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 ufd2 Δ yeast was not as severe as that observed in ubc7 Δ and hrd1 Δ doa10 Δ yeast. Hygromycin B sensitivity of ufd2 Δ yeast derived from a distinct genetic background further validates a role for this E4 in combatting proteotoxic stress ( Figure 1C ).

Our finding that ufd2 Δ yeast are hypersensitive to hygromycin B is consistent with a general role for this E4 in protein quality control. Although plasmid-based complementation of ufd2 Δ yeast would provide additional confirmation, reproducible observation of hygromycin B sensitivity in independently generated ufd2 Δ 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.

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.

Yeast strains used in this study.

Experiments to determine sensitivity of ufd2 Δ 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.