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.
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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.
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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.
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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.
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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)
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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
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DOI: https://doi.org/10.1007/s00294-022-01227-1