids used in this study.
Name | Description | Figure | Reference |
pVJ39 (pRS314) | empty vector (CEN, TRP1, AmpR) | 1D | Sikorski & Hieter, 1989 |
pVJ653 (p414-MET25-Cue1-3xFLAG) | 3xFlag-tagged Cue1 driven by MET25 promoter (CEN, TRP1, AmpR) | 1D | Mehrtash & Hochstrasser, 2022 |
Cellular and organismal health depends on protein quality control (Badawi et al., 2023; Guerriero & Brodsky, 2012; Higuchi-Sanabria et al., 2018). Aberrant and excess endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed by ER-associated degradation (ERAD) and INM-associated degradation (INMAD), respectively (reviewed in (Mehrtash & Hochstrasser, 2019)). In Saccharomyces cerevisiae, the Hrd1 and Doa10 ubiquitin ligases (E3s) mediate ERAD of soluble, transmembrane, and translocon-clogging proteins at the ER membrane (Carvalho et al., 2006; Huyer et al., 2004; Metzger et al., 2008; Rubenstein et al., 2012; Runnebohm, Richards, et al., 2020; Sato et al., 2009; Swanson et al., 2001) (Figure 1A). Doa10 also localizes to the INM, where it and the heterotrimeric E3 Asi (comprised of Asi1, Asi2, and Asi3) catalyze INMAD of transmembrane INM and soluble nucleoplasmic proteins (Deng & Hochstrasser, 2006; Foresti et al., 2014; Khmelinskii et al., 2014; Swanson et al., 2001). Additional E3s, including Ubr1, Ltn1, and the anaphase-promoting complex, contribute to ER and INM protein quality control (Arakawa et al., 2016; Crowder et al., 2015; Koch et al., 2019; Ruggiano et al., 2016; Stolz et al., 2013).
Ubiquitin-conjugating enzymes (E2s) deliver ubiquitin molecules to E3s for substrate ligation. Hrd1 functions with the E2 Ubc7, while Doa10 and Asi work with two E2s, Ubc6 and Ubc7 (Bays et al., 2001; Foresti et al., 2014; Khmelinskii et al., 2014; Lips et al., 2020; Plemper et al., 1999; Swanson et al., 2001). Ubc7 is anchored at the membrane, stabilized, and activated by the transmembrane protein Cue1 (Biederer et al., 1997; Kostova et al., 2009; Ravid & Hochstrasser, 2007). While a direct Cue1 homolog is not present in mammals, E3s and E3-accessory proteins anchor, stabilize, and activate the Ubc7 homolog UBE2G2 to ensure functional ERAD in mammalian cells (Chen et al., 2006; Das et al., 2009; Smith et al., 2021).
The aminoglycoside hygromycin B distorts ribosome A sites (Brodersen et al., 2000; Ganoza & Kiel, 2001), likely increasing synthesis of the types of aberrant proteins that contribute to age-related neurodegenerative and other diseases (Akbergenov et al., 2025). We and others have demonstrated that several genes required for efficient protein quality control enable cells to resist stress caused by hygromycin B (Akoto et al., 2025; Bengtson & Joazeiro, 2010; Chuang & Madura, 2005; Daraghmi et al., 2023; Flagg et al., 2023; Jaeger et al., 2018; Niekamp et al., 2019; Verma et al., 2013). Deletion of genes encoding ERAD and INMAD E2s and E3s profoundly sensitizes yeast to hygromycin B (Crowder et al., 2015; Doss et al., 2023; Runnebohm, Evans, et al., 2020; Turk et al., 2023; Woodruff et al., 2021). Simultaneous loss of E3s Hrd1, Doa10, and Asi1 causes greater hygromycin B sensitivity than loss of E2s Ubc6 and Ubc7 (Owutey et al., 2024), consistent with compensatory contributions by other E2s to ERAD and INMAD (Bays et al., 2001). Large-scale genetic analyses indicated CUE1 deletion reduces hygromycin B tolerance (Brown et al., 2006; Dudley et al., 2005), but this result has not been validated in targeted, small-scale studies.
We tested the hypothesis that Cue1 is required for proteotoxic stress resistance. We compared cellular fitness of wild type yeast, two independent CUE1 knockouts, yeast lacking the E2 Ubc7, and yeast lacking the three major ERAD and INMAD E3s (hrd1Δ doa10Δ asi1Δ) (Figure 1B) on media lacking or containing hygromycin B. Deletion of CUE1 and UBC7 sensitized yeast to hygromycin B to a similar extent, consistent with a shared role in protein quality control. As previously observed (Owutey et al., 2024), deletion of genes encoding ERAD and INMAD E3s caused a more profound growth defect in the presence of drug. Hygromycin B sensitivity of cue1Δ yeast from a distinct genetic background (Figure 1C) and CUE1 plasmid complementation (Figure 1D) validate this result.
Hypersensitivity of cue1Δ and ubc7Δ yeast to proteotoxic stress is consistent with function of a Cue1-Ubc7 subcomplex in ERAD and INMAD (Biederer et al., 1997; Buchanan et al., 2016; Khmelinskii et al., 2014; Pantazopoulou et al., 2016). Future experiments may be conducted to assess whether cue1Δ yeast are sensitive to other proteotoxic stressors; however, large-scale studies have demonstrated that CUE1 promotes resistance to a range of stressors, including transition metals, which oxidatively damage proteins (Ruotolo et al., 2008; Zhao et al., 2020), genotoxic agents (Alamgir et al., 2010; Gaytan et al., 2013; Kapitzky et al., 2010; Ogbede et al., 2021), and sterol biosynthesis disruption (Kapitzky et al., 2010). Our results support a crucial function for Cue1 in maintaining proteostasis in yeast.
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Inner Nuclear Membrane Asi Ubiquitin Ligase Catalytic Subunits Asi1p and Asi3p, but not Asi2p, confer resistance to aminoglycoside hygromycin B in Saccharomyces cerevisiae. MicroPubl Biol. 2021 43.","pubmedId":"34095778","doi":"10.17912/micropub.biology.000403"},{"reference":"Zhao YY, Cao CL, Liu YL, Wang J, Li SY, Li J, Deng Y. 2020. Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast. Biochim Biophys Acta Gen Subj. 1864: 129516. 50.","pubmedId":"31904504","doi":"10.1016/j.bbagen.2020.129516"}],"title":"Ubiquitin-conjugating enzyme membrane anchor Cue1 confers resistance to hygromycin B in Saccharomyces cerevisiae
","reviews":[{"reviewer":{"displayName":"Oliver Kerscher"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"0f2b86b2-9ee6-432e-9a7c-522ec53a6655","decision":"publish","abstract":"Aberrant and excess proteins are destroyed by compartment-specific protein quality control mechanisms. In Saccharomyces cerevisiae, endoplasmic reticulum (ER)-associated degradation (ERAD) and inner nuclear membrane (INM)-associated degradation (INMAD) requires the Ubc6 and Ubc7 ubiquitin-conjugating enzymes. Ubc6 is an integral membrane protein. By contrast, Ubc7 is a soluble protein tethered to the ER and INM membranes by the transmembrane protein Cue1. Here, we assessed the requirement of Cue1 in resisting proteotoxic stress. CUE1 loss sensitized cells to hygromycin B to a similar extent as UBC7 deletion, consistent with a shared role for Cue1 and Ubc7 in ER and INM protein quality control.
","acknowledgements":"Experiments to determine sensitivity of cue1Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Kalie Adams, Reva Allam, Alexis Baynes, Dilynn Blair, Olivia Coltharp, Piper Conway, La’Naviya Farries, Morgan Hensley, Sydney Hrehor, Madyson Jones, Zachary Mann, Abigail McLaughlin, Dailey Morris, Skyler Petro, Ken Reed, Mercedes Sanders, Alonna Williams) and high school students in the Spring 2025 Genetics course at the Indiana Academy for Science, Mathematics, and Humanities (Sylvester Acherekoh, Sebastian Bilbo, Jackson Booth, Sophia Bortolotti, Natalie Garringer, MaKaela Gist, Ella Johnson, Victoria Johnson, Emma-Lee Kennedy, Elizabeth Long, Cole McDermott, Jayden McDole, Kha Nguyen, Mallory Saldana). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model, as described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Christopher Hickey for generously sharing yeast strains and plasmids. We thank the Saccharomyces Genome Database for serving as an invaluable repository for yeast genetic information (Wong et al., 2023). We thank the Ball State University Division of Online and Strategic Learning for supporting an initiative to transform undergraduate laboratory courses into authentic research-based learning experiences.
This manuscript is dedicated to the memory of our friend, Ian Tesch, whose generosity of spirit was unmatched. Thank you for being a part of our team.
","authors":[{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","investigation","validation","writing_reviewEditing"],"email":"james.avaala@bsu.edu","firstName":"James A","lastName":"Avaala","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0008-4575-7329"},{"affiliations":["Ball State University","The Indiana Academy for Science, Mathematics, and Humanities"],"departments":["Department of Biology",""],"credit":["investigation","writing_reviewEditing","validation"],"email":"melissa.palackel@bsu.edu","firstName":"Melissa","lastName":"Palackel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0007-2695-8603"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["resources","investigation"],"email":"IanTesch90@gmail.com","firstName":"Ian M","lastName":"Tesch","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Ball State University","Midwestern University"],"departments":["Department of Biology","Chicago College of Osteopathic Medicine"],"credit":["supervision","writing_reviewEditing"],"email":"joseph.gumina@midwestern.edu","firstName":"Joseph","lastName":"Gumina","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"chance.creviston@bsu.edu","firstName":"Chance S","lastName":"Creviston","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0005-8785-1581"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"affiliations":["The Indiana Academy for Science, Mathematics, and Humanities","Ball State University"],"departments":["Division of Natural, Physical, and Computer Sciences","Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"justin.crowder@bsu.edu","firstName":"Justin J","lastName":"Crowder","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-6327-6888"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["fundingAcquisition","supervision","writing_originalDraft"],"email":"emrubenstein@bsu.edu","firstName":"Eric M","lastName":"Rubenstein","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0003-4983-1430"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was funded by NIH grant R15 GM111713 (EMR). JAA was supported by a Ball State University Aspire student research grant. Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology and the Indiana Academy for Science, Mathematics, and Humanities. This project was conceived while EMR was supported in part by a Ball State University Excellence in Teaching award (sponsored by the Ball State University Division of Online and Strategic Learning and the Office of the Provost).
","image":{"url":"https://portal.micropublication.org/uploads/7f9217398ef1e2d02047c056d638ecd1.png"},"imageCaption":"(A) By anchoring the ubiquitin-conjugating enzyme Ubc7 to the endoplasmic reticulum and inner nuclear membranes, Cue1 contributes to protein degradation mediated by the Hrd1, Doa10, and Asi ubiquitin ligases. See text for details. (B-D) Sixfold serial dilutions of yeast of the indicated genotypes were spotted on medium lacking (No Drug) or containing hygromycin B at the indicated concentrations. Strains in (D) were transformed with an empty vector or a plasmid encoding Cue1. Strains used in the experiments in (B and D) are derived from the MHY500 genetic background, whereas strains in (C) are derived from BY4741. cue1Δ #1 and cue1Δ #2 are independent clones resulting from homologous recombination-mediated gene replacement. Plates were incubated at 30°C and imaged at the indicated times. Experiments in (B and C) were performed three times. The experiment in (D) was performed twice.
","imageTitle":"CUE1 confers resistance to hygromycin B
","methods":"CUE1 gene replacement
To generate yeast strains VJY336 and VJY337, CUE1 was replaced by natMX4 through homologous recombination. A 1464-bp nat4MX4 cassette with termini possessing sequences flanking the CUE1 gene was PCR-amplified from pAG25 (Goldstein & McCusker, 1999) using primers VJR253 (5’ CGCCATAAAGCATTACAATCTACGATCGCGCAAACTTTTTTCTTTTGGCCCACATACGATTTAGGTGACAC) and VJR254 (5’ TTATGCGCATTATGGGCACACTTGCGTGTTCCCGACAAGCACTTAAGCGTAATACGACTCACTATAGGGAG 3’). The natMX4 cassette was introduced into VJY6 yeast by lithium acetate transformation (Guthrie & Fink, 2004), followed by selection on medium containing nourseothricin. Successful integration was verified by PCR at 5’ and 3’ recombination junctions.
Growth assays
Sixfold serial dilutions of indicated yeast strains were spotted onto yeast extract-peptone-dextrose medium lacking or containing hygromycin B (Gibco) at the indicated concentrations (Guthrie & Fink, 2004) followed by incubation at 30°C for the indicated time, as described in (Watts et al., 2015).
","reagents":"Yeast strains used in this study.
Name | Genotype | Figure | Reference |
VJY6 (alias MHY500) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 | 1B, 1D | Chen et al., 1993 |
VJY50 (alias MHY551) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc7Δ::LEU2 | 1B | Chen et al., 1993 |
VJY324 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 cue1Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY336 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 1 | 1B, 1D | This study |
VJY337 | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 cue1Δ::natMX4 Clone 2 | 1B | This study |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1C | Tong et al., 2001 |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1C | Tong et al., 2001 |
VJY1098 (MHY11132, ABM297) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 asi1Δ::kanMX6 | 1B | Mehrtash & Hochstrasser, 2023 |
Plasmids used in this study.
Name | Description | Figure | Reference |
pVJ39 (pRS314) | empty vector (CEN, TRP1, AmpR) | 1D | Sikorski & Hieter, 1989 |
pVJ653 (p414-MET25-Cue1-3xFLAG) | 3xFlag-tagged Cue1 driven by MET25 promoter (CEN, TRP1, AmpR) | 1D | Mehrtash & Hochstrasser, 2022 |
Cellular and organismal health depends on protein quality control (Badawi et al., 2023; Guerriero & Brodsky, 2012; Higuchi-Sanabria et al., 2018). Aberrant and excess endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed by ER-associated degradation (ERAD) and INM-associated degradation (INMAD), respectively (reviewed in (Mehrtash & Hochstrasser, 2019)). In Saccharomyces cerevisiae, the Hrd1 and Doa10 ubiquitin ligases (E3s) mediate ERAD of soluble, transmembrane, and translocon-clogging proteins at the ER membrane (Carvalho et al., 2006; Huyer et al., 2004; Metzger et al., 2008; Rubenstein et al., 2012; Runnebohm, Richards, et al., 2020; Sato et al., 2009; Swanson et al., 2001) (Figure 1A). Doa10 also localizes to the INM, where it and the heterotrimeric E3 Asi (comprised of Asi1, Asi2, and Asi3) catalyze INMAD of transmembrane INM and soluble nucleoplasmic proteins (Deng & Hochstrasser, 2006; Foresti et al., 2014; Khmelinskii et al., 2014; Swanson et al., 2001). Additional E3s, including Ubr1, Ltn1, and the anaphase-promoting complex, contribute to ER and INM protein quality control (Arakawa et al., 2016; Crowder et al., 2015; Koch et al., 2019; Ruggiano et al., 2016; Stolz et al., 2013).
Ubiquitin-conjugating enzymes (E2s) deliver ubiquitin molecules to E3s for substrate ligation. Hrd1 functions with the E2 Ubc7, while Doa10 and Asi work with two E2s, Ubc6 and Ubc7 (Bays et al., 2001; Foresti et al., 2014; Khmelinskii et al., 2014; Lips et al., 2020; Plemper et al., 1999; Swanson et al., 2001). Ubc7 is anchored at the membrane, stabilized, and activated by the transmembrane protein Cue1 (Biederer et al., 1997; Kostova et al., 2009; Ravid & Hochstrasser, 2007). While a direct Cue1 homolog is not present in mammals, E3s and E3-accessory proteins anchor, stabilize, and activate the Ubc7 homolog UBE2G2 to ensure functional ERAD in mammalian cells (Chen et al., 2006; Das et al., 2009; Smith et al., 2021).
The aminoglycoside hygromycin B distorts ribosome A sites (Brodersen et al., 2000; Ganoza & Kiel, 2001), likely increasing synthesis of the types of aberrant proteins that contribute to age-related neurodegenerative and other diseases (Akbergenov et al., 2025). We and others have demonstrated that several genes required for efficient protein quality control enable cells to resist stress caused by hygromycin B (Akoto et al., 2025; Bengtson & Joazeiro, 2010; Chuang & Madura, 2005; Daraghmi et al., 2023; Flagg et al., 2023; Jaeger et al., 2018; Niekamp et al., 2019; Verma et al., 2013). Deletion of genes encoding ERAD and INMAD E2s and E3s profoundly sensitizes yeast to hygromycin B (Crowder et al., 2015; Doss et al., 2023; Runnebohm, Evans, et al., 2020; Turk et al., 2023; Woodruff et al., 2021). Simultaneous loss of E3s Hrd1, Doa10, and Asi1 causes greater hygromycin B sensitivity than loss of E2s Ubc6 and Ubc7 (Owutey et al., 2024), consistent with compensatory contributions by other E2s to ERAD and INMAD (Bays et al., 2001). Large-scale genetic analyses indicated CUE1 deletion reduces hygromycin B tolerance (Brown et al., 2006; Dudley et al., 2005), but this result has not been validated in targeted, small-scale studies.
We tested the hypothesis that Cue1 is required for proteotoxic stress resistance. We compared cellular fitness of wild type yeast, two independent CUE1 knockouts, yeast lacking the E2 Ubc7, and yeast lacking the three major ERAD and INMAD E3s (hrd1Δ doa10Δ asi1Δ) (Figure 1B) on media lacking or containing hygromycin B. Deletion of CUE1 and UBC7 sensitized yeast to hygromycin B to a similar extent, consistent with a shared role in protein quality control. As previously observed (Owutey et al., 2024), deletion of genes encoding ERAD and INMAD E3s caused a more profound growth defect in the presence of drug. Hygromycin B sensitivity of cue1Δ yeast from a distinct genetic background (Figure 1C) and CUE1 plasmid complementation (Figure 1D) validate this result.
Hypersensitivity of cue1Δ and ubc7Δ yeast to proteotoxic stress is consistent with function of a Cue1-Ubc7 subcomplex in ERAD and INMAD (Biederer et al., 1997; Buchanan et al., 2016; Khmelinskii et al., 2014; Pantazopoulou et al., 2016). Future experiments may be conducted to assess whether cue1Δ yeast are sensitive to other proteotoxic stressors; however, large-scale studies have demonstrated that CUE1 promotes resistance to a range of stressors, including transition metals, which oxidatively damage proteins (Ruotolo et al., 2008; Zhao et al., 2020), genotoxic agents (Alamgir et al., 2010; Gaytan et al., 2013; Kapitzky et al., 2010; Ogbede et al., 2021), and sterol biosynthesis disruption (Kapitzky et al., 2010). Our results support a crucial function for Cue1 in maintaining proteostasis in yeast.
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