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. 2024 Jul 29;2024:10.17912/micropub.biology.001276. doi: 10.17912/micropub.biology.001276

Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes Ubc6 and Ubc7 confer resistance to hygromycin B in Saccharomyces cerevisiae

Sophia L Owutey 1,#, Katrina A Procuniar 1,#, Emmanuel Akoto 1, Jacob C Davis 1,2, Rachel M Vachon 1, LiLi F O'Malley 1, Hayden O Schneider 1,3, Philip J Smaldino 1, Jason D True 1, Ashley L Kalinski 1, Eric M Rubenstein 1,§
Reviewed by: Anonymous
PMCID: PMC11320122  PMID: 39139584

Abstract

Aberrant endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed through ER-associated degradation (ERAD) and INM-associated degradation (INMAD). We previously showed the Hrd1, Doa10, and Asi ERAD and INMAD ubiquitin ligases (E3s) in Saccharomyces cerevisiae confer resistance to hygromycin B, which distorts the ribosome decoding center. Here, we assessed the requirement of Ubc6 and Ubc7, the primary ERAD and INMAD ubiquitin-conjugating enzymes (E2s) for hygromycin B resistance. Loss of either E2 sensitized cells to hygromycin B, with UBC7 deletion having a greater impact, consistent with characterized roles for Ubc6 and Ubc7 in ER and INM protein quality control.

Figure 1. UBC6 and UBC7 confer resistance to hygromycin B .

Figure 1. UBC6 and UBC7 confer resistance to hygromycin B

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(A) Endoplasmic Reticulum (ER)-Associated Degradation and Inner Nuclear Membrane (INM)-Associated Degradation pathways. In conjunction with the E2 Ubc7, the E3 Hrd1 promotes degradation of aberrant ER luminal and transmembrane proteins as well as proteins that clog ER translocons. The E3 Doa10 functions with two E2s, Ubc6 and Ubc7, to mediate degradation of aberrant transmembrane proteins at the ER or INM in addition to soluble cytosolic or nucleoplasmic proteins. The trimeric Asi E3 complex (Asi1, Asi2, and Asi3) works with Ubc6 and Ubc7 to target aberrant transmembrane INM and soluble nucleoplasmic proteins. Ubc7 is anchored at the ER membrane through interaction with Cue1. Ub, ubiquitin. (B) and (C) Sixfold serial dilutions of yeast of the indicated genotype were spotted on medium lacking (No Drug) or containing increasing concentrations of hygromycin B. Plates were incubated at 30°C and imaged after 1-2 days. Experiments were performed three or more times.

Description

Degradation of misfolded, excess, and otherwise aberrant proteins is critical for cellular homeostasis. The ability to recognize and destroy faulty proteins declines with age, and disruptions to enzymes contributing to protein quality control (PQC) contribute to several diseases (Badawi et al., 2023; Guerriero & Brodsky, 2012) . Eukaryotic cells possess compartment-specific PQC mechanisms, including those dedicated to the turnover of aberrant proteins at the physically continuous endoplasmic reticulum (ER) membrane and inner nuclear membrane (INM) (Mehrtash & Hochstrasser, 2019) . ER-associated degradation (ERAD) promotes turnover of aberrant ER luminal, transmembrane, and translocon-clogging proteins as well as cytosolic polypeptides that contact the ER surface. INM-associated degradation (INMAD) mediates proteolysis of faulty INM transmembrane and INM-abutting soluble nucleoplasmic proteins. ERAD and INMAD both employ ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) to polyubiquitylate proteins ( Figure 1A ), destining them for destruction by cytosolic or nucleoplasmic proteasomes.

The highly conserved transmembrane Hrd1 and Doa10 E3s mediate ERAD in Saccharomyces cerevisiae , targeting distinct classes of aberrant proteins for degradation based on the location and nature of the degradation signals (degrons) (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) . Doa10 also functions in INMAD alongside the heterotrimeric Asi E3 (composed of Asi1, Asi2, and Asi3) (Deng & Hochstrasser, 2006; Foresti et al., 2014; Khmelinskii et al., 2014) . Loss of either Asi1 or Asi3 abolishes Asi PQC function (Foresti et al., 2014; Woodruff et al., 2021) . Hrd1, Doa10, and the Asi complex have partially overlapping E2 dependencies. Hrd1 functions primarily with the soluble E2 Ubc7 (human homolog, UBE2G2), which is anchored at the membrane by the transmembrane protein Cue1 (Bays et al., 2001; Lips et al., 2020; Plemper et al., 1999) . By contrast, Doa10 and Asi use two E2s, Ubc7 and the transmembrane Ubc6 (human homolog, UBE2J2) (Foresti et al., 2014; Khmelinskii et al., 2014; Swanson et al., 2001) . Ubc6 and Ubc7 participate in a sequential ubiquitylation mechanism, with Ubc6 “priming” substrates with an initial ubiquitin molecule and Ubc7 elongating polyubiquitin chains (Lips et al., 2020; Weber et al., 2016) . It is likely that additional E2s contribute to a lesser extent to ERAD and INMAD. For example, in some circumstances, the E2 Ubc1 partially compensates for impaired Ubc7 function in promoting Hrd1 substrate ubiquitylation (Bays et al., 2001) .

The aminoglycoside hygromycin B binds to and distorts the ribosome A site, thereby likely increasing the frequency of mistranslation and generation of PQC substrates (Brodersen et al., 2000; Ganoza & Kiel, 2001) . Mutation of genes encoding several proteins with documented or predicted PQC function causes hygromycin B hypersensitivity (Bengtson & Joazeiro, 2010; Chuang & Madura, 2005; Daraghmi et al., 2023; Flagg et al., 2023; Jaeger et al., 2018; Turk et al., 2023; Verma et al., 2013) . Indeed, we have previously shown that loss of several ubiquitin ligases, including Hrd1, Doa10, Asi1, or Asi3, sensitizes cells to hygromycin B (Crowder et al., 2015; Doss et al., 2022; Niekamp et al., 2019; Runnebohm, Evans, et al., 2020; Woodruff et al., 2021) . A role for Ubc6 and Ubc7 in combatting hygromycin B-induced proteotoxic stress has not been demonstrated. Given their functions as the major characterized E2s in ERAD and INMAD, we predicted loss of either enzyme would reduce fitness in the presence of this drug.

To assess the roles of Ubc6 and Ubc7 in combatting proteotoxic stress caused by hygromycin B, we cultured serial dilutions of wild type yeast, yeast lacking UBC6 and UBC7 individually or in concert, as well as a yeast strain rendered broadly defective for ERAD and INMAD by simultaneous deletion of HRD1 , DOA10 , and ASI1 ( Figure 1B ). All strains grew similarly in the absence of hygromycin B. Loss of either UBC6 or UBC7 sensitized yeast to hygromycin B, with UBC7 deletion having a stronger impact. Combined deletion of both UBC6 and UBC7 caused a greater growth defect than individual absence of either E2-encoding gene. Finally, hrd1 Δ doa10 Δ asi1 Δ yeast exhibited a more profound growth defect than any E2 mutant.

To validate these results, we assessed hygromycin B sensitivity of ubc6 Δ and ubc7 Δ yeast strains in a distinct genetic background, as well as three double mutants lacking catalytic components of the ERAD or INMAD E3s ( Figure 1C ). As before, loss of either E2 sensitized yeast to hygromycin B, with cells lacking Ubc7 faring more poorly than those without Ubc6. Loss of any two ERAD or INMAD E3s approximately phenocopied ubc7 Δ yeast.

A greater role for Ubc7 than Ubc6 in combatting proteotoxicity likely reflects broader Ubc7 participation in ERAD and INMAD. Loss of Ubc6 is expected to compromise Doa10 and Asi function, while UBC7 deletion is predicted to abolish all three major branches of ERAD and INMAD. The observation that ubc6 Δ ubc7 Δ double mutant yeast exhibit a stronger growth defect than either ubc6 Δ or ubc7 Δ single mutant suggests independent functions for both Ubc6 and Ubc7. Identification of Ubc6-dependent, Ubc7-independent PQC substrates would support this model. Further, an enhanced growth defect of hrd1 Δ doa10 Δ asi1 Δ compared to ubc6 Δ ubc7 Δ yeast is in agreement with other reports indicating additional E2s (such as Ubc1) may function with ERAD and INMAD E3s, when the primary E2s are unavailable.

Our data are consistent with a previous study demonstrating overexpression of genes encoding either E2 enhances resistence to multiple stresses, including heat stress, oxidative stress, and presence of the toxic amino acid analog canavanine (Hiraishi et al., 2006) . Conversely, previous work showed that ubc7 Δ and hrd1 Δ doa10 Δ yeast exhibited similar hypersensitivity to cadmium (Swanson et al., 2001) . Large-scale analyses indicated loss of UBC7 reduces tolerance to multiple transition metals, which oxidatively damage a range of biological macromolecules, including proteins (Bleackley et al., 2011; Ruotolo et al., 2008; Zhao et al., 2020) , and genotoxic agents (Alamgir et al., 2010; Brown et al., 2006; Gaytan et al., 2013; Kapitzky et al., 2010) . We note hygromycin B hypersensitivity was not observed for ubc6 Δ or ubc7 Δ yeast in a previous report (Chuang & Madura, 2005) . This may be due to differences in effective drug concentrations in culture medium. In alignment with our results, we have also recently shown that loss of Doa10, Hrd1, and Ubc7 homologs sensitizes the pathogenic fungi Candida albicans to hygromycin B (Doss et al., 2023) . Overall, our work supports a critical and conserved function for endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes in protein quality control.

Methods

Growth assays

Yeast growth was analyzed as previously described (Watts et al., 2015) . Briefly, sixfold dilutions of each yeast strain were spotted onto yeast extract-peptone-dextrose medium (Guthrie & Fink, 2004) lacking or containing hygromycin B (Gibco) at the indicated concentrations and incubated at 30°C for the indicated amount of time.

ASI3 gene replacement

To generate yeast strains VJY409 and VJY410, ASI3 was replaced by natMX4 through homologous recombination. A 1464-bp nat4MX4 cassette with termini possessing sequences flanking the ASI3 gene was PCR-amplified from pAG25 (Goldstein & McCusker, 1999) using primers VJR274 (5’ AGGAACAGTCATTACGTAGGGATTTTCAAAAGTTTGACTGCACATACGATTTAGGTGACAC) and VJR275 (5’ TCCTATGATGTCTTAAATACGTATACCTAATAAAATAATTAATACGACTCACTATAGGGAG 3’). The natMX4 cassette was introduced into VJY22 ( hrd1 Δ ::kanMX4 ) yeast and VJY102 ( doa10 Δ ::kanMX4 ) by lithium acetate transformation (Guthrie & Fink, 2004) . Successful integration in nourseothricin-resistant clones were verified by PCR at the 5’ and 3’ recombination junctions, and genotypes at the DOA10 , HRD1 , and ASI3 loci were PCR validated for both strains.

Reagents

Yeast strains used in this study.

Name

Genotype

Figure or purpose

Reference

VJY6 (alias MHY500)

MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2

1B

(Chen et al., 1993)

VJY22

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3Δ0 hrd1 Δ:: kanMX4

Used to generate VJY409

(Tong et al., 2001)

VJY44 (alias MHY496)

MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc6- Δ 1 :: HIS3

1B

(Sommer & Jentsch, 1993)

VJY50 (alias MHY551)

MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc7 Δ:: LEU2

1B

(Chen et al., 1993)

VJY102

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 doa10 Δ:: kanMX4

Used to generate VJY410

(Tong et al., 2001)

VJY305 (alias SKY252)

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 doa10 Δ:: kanMX4 hrd1 Δ:: kanMX4

1C

(Habeck et al., 2015)

VJY409

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 hrd1 Δ:: kanMX4 asi3 Δ:: natMX4

1C

This study

VJY410

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 doa10 Δ:: kanMX4 asi3 Δ:: natMX4

1C

This study

VJY476 (alias BY4741)

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0

1C

(Tong et al., 2001)

VJY723

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 ubc6 Δ:: kanMX4

1C

(Hickey et al., 2021)

VJY1075

MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 ubc7 Δ:: kanMX4

1C

(Tong et al., 2001)

VJY1096 (alias MHY553)

MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 ubc6 Δ:: HIS3 ubc7 Δ:: LEU2

1B

(Chen et al., 1993)

VJY1098 (MHY11132, ABM297)

MATa his3- Δ 200 leu2-3,112 ura3-52 lys2-801 trp1-1 gal2 doa10 Δ:: HIS3 hrd1 Δ:: LEU2 asi1 Δ:: kanMX6

1B

(Mehrtash & Hochstrasser, 2023)

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Acknowledgments

Acknowledgments

Experiments to determine sensitivity of ubc6 Δ yeast to hygromycin B were piloted by undergraduate students in the Fall 2022 Methods in Cell Biology (BIO 315) Course at Ball State University (Noah Bische, James Carty, Kieran Claypool, Natalie Coomer, Alexandria Deel, Emily Desai, Allison Dittmer, Grace Gilbert, Evan Gosnell, Bryce Harman, Kerrigan Huffman, Breanna Long, Dorcas Macanthony, Mildred Obungu, Dylan Seiler, Cole Strassburger) and validated in the research laboratory of EMR. We thank Evan Rogers for serving as a Teaching Assistant in BIO 315. We thank Christopher Hickey, Mark Hochstrasser, Stefan Kreft, and Adrian Mehrtash for generously sharing yeast strains. We thank the Saccharomyces Genome Database for thorough, organized, and up-to-date curation of 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. We dedicate this manuscript to Susan Calvin – thank you for you all you have done to promote student success (and to save reagents from failing freezers!). All the best for a fun and fulfilling retirement!

Funding Statement

<p>This work was funded by NIH grant R15 GM111713 (EMR). Work in the lab of PJS is funded by NIH grant R15 G067291 and NIH grant R15 CA252996. Work in the lab of ALK is funded by NINDS-1R15NS128837 and John’s Hopkins Merkin PNNR Seed Funds. KAP was supported by the Ball State University Teacher-Scholar Program and a Ball State University Pepsi Scholarship Summer Research Award. RMV was supported by a Research Grant from the Ball State University Chapter of Sigma Xi. SLO was supported by a Ball State University Graduate School Capstone Completion Fellowship. LFO and SLO were supported by Ball State University Aspire Student Research grants. Preliminary studies conducted in BIO 315 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).</p>

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