Abstract
Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61-2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61-2 grow at 25 °C because the protein is stable, but sec61-2 yeast are inviable at 38 °C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61-2 and hence sec61-2hrd1∆ double mutants are viable at 38 °C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1’s transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.
Similar content being viewed by others
References
Adle DJ, Wei W, Smith N, Bies JJ, Lee J (2009) Cadmium-mediated rescue from ER-associated degradation induces expression of its exporter. Proc Natl Acad Sci USA 106:10189–10194. https://doi.org/10.1073/pnas.0812114106
Bagola K, Mehnert M, Jarosch E, Sommer T (2011) Protein dislocation from the ER. Biochim Biophys Acta 1808:925–936. https://doi.org/10.1016/j.bbamem.2010.06.025
Bahler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951. https://doi.org/10.1002/(SICI)1097-0061(199807)14:10%3c943::AID-YEA292%3e3.0.CO;2-Y
Baldridge RD, Rapoport TA (2016) Autoubiquitination of the Hrd1 ligase triggers protein retrotranslocation in ERAD. Cell 166:394–407. https://doi.org/10.1016/j.cell.2016.05.048
Betegon M, Brodsky JL (2020) Unlocking the door for ERAD. Nat Cell Biol 22:263–265. https://doi.org/10.1038/s41556-020-0476-1
Biederer T, Volkwein C, Sommer T (1996) Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway. EMBO J 15:2069–2076
Bordallo J, Wolf DH (1999) A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae. FEBS Lett 448:244–248. https://doi.org/10.1016/s0014-5793(99)00362-2
Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9:209–222. https://doi.org/10.1091/mbc.9.1.209
Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132. https://doi.org/10.1002/(SICI)1097-0061(19980130)14:2%3c115::AID-YEA204%3e3.0.CO;2-2
Carroll SM, Hampton RY (2010) Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase. J Biol Chem 285:5146–5156. https://doi.org/10.1074/jbc.M109.067876
Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126:361–373. https://doi.org/10.1016/j.cell.2006.05.043
Christianson JC, Olzmann JA, Shaler TA, Sowa ME, Bennett EJ, Richter CM, Tyler RE, Greenblatt EJ, Harper JW, Kopito RR (2011) Defining human ERAD networks through an integrative mapping strategy. Nat Cell Biol 14:93–105. https://doi.org/10.1038/ncb2383
Cohen I, Wiener R, Reiss Y, Ravid T (2015) Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases. Proc Natl Acad Sci USA 112:E625-632. https://doi.org/10.1073/pnas.1415621112
Denic V, Quan EM, Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126:349–359. https://doi.org/10.1016/j.cell.2006.05.045
Egner R, Rosenthal FE, Kralli A, Sanglard D, Kuchler K (1998) Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter. Mol Biol Cell 9:523–543. https://doi.org/10.1091/mbc.9.2.523
Fang S, Ferrone M, Yang C, Jensen JP, Tiwari S, Weissman AM (2001) The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc Natl Acad Sci USA 98:14422–14427. https://doi.org/10.1073/pnas.251401598
Finger A, Knop M, Wolf DH (1993) Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur J Biochem 218:565–574. https://doi.org/10.1111/j.1432-1033.1993.tb18410.x
Finley D, Ulrich HD, Sommer T, Kaiser P (2012) The ubiquitin-proteasome system of Saccharomyces cerevisiae. Genetics 192:319–360. https://doi.org/10.1534/genetics.112.140467
Flury I, Garza R, Shearer A, Rosen J, Cronin S, Hampton RY (2005) INSIG: a broadly conserved transmembrane chaperone for sterol-sensing domain proteins. EMBO J 24:3917–3926. https://doi.org/10.1038/sj.emboj.7600855
Friedlander R, Jarosch E, Urban J, Volkwein C, Sommer T (2000) A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat Cell Biol 2:379–384. https://doi.org/10.1038/35017001
Gardner RG, Swarbrick GM, Bays NW, Cronin SR, Wilhovsky S, Seelig L, Kim C, Hampton RY (2000) Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p. J Cell Biol 151:69–82. https://doi.org/10.1083/jcb.151.1.69
Gauss R, Sommer T, Jarosch E (2006) The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment. EMBO J 25:1827–1835. https://doi.org/10.1038/sj.emboj.7601088
Guerriero CJ, Brodsky JL (2012) The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiol Rev 92:537–576. https://doi.org/10.1152/physrev.00027.2011
Hampton RY, Gardner RG, Rine J (1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell 7:2029–2044. https://doi.org/10.1091/mbc.7.12.2029
Hentges P, Van Driessche B, Tafforeau L, Vandenhaute J, Carr AM (2005) Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast 22:1013–1019. https://doi.org/10.1002/yea.1291
Horn SC, Hanna J, Hirsch C, Volkwein C, Schutz A, Heinemann U, Sommer T, Jarosch E (2009) Usa1 functions as a scaffold of the HRD-ubiquitin ligase. Mol Cell 36:782–793. https://doi.org/10.1016/j.molcel.2009.10.015
Knop M, Finger A, Braun T, Hellmuth K, Wolf DH (1996) Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J 15:753–763
Lips C, Ritterhoff T, Weber A, Janowska MK, Mustroph M, Sommer T, Klevit RE (2020) Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation. EMBO J 39:e104863. https://doi.org/10.15252/embj.2020104863
Mehnert M, Sommer T, Jarosch E (2014) Der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane. Nat Cell Biol 16:77–86. https://doi.org/10.1038/ncb2882
Metzger MB, Liang YH, Das R, Mariano J, Li S, Li J, Kostova Z, Byrd RA, Ji X, Weissman AM (2013) A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms. Mol Cell 50:516–527. https://doi.org/10.1016/j.molcel.2013.04.004
Nakatsukasa K, Brodsky JL, Kamura T (2013) A stalled retrotranslocation complex reveals physical linkage between substrate recognition and proteasomal degradation during ER-associated degradation. Mol Biol Cell 24(1765–1775):S1761-1768. https://doi.org/10.1091/mbc.E12-12-0907
Nakatsukasa K, Nishimura T, Byrne SD, Okamoto M, Takahashi-Nakaguchi A, Chibana H, Okumura F, Kamura T (2015) The ubiquitin ligase SCF(Ucc1) acts as a metabolic switch for the glyoxylate cycle. Mol Cell 59:22–34. https://doi.org/10.1016/j.molcel.2015.04.013
Nakatsukasa K, Sone M, Alemayehu DH, Okumura F, Kamura T (2018) The HECT-type ubiquitin ligase Tom1 contributes to the turnover of Spo12, a component of the FEAR network, in G2/M phase. FEBS Lett 592:1716–1724. https://doi.org/10.1002/1873-3468.13066
Nakatsukasa K, Kawarasaki T, Moriyama A (2019) Heterologous expression and functional analysis of the F-box protein Ucc1 from other yeast species in Saccharomyces cerevisiae. J Biosci Bioeng 128:704–709. https://doi.org/10.1016/j.jbiosc.2019.06.003
Needham PG, Guerriero CJ, Brodsky JL (2019) Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases. Cold Spring Harb Perspect Biol. https://doi.org/10.1101/cshperspect.a033928
Peterson BG, Glaser ML, Rapoport TA, Baldridge RD (2019) Cycles of autoubiquitination and deubiquitination regulate the ERAD ubiquitin ligase Hrd1. Elife. https://doi.org/10.7554/eLife.50903
Plemper RK, Egner R, Kuchler K, Wolf DH (1998) Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome. J Biol Chem 273:32848–32856. https://doi.org/10.1074/jbc.273.49.32848
Preston GM, Brodsky JL (2017) The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation. Biochem J 474:445–469. https://doi.org/10.1042/BCJ20160582
Ravid T, Kreft SG, Hochstrasser M (2006) Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. EMBO J 25:533–543. https://doi.org/10.1038/sj.emboj.7600946
Sato BK, Schulz D, Do PH, Hampton RY (2009) Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase. Mol Cell 34:212–222. https://doi.org/10.1016/j.molcel.2009.03.010
Schmidt CC, Vasic V, Stein A (2020) Doa10 is a membrane protein retrotranslocase in ER-associated protein degradation. Elife. https://doi.org/10.7554/eLife.56945
Schoebel S, Mi W, Stein A, Ovchinnikov S, Pavlovicz R, DiMaio F, Baker D, Chambers MG, Su H, Li D, Rapoport TA, Liao M (2017) Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3. Nature 548:352–355. https://doi.org/10.1038/nature23314
Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27
Vashist S, Ng DT (2004) Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J Cell Biol 165:41–52. https://doi.org/10.1083/jcb.200309132
Vashist S, Kim W, Belden WJ, Spear ED, Barlowe C, Ng DT (2001) Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J Cell Biol 155:355–368. https://doi.org/10.1083/jcb.200106123
Vashistha N, Neal SE, Singh A, Carroll SM, Hampton RY (2016) Direct and essential function for Hrd3 in ER-associated degradation. Proc Natl Acad Sci USA 113:5934–5939. https://doi.org/10.1073/pnas.1603079113
Vasic V, Denkert N, Schmidt CC, Riedel D, Stein A, Meinecke M (2020) Hrd1 forms the retrotranslocation pore regulated by auto-ubiquitination and binding of misfolded proteins. Nat Cell Biol 22:274–281. https://doi.org/10.1038/s41556-020-0473-4
Veit G, Roldan A, Hancock MA, Da Fonte DF, Xu H, Hussein M, Frenkiel S, Matouk E, Velkov T, Lukacs GL (2020) Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination. JCI Insight. https://doi.org/10.1172/jci.insight.139983
Wangeline MA, Vashistha N, Hampton RY (2017) Proteostatic tactics in the strategy of sterol regulation. Annu Rev Cell Dev Biol 33:467–489. https://doi.org/10.1146/annurev-cellbio-111315-125036
Wu X, Rapoport TA (2018) Mechanistic insights into ER-associated protein degradation. Curr Opin Cell Biol 53:22–28. https://doi.org/10.1016/j.ceb.2018.04.004
Wu X, Rapoport TA (2021) Translocation of proteins through a distorted lipid bilayer. Trends Cell Biol 31:473–484. https://doi.org/10.1016/j.tcb.2021.01.002
Wu X, Siggel M, Ovchinnikov S, Mi W, Svetlov V, Nudler E, Liao M, Hummer G, Rapoport TA (2020) Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex. Science. https://doi.org/10.1126/science.aaz2449
Xie W, Ng DT (2010) ERAD substrate recognition in budding yeast. Semin Cell Dev Biol 21:533–539. https://doi.org/10.1016/j.semcdb.2010.02.007
Acknowledgements
We thank the members of the Nakatsukasa lab for discussions and critical comments on the manuscript, and Dr. Kazuya Nishio and Dr. Tsunehiro Mizushima for help with structural graphics. This work was supported by the Toray Science Foundation to K.N., the Toyoaki Scholarship Foundation to K.N., and JSPS grants KAKENHI to K.N. (Grant Numbers 15K18503, 18K19306, and 19H02923), as well as by a National Institutes of Health grant GM131732 to J.L.B. We would like to thank the National Bio-Resource Project (NBRP) of the MEXT program, Japan, for strains and plasmids.
Funding
This work was supported by Toray Science Foundation to K.N., Toyoaki Scholarship Foundation to K.N., and JSPS KAKENHI to K.N. (Grant Numbers 15K18503, 18K19306, and 19H02923), as well as by National Institutes of Health grant GM131732 to J.L.B. We would like to thank the National Bio-Resource Project (NBRP) of the MEXT program, Japan, for strains and plasmids.
Author information
Authors and Affiliations
Contributions
KN and JLB designed the entire research program. KN and JLB wrote the original draft and edited the manuscript. KN and SW performed most experiments. YT and ToK performed some experiments. TaK supported biochemical and genetic analysis. All authors have read and agreed to the final version of the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors have no relevant financial or non-financial interest to disclose.
Additional information
Communicated by Michael Polymenis.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
294_2022_1227_MOESM1_ESM.pdf
Supplementary file1. Figure S1 The levels of expression of Hrd1-3HA or the mutant variants that rescued the temperature sensitivity of the sec61-2 allele were analyzed by western blotting with anti-HA antibody. Hrd1-3HA and the mutant variants were expressed under the control of HRD1 promoter from CEN/ARS plasmids. Cells were incubated in synthetic medium at 26 °C and shifted to 38 °C for 1 h. Cells were collected and the total lysate was subjected to western blot analysis. Figure S2 A Quantification of the results of independent cycloheximide chase analyses of CPY*, as shown in one example in Fig. 6D, are presented. The amount of CPY* at time = 0 was set to 100%. Values represent the means of the data ± SD (n = 3). Where indicated, p values were determined using a Student’s t-test by comparing the amount of CPY* in Hrd1 mutant cells to that in wild-type cells. Significance was indicated as follows: n.s. not significant; *p < 0.05, **p < 0.01. B Quantification of the results of independent cycloheximide chase analyses of Hrd1, as shown in one example in Fig. 6D, are plotted. The amount of wild-type Hrd1 at time = 0 was set to 100%. Please note that the relative amount of Hrd1 at time = 0 (steady state level of Hrd1) differs between wild-type and mutants. Values are mean ± SD (n = 3). Where indicated, p values were determined using a Student’s t-test by comparing the amount of mutant Hrd1 to wild-type Hrd1. Significance was indicated as follows: ***p < 0.001, ****p < 0.0001. C CPY* degradation in cells expressing the isolated Hrd1 mutants was assessed by cycloheximide chase analysis as in Fig. 6D except that cells were grown at 26 °C instead of 30 °C. CPY* and Hrd1 were detected by western blotting with anti-HA antibody and anti-Hrd1 antibody, respectively. Coomassie Brilliant Blue (CBB) staining of membrane served as a loading control (PDF 1392 kb)
Rights and permissions
About this article
Cite this article
Nakatsukasa, K., Wigge, S., Takano, Y. et al. A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD. Curr Genet 68, 227–242 (2022). https://doi.org/10.1007/s00294-022-01227-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-022-01227-1