Ubiquitin-elongating enzyme Ufd2 confers resistance to hygromycin B in Saccharomyces cerevisiae

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.

Figure 1. UFD2 confers resistance to hygromycin B .

(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.

Description

Protein quality control and regulated protein degradation through the ubiquitin-proteasome system (UPS) are 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.

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- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2	1C	(Chen et al., 1993)
VJY8 (alias MHY1702)	MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10 Δ ::HIS3 hrd1 Δ ::LEU2	1C	(Huyer et al., 2004)
VJY305 (aka SKY242)	MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 doa10 Δ ::kanMX4 hrd1 Δ ::kanMX4	1B	(Habeck et al., 2015)
VJY476 (alias BY4741)	MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0	1B	(Tong et al., 2001)
VJY645	MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 ufd2 Δ ::kanMX4	1B	(Tong et al., 2001)
VJY1075	MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 ubc7 Δ ::kanMX4	1B	(Tong et al., 2001)
VJY1249 (alias MHY9433)	MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ufd2 Δ ::kanMX6	1C	Gift of Mark Hochstrasser and Adrian Mehrtash

Funding Statement

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). This work was supported by a generous gift from Drs. Thomas E. and Karen Bumb Lauer.