Aberrant proteins are targeted for proteasomal degradation by polyubiquitylation catalyzed by sequential action of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). A subset of proteasomal substrates require ubiquitin chain extension by ubiquitin-elongating enzymes (E4s) prior to proteolysis. We tested the requirement of Ufd2, the founding member of the E4 enzyme family, in resisting proteotoxic stress caused by the aminoglycoside hygromycin B in Saccharomyces cerevisiae. The human homolog, UBE4B, is a potential therapeutic target for neurological disease and cancer. UFD2 deletion sensitized yeast to hygromycin B, consistent with a role for the E4 in protein quality control.
","acknowledgements":"Experiments to determine sensitivity of ufd2Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Olivia Carolan, Ashland Kahalekomo, Lucas Liapes, Hal Plummer, and Brittney Reece). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Zane Johnson 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. EMR dedicates this manuscript to the memory of Dr. Arnold Sodergren, whose kindness, mentorship, and early encouragement to pursue research profoundly shaped my career and continue to guide my work.
","authors":[{"affiliations":["Ball State University"],"departments":["Biology"],"credit":["investigation","validation","writing_reviewEditing"],"email":"kmpaxton@bsu.edu","firstName":"Kaikeyi M","lastName":"Paxton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-1423-602X"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","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"],"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","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":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"affiliations":["Ball State University"],"departments":["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":[{"awardId":"GM111713","funderName":"National Institutes of Health (United States)","awardRecipient":"Eric M Rubenstein"}],"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). Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology. 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/4b595d2c0b1d36bac8b4e48bca700aad.png"},"imageCaption":"(A) The E3s Hrd1 and Doa10 mediate protein turnover at the endoplasmic reticulum (ER) via ER-associated degradation (ERAD). Hrd1 functions with the E2 Ubc7, which is tethered to the ER by Cue1. Doa10 works with two E2s, Ubc6 and Ubc7. Following Cdc48-dependent extraction from the ER, the E4 Ufd2 extends the polyubiquitin chains of a subset of ERAD substrates to accelerate proteasomal degradation. (B, C) Sixfold serial dilutions of yeast of the indicated genotypes were pipetted on medium lacking or containing hygromycin. Plates were incubated at 30°C and imaged at the indicated times. Strains used in the experiment in (B) are derived from the BY4741 genetic background, whereas strains in (C) are derived from MHY500. Experiments were performed three or more times.
","imageTitle":"UFD2 confers resistance to hygromycin B
","methods":"Growth assays
Serial sixfold dilutions of yeast were pipetted onto agar plates with yeast extract-peptone-dextrose (YPD) medium (Guthrie & Fink, 2004) lacking or containing hygromycin B (Gibco), as described in (Watts et al., 2015). Plates were incubated at 30°C.
","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 | 1C | (Chen et al., 1993) |
VJY8 (alias | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 | 1C |
|
VJY305 (aka SKY242) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 doa10Δ::kanMX4 hrd1Δ::kanMX4 | 1B | (Habeck et al., 2015) |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1B | (Tong et al., 2001) |
VJY645 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ufd2Δ::kanMX4 | 1B | (Tong et al., 2001) |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1B | (Tong et al., 2001) |
VJY1249 (alias MHY9433) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ufd2Δ::kanMX6 | 1C | Gift of Mark Hochstrasser and Adrian Mehrtash |
","patternDescription":"
Protein quality control and regulated protein degradation through the ubiquitin-proteasome system (UPS) is required for cellular and organismal homeostasis. Through the sequential activity of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), polymers of the small protein ubiquitin are covalently linked to proteins destined for proteasomal destruction (Kleiger & Mayor, 2014). In some cases, further elaboration of poly-ubiquitin chains by ubiquitin-elongating enzymes (E4s) accelerates protein degradation (Muller & Hoppe, 2024).
The Saccharomyces cerevisiae protein Ufd2 is the founding member of the E4 family of enzymes. It is structurally similar to the Really Interesting New Gene (RING) class of ubiquitin ligases (Koegl et al., 1999). Ufd2 contributes broadly to protein degradation, promoting turnover mediated by endoplasmic reticulum (ER)-associated degradation (ERAD) E3s (Figure 1A), the anaphase-promoting complex (APC), Skp1-Cullin-F-Box (SCF) enzymes, and other E3s (Anton et al., 2023; Liu et al., 2011; Liu et al., 2010; Nakatsukasa et al., 2008; Richly et al., 2005; Smith et al., 2016). Consistent with its broad substrate range, Ufd2 regulates homeostasis in multiple compartments, including the ER and mitochondria (Altin et al., 2025; Liu et al., 2010; Richly et al., 2005), and loss of Ufd2 sensitizes yeast to heat, ethanol, and cadmium stress in the context of dampened proteasome function (Koegl et al., 1999).
Function of UBE4B, the human homolog of Ufd2, is implicated in neurological health and tumor development. UBE4B plays a potentially protective role in the neurological disorder Machado-Joseph disease, as it promotes turnover of polyglutamine-expanded, pathogenic forms of ataxin-3 (Matsumoto et al., 2004). By contrast, UBE4B constitutively targets Charcot-Marie-Tooth disease variants of mitofusin, likely contributing to dysregulated mitochondrial dynamics and symptom progression in afflicted patients (Anton et al., 2023). Moreover, UBE4B also facilitates Mdm2-dependent degradation of the tumor suppressor p53 (Wu et al., 2011). Thus, strategies that modulate UBE4B activity may be therapeutic or detrimental, depending on the condition.
Hygromycin B, an aminoglycoside produced by Streptomyces hygroscopicus, distorts ribosome A sites, resulting in mistranslation and impaired ribosomal translocation (Cabanas et al., 1978), likely increasing the abundance of aberrant proteins. We and others have demonstrated that mutations in several genes with demonstrated or predicted roles in protein quality control cause hygromycin B sensitivity (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). For example, loss of genes encoding ERAD enzymes causes a profound growth defect in the presence of hygromycin B (Crowder et al., 2015; Doss et al., 2023; Runnebohm et al., 2020; Turk et al., 2023; Woodruff et al., 2021).
Given its requirement for degradation of select ERAD substrates, we hypothesized that Ufd2 confers resistance to proteotoxic stress, such as that caused by hygromycin B. We assessed the fitness of wild type yeast, yeast lacking Ufd2, and yeast lacking ERAD components (either the ERAD E2, Ubc7, or two major ERAD E3s, Hrd1 and Doa10 (Mehrtash & Hochstrasser, 2019)) in the presence of sublethal concentrations of hygromycin B. As in previous reports, deletion of UBC7 or of HRD1 and DOA10 substantially impeded growth when hygromycin B was included in the growth medium (Crowder et al., 2015; Owutey et al., 2024). Indeed, loss of Ufd2 caused a marked growth defect in the presence of hygromycin B. Growth impairment in ufd2Δ yeast was not as severe as that observed in ubc7Δ and hrd1Δ doa10Δ yeast. Hygromycin B sensitivity of ufd2Δ yeast derived from a distinct genetic background further validates a role for this E4 in combatting proteotoxic stress (Figure 1C).
Our finding that ufd2Δ yeast are hypersensitive to hygromycin B is consistent with a general role for this E4 in protein quality control. Although plasmid-based complementation of ufd2Δ would provide additional confirmation, reproducible observation of hygromycin B sensitivity in independently generated ufd2Δ strains from distinct genetic backgrounds (BY4741 (Brachmann et al., 1998) in Figure 1B and MHY500 (Chen et al., 1993) in Figure 1C) increases confidence in this result. Future experiments will assess the full range of stressors to which UFD2 confers resistance. Hygromycin B sensitivity is not a general feature of E4 enzymes, as we previously observed that deletion of the gene encoding E4 Hul5 does not sensitize yeast to the drug (Woodruff et al., 2021). While targeting human UBE4B may be therapeutic for neurological conditions or cancer, our results suggest caution is warranted, as important quality control functions may be impacted upon enzyme inhibition.
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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 25.","pubmedId":"34095778","doi":"10.17912/micropub.biology.000403"},{"reference":"Wu H, Pomeroy SL, Ferreira M, Teider N, Mariani J, Nakayama KI, et al., Leng RP. 2011. UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53. Nat Med. 17: 347–55. 12.","pubmedId":"21317885","doi":"10.1038/nm.2283"}],"title":"Ubiquitin-Elongating Enzyme Ufd2 confers resistance to hygromycin B in Saccharomyces cerevisiae
","reviews":[{"reviewer":{"displayName":"Prasanna Satpute-Krishnan"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Caroline (Lina) Lund Dahlberg"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"ee8e144d-73cb-4c21-8dab-8c966973174c","decision":"accept","abstract":"Aberrant proteins are targeted for proteasomal degradation by polyubiquitylation catalyzed by sequential action of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). A subset of proteasomal substrates require ubiquitin chain extension by ubiquitin-elongating enzymes (E4s) prior to proteolysis. We tested the requirement of Ufd2, the founding member of the E4 enzyme family, in resisting proteotoxic stress caused by the aminoglycoside hygromycin B in Saccharomyces cerevisiae. The human homolog, UBE4B, is a potential therapeutic target for neurological disease and cancer. UFD2 deletion sensitized yeast to hygromycin B, consistent with a role for the E4 in protein quality control.
","acknowledgements":"Experiments to determine sensitivity of ufd2Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Gabriel Baker, Aidan Bane, Matt Bell, Duncan Bowman, Camdyn Cantrell, Olivia Carolan, Kaiya Godar, Gus Gullion, Ashland Kahalekomo, Lucas Liapes, Hal Plummer, Brittney Reece, Avery Renshaw, Elise Whitehurst, and Tony Williamson). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Zane Johnson 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. EMR dedicates this manuscript to the memory of Dr. Arnold Sodergren, whose kindness, mentorship, and early encouragement to pursue research profoundly shaped my career and continue to guide my work.
","authors":[{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["investigation","validation","writing_reviewEditing"],"email":"kmpaxton@bsu.edu","firstName":"Kaikeyi M","lastName":"Paxton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-1423-602X"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"james.avaala@bsu.edu","firstName":"James A","lastName":"Avaala","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0008-4575-7329"},{"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","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":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"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":[{"awardId":"GM111713","funderName":"National Institutes of Health (United States)","awardRecipient":"Eric M Rubenstein"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology. 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/4b595d2c0b1d36bac8b4e48bca700aad.png"},"imageCaption":"(A) The E3s Hrd1 and Doa10 mediate protein turnover at the endoplasmic reticulum (ER) via ER-associated degradation (ERAD). Hrd1 functions with the E2 Ubc7, which is tethered to the ER by Cue1. Doa10 works with two E2s, Ubc6 and Ubc7. Following Cdc48-dependent extraction from the ER, the E4 Ufd2 extends the polyubiquitin chains of a subset of ERAD substrates to accelerate proteasomal degradation. (B, C) Sixfold serial dilutions of yeast of the indicated genotypes were pipetted on medium lacking or containing hygromycin B. Plates were incubated at 30°C and imaged at the indicated times. Strains used in the experiment in (B) are derived from the BY4741 genetic background, whereas strains in (C) are derived from MHY500. Experiments were performed three or more times.
","imageTitle":"UFD2 confers resistance to hygromycin B
","methods":"Growth assays
Serial sixfold dilutions of S. cerevisiae were pipetted onto agar plates with yeast extract-peptone-dextrose (YPD) medium (Guthrie & Fink, 2004) lacking or containing hygromycin B (Gibco), as described in (Watts et al., 2015). Plates were incubated at 30°C. Hygromycin B concentrations were selected empirically to produce sublethal growth inhibition that permitted discrimination between strain fitness.
","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 | 1C | (Chen et al., 1993) |
VJY8 (alias MHY1702) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 | 1C | (Huyer et al., 2004) |
VJY305 (aka SKY242) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 doa10Δ::kanMX4 hrd1Δ::kanMX4 | 1B | (Habeck et al., 2015) |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1B | (Tong et al., 2001) |
VJY645 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ufd2Δ::kanMX4 | 1B | (Tong et al., 2001) |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1B | (Tong et al., 2001) |
VJY1249 (alias MHY9433) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ufd2Δ::kanMX6 | 1C | Gift of Mark Hochstrasser and Adrian Mehrtash |
","patternDescription":"
Protein quality control and regulated protein degradation through the ubiquitin-proteasome system (UPS) is required for cellular and organismal homeostasis. Through the sequential activity of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), polymers of the small protein ubiquitin are covalently linked to proteins destined for proteasomal destruction (Kleiger & Mayor, 2014). In some cases, further elaboration of poly-ubiquitin chains by ubiquitin-elongating enzymes (E4s) accelerates protein degradation (Muller & Hoppe, 2024).
The Saccharomyces cerevisiae protein Ufd2 is the founding member of the E4 family of enzymes. It is structurally similar to the Really Interesting New Gene (RING) class of ubiquitin ligases (Koegl et al., 1999). Ufd2 contributes broadly to protein degradation, promoting turnover mediated by endoplasmic reticulum (ER)-associated degradation (ERAD) E3s (Figure 1A), the anaphase-promoting complex (APC), Skp1-Cullin-F-Box (SCF) enzymes, and other E3s (Anton et al., 2023; Liu et al., 2011; Liu et al., 2010; Nakatsukasa et al., 2008; Richly et al., 2005; Smith et al., 2016). Consistent with its broad substrate range, Ufd2 regulates homeostasis in multiple compartments, including the ER and mitochondria (Altin et al., 2025; Liu et al., 2010; Richly et al., 2005). Loss of Ufd2 sensitizes yeast to heat, ethanol, and cadmium stress in the context of dampened proteasome function caused by deletion of RPN10, which encodes a 19S regulatory particle subunit (Koegl et al., 1999).
UBE4B, the human homolog of Ufd2, is implicated in neurological health and tumor development. UBE4B plays a potentially protective role in the neurological disorder Machado-Joseph disease, as it promotes turnover of polyglutamine-expanded, pathogenic forms of ataxin-3 (Matsumoto et al., 2004). By contrast, UBE4B constitutively targets Charcot-Marie-Tooth disease variants of mitofusin, likely contributing to dysregulated mitochondrial dynamics and symptom progression in afflicted patients (Anton et al., 2023). Moreover, UBE4B also facilitates Mdm2-dependent degradation of the tumor suppressor p53 and inhibits tumor cell apoptosis (Wu et al., 2011). Thus, strategies that modulate UBE4B activity may be therapeutic or detrimental, depending on the condition.
Hygromycin B, an aminoglycoside produced by Streptomyces hygroscopicus, distorts ribosome A sites, resulting in mistranslation and impaired ribosomal translocation (Cabanas et al., 1978), likely increasing the abundance of aberrant proteins. We and others have demonstrated that mutations in several genes with demonstrated or predicted roles in protein quality control cause hygromycin B sensitivity (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). For example, loss of genes encoding ERAD enzymes causes a profound growth defect in the presence of hygromycin B (Avaala et al., 2026; Crowder et al., 2015; Doss et al., 2023; Runnebohm et al., 2020; Turk et al., 2023; Woodruff et al., 2021).
Given its requirement for degradation of select ERAD substrates, we hypothesized that Ufd2 confers resistance to proteotoxic stress, such as that caused by hygromycin B. We assessed the fitness of wild type yeast, yeast lacking Ufd2, and yeast lacking ERAD components (either the ERAD E2, Ubc7, or two major ERAD E3s, Hrd1 and Doa10 (Mehrtash & Hochstrasser, 2019)) in the presence of sublethal concentrations of hygromycin B. As in previous reports, deletion of UBC7 or of HRD1 and DOA10 substantially impeded growth when hygromycin B was included in the growth medium (Crowder et al., 2015; Owutey et al., 2024). Indeed, loss of Ufd2 caused a marked growth defect in the presence of hygromycin B. Growth impairment in ufd2Δ yeast was not as severe as that observed in ubc7Δ and hrd1Δ doa10Δ yeast. Hygromycin B sensitivity of ufd2Δ yeast derived from a distinct genetic background further validates a role for this E4 in combatting proteotoxic stress (Figure 1C).
Our finding that ufd2Δ yeast are hypersensitive to hygromycin B is consistent with a general role for this E4 in protein quality control. Although plasmid-based complementation of ufd2Δ yeast would provide additional confirmation, reproducible observation of hygromycin B sensitivity in independently generated ufd2Δ strains from distinct genetic backgrounds (BY4741 (Brachmann et al., 1998) in Figure 1B and MHY500 (Chen et al., 1993) in Figure 1C) increases confidence in this result. Future experiments will extend these analyses to define the spectrum of global and organelle-specific protein homeostasis stressors to which UFD2 confers resistance. Hygromycin B sensitivity is not a general feature of E4 enzymes, as we previously observed that deletion of the gene encoding E4 Hul5 does not sensitize yeast to the drug (Woodruff et al., 2021). Together, these findings support an important role for Ufd2 in maintaining protein homeostasis and suggest that any future therapeutic strategies targeting the human homolog UBE4B should consider the potential consequences for protein quality control.
","references":[{"reference":"Akoto E, Doss EM, Claypool KP, Owutey SL, Richards KA, Lehman KM, et al., Rubenstein EM. 2025. The kinesin Kar3 is required for endoplasmic reticulum-associated degradation. Mol Biol Cell. 36: br9. 21.","pubmedId":"39841550","doi":"10.1091/mbc.E24-10-0437"},{"reference":"Altin S, Simoes T, Behrendt C, Anton V, Domke D, Voltzke KM, et al., Escobar Henriques M. 2025. Ubiquitin precursor with C-terminal extension promotes proteostasis and longevity. Mol Cell. 85: 3677–3693 e7. 10.","pubmedId":"40992376","doi":"10.1016/j.molcel.2025.08.032"},{"reference":"Anton V, Buntenbroich I, Simoes T, Joaquim M, Muller L, Buettner R, et al., Escobar Henriques M. 2023. E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response. Mol Cell. 83: 2976–2990 e9. 4.","pubmedId":"37595558","doi":"10.1016/j.molcel.2023.07.021"},{"reference":"Avaala JA, Palackel M, Tesch IM, Gumina J, Creviston CS, True JD, Crowder JJ, Rubenstein EM. 2026. Ubiquitin-conjugating enzyme membrane anchor Cue1 confers resistance to hygromycin B in Saccharomyces cerevisiae. MicroPubl Biol. 2026 1.
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","reviews":[],"curatorReviews":[]},{"id":"3a59f998-74ba-4131-8e1a-ec107bf33f64","decision":"publish","abstract":"Aberrant proteins are targeted for proteasomal degradation by polyubiquitylation catalyzed by sequential action of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). A subset of proteasomal substrates require ubiquitin chain extension by ubiquitin-elongating enzymes (E4s) prior to proteolysis. We tested the requirement of Ufd2, the founding member of the E4 enzyme family, in resisting proteotoxic stress caused by the aminoglycoside hygromycin B in Saccharomyces cerevisiae. The human homolog, UBE4B, is a potential therapeutic target for neurological disease and cancer. UFD2 deletion sensitized yeast to hygromycin B, consistent with a role for the E4 in protein quality control.
","acknowledgements":"Experiments to determine sensitivity of ufd2Δ yeast to hygromycin B were piloted by undergraduate students in the Spring 2025 Methods in Cell Biology (BIO 315) course at Ball State University (Gabriel Baker, Aidan Bane, Matt Bell, Duncan Bowman, Camdyn Cantrell, Olivia Carolan, Kaiya Godar, Gus Gullion, Ashland Kahalekomo, Lucas Liapes, Hal Plummer, Brittney Reece, Avery Renshaw, Elise Whitehurst, and Tony Williamson). Results were validated in the research laboratory of EMR, using the CURE-to-PIRL workflow model described in (Rubenstein et al., 2024). We thank Mark Hochstrasser, Adrian Mehrtash, and Zane Johnson 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. EMR dedicates this manuscript to the memory of Dr. Arnold Sodergren, whose kindness, mentorship, and early encouragement to pursue research profoundly shaped my career and continue to guide my work.
","authors":[{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["investigation","validation","writing_reviewEditing"],"email":"kmpaxton@bsu.edu","firstName":"Kaikeyi M","lastName":"Paxton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-1423-602X"},{"affiliations":["Ball State University"],"departments":["Department of Biology"],"credit":["supervision","writing_reviewEditing"],"email":"james.avaala@bsu.edu","firstName":"James A","lastName":"Avaala","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0008-4575-7329"},{"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","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":"jdtrue@bsu.edu","firstName":"Jason D","lastName":"True","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0002-9320-440X"},{"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":[{"awardId":"GM111713","funderName":"National Institutes of Health (United States)","awardRecipient":"Eric M Rubenstein"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Preliminary studies conducted in teaching labs were funded by the Ball State University Department of Biology. 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/4b595d2c0b1d36bac8b4e48bca700aad.png"},"imageCaption":"(A) The E3s Hrd1 and Doa10 mediate protein turnover at the endoplasmic reticulum (ER) via ER-associated degradation (ERAD). Hrd1 functions with the E2 Ubc7, which is tethered to the ER by Cue1. Doa10 works with two E2s, Ubc6 and Ubc7. Following Cdc48-dependent extraction from the ER, the E4 Ufd2 extends the polyubiquitin chains of a subset of ERAD substrates to accelerate proteasomal degradation. (B, C) Sixfold serial dilutions of yeast of the indicated genotypes were pipetted on medium lacking or containing hygromycin B. Plates were incubated at 30°C and imaged at the indicated times. Strains used in the experiment in (B) are derived from the BY4741 genetic background, whereas strains in (C) are derived from MHY500. Experiments were performed three or more times.
","imageTitle":"UFD2 confers resistance to hygromycin B
","methods":"Growth assays
Serial sixfold dilutions of S. cerevisiae were pipetted onto agar plates with yeast extract-peptone-dextrose (YPD) medium (Guthrie & Fink, 2004) lacking or containing hygromycin B (Gibco), as described in (Watts et al., 2015). Plates were incubated at 30°C. Hygromycin B concentrations were selected empirically to produce sublethal growth inhibition that permitted discrimination between strain fitness.
","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 | 1C | (Chen et al., 1993) |
VJY8 (alias MHY1702) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10Δ::HIS3 hrd1Δ::LEU2 | 1C | (Huyer et al., 2004) |
VJY305 (aka SKY242) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 doa10Δ::kanMX4 hrd1Δ::kanMX4 | 1B | (Habeck et al., 2015) |
VJY476 (alias BY4741) | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | 1B | (Tong et al., 2001) |
VJY645 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ufd2Δ::kanMX4 | 1B | (Tong et al., 2001) |
VJY1075 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc7Δ::kanMX4 | 1B | (Tong et al., 2001) |
VJY1249 (alias MHY9433) | MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ufd2Δ::kanMX6 | 1C | Gift of Mark Hochstrasser and Adrian Mehrtash |
","patternDescription":"
Protein quality control and regulated protein degradation through the ubiquitin-proteasome system (UPS) is required for cellular and organismal homeostasis. Through the sequential activity of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), polymers of the small protein ubiquitin are covalently linked to proteins destined for proteasomal destruction (Kleiger & Mayor, 2014). In some cases, further elaboration of poly-ubiquitin chains by ubiquitin-elongating enzymes (E4s) accelerates protein degradation (Muller & Hoppe, 2024).
The Saccharomyces cerevisiae protein Ufd2 is the founding member of the E4 family of enzymes. It is structurally similar to the Really Interesting New Gene (RING) class of ubiquitin ligases (Koegl et al., 1999). Ufd2 contributes broadly to protein degradation, promoting turnover mediated by endoplasmic reticulum (ER)-associated degradation (ERAD) E3s (Figure 1A), the anaphase-promoting complex (APC), Skp1-Cullin-F-Box (SCF) enzymes, and other E3s (Anton et al., 2023; Liu et al., 2011; Liu et al., 2010; Nakatsukasa et al., 2008; Richly et al., 2005; Smith et al., 2016). Consistent with its broad substrate range, Ufd2 regulates homeostasis in multiple compartments, including the ER and mitochondria (Altin et al., 2025; Liu et al., 2010; Richly et al., 2005). Loss of Ufd2 sensitizes yeast to heat, ethanol, and cadmium stress in the context of dampened proteasome function caused by deletion of RPN10, which encodes a 19S regulatory particle subunit (Koegl et al., 1999).
UBE4B, the human homolog of Ufd2, is implicated in neurological health and tumor development. UBE4B plays a potentially protective role in the neurological disorder Machado-Joseph disease, as it promotes turnover of polyglutamine-expanded, pathogenic forms of ataxin-3 (Matsumoto et al., 2004). By contrast, UBE4B constitutively targets Charcot-Marie-Tooth disease variants of mitofusin, likely contributing to dysregulated mitochondrial dynamics and symptom progression in afflicted patients (Anton et al., 2023). Moreover, UBE4B also facilitates Mdm2-dependent degradation of the tumor suppressor p53 and inhibits tumor cell apoptosis (Wu et al., 2011). Thus, strategies that modulate UBE4B activity may be therapeutic or detrimental, depending on the condition.
Hygromycin B, an aminoglycoside produced by Streptomyces hygroscopicus, distorts ribosome A sites, resulting in mistranslation and impaired ribosomal translocation (Cabanas et al., 1978), likely increasing the abundance of aberrant proteins. We and others have demonstrated that mutations in several genes with demonstrated or predicted roles in protein quality control cause hygromycin B sensitivity (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). For example, loss of genes encoding ERAD enzymes causes a profound growth defect in the presence of hygromycin B (Avaala et al., 2026; Crowder et al., 2015; Doss et al., 2023; Runnebohm et al., 2020; Turk et al., 2023; Woodruff et al., 2021).
Given its requirement for degradation of select ERAD substrates, we hypothesized that Ufd2 confers resistance to proteotoxic stress, such as that caused by hygromycin B. We assessed the fitness of wild type yeast, yeast lacking Ufd2, and yeast lacking ERAD components (either the ERAD E2, Ubc7, or two major ERAD E3s, Hrd1 and Doa10 (Mehrtash & Hochstrasser, 2019)) in the presence of sublethal concentrations of hygromycin B (Figure 1B). As in previous reports, deletion of UBC7 or of HRD1 and DOA10 substantially impeded growth when hygromycin B was included in the growth medium (Crowder et al., 2015; Owutey et al., 2024). Indeed, loss of Ufd2 caused a marked growth defect in the presence of hygromycin B. Growth impairment in ufd2Δ yeast was not as severe as that observed in ubc7Δ and hrd1Δ doa10Δ yeast. Hygromycin B sensitivity of ufd2Δ yeast derived from a distinct genetic background further validates a role for this E4 in combatting proteotoxic stress (Figure 1C).
Our finding that ufd2Δ yeast are hypersensitive to hygromycin B is consistent with a general role for this E4 in protein quality control. Although plasmid-based complementation of ufd2Δ yeast would provide additional confirmation, reproducible observation of hygromycin B sensitivity in independently generated ufd2Δ strains from distinct genetic backgrounds (BY4741 (Brachmann et al., 1998) in Figure 1B and MHY500 (Chen et al., 1993) in Figure 1C) increases confidence in this result. Future experiments will extend these analyses to define the spectrum of global and organelle-specific protein homeostasis stressors to which UFD2 confers resistance. Hygromycin B sensitivity is not a general feature of E4 enzymes, as we previously observed that deletion of the gene encoding E4 Hul5 does not sensitize yeast to the drug (Woodruff et al., 2021). Together, these findings support an important role for Ufd2 in maintaining protein homeostasis and suggest that any future therapeutic strategies targeting the human homolog UBE4B should consider the potential consequences for protein quality control.
","references":[{"reference":"Akoto E, Doss EM, Claypool KP, Owutey SL, Richards KA, Lehman KM, et al., Rubenstein EM. 2025. The kinesin Kar3 is required for endoplasmic reticulum-associated degradation. Mol Biol Cell. 36: br9. 21.","pubmedId":"39841550","doi":"10.1091/mbc.E24-10-0437"},{"reference":"Altin S, Simoes T, Behrendt C, Anton V, Domke D, Voltzke KM, et al., Escobar Henriques M. 2025. Ubiquitin precursor with C-terminal extension promotes proteostasis and longevity. Mol Cell. 85: 3677–3693 e7. 10.","pubmedId":"40992376","doi":"10.1016/j.molcel.2025.08.032"},{"reference":"Anton V, Buntenbroich I, Simoes T, Joaquim M, Muller L, Buettner R, et al., Escobar Henriques M. 2023. E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response. Mol Cell. 83: 2976–2990 e9. 4.","pubmedId":"37595558","doi":"10.1016/j.molcel.2023.07.021"},{"reference":"Avaala JA, Palackel M, Tesch IM, Gumina J, Creviston CS, True JD, Crowder JJ, Rubenstein EM. 2026. Ubiquitin-conjugating enzyme membrane anchor Cue1 confers resistance to hygromycin B in Saccharomyces cerevisiae. MicroPubl Biol. 2026 1.
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