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HRD1-mediated ERAD tuning of ER-bound E2 is conserved between plants and mammals

Nature Plants volume 2, Article number: 16094 (2016) Cite this article

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Abstract

When membrane proteins and secretory proteins are misfolded or incompletely folded, they are retained in the endoplasmic reticulum (ER) for further folding or degradation. The HMG-COA reductase degradation 1 (HRD1) and degradation of alpha2 10 (DOA10) complexes are two major components involved in the ER-associated protein degradation (ERAD) system in eukaryotic organisms1–4. However, the relationship between these two complexes is largely unknown, especially in higher eukaryotes. Here, we report that the plant ubiquitin-conjugating enzyme 32 (UBC32), an ER-bound E2 working in the DOA10 complex, is maintained at low levels under standard conditions by proteasome-dependent degradation mediated by the HRD1 complex, the other E3 complex involved in ERAD. Loss of this negative regulation under ER stress increases capacity for degradation of misfolded proteins retained in the ER. Consistently, UBE2J1, the homologue of UBC32 in mammals, was also identified to be targeted by HRD1 for degradation. Taken together, these results suggest that the regulation of UBC32 (or UBE2J1) by the HRD1 complex is conserved between plants and mammals.

From yeast to metazoan, HRD3A and its homologies are conserved adaptors functioning in the HRD1 complex11. Therefore, we checked the protein status of UBC32 in hrd1 mutants. There are two HRD1 homologous genes in Arabidopsis, termed HRD1A and HRD1B, and they function redundantly in the plant ERAD pathway5. The result showed that, compared with the WT, the UBC32 protein level was slightly higher in both hrd1a and hrd1b single mutants but was dramatically increased in the hrd1a hrd1b double mutant (Fig. 1b). In addition, the mRNA level of UBC32 has no significant difference among those different genotypes (Supplementary Fig. 1). To test the specificity of this accumulation, mutants of other E3 ligases in the ERAD pathway or located on the ER membrane were checked, including RMA1, DOA10A, DOA10B, SDIR112 and CHIP13. The results showed that UBC32 specifically accumulated in hrd1a hrd1b compared with other E3 mutants (Fig. 1c).

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Figure 1: UBC32 is stabilized by knockout of key components of the HRD1 complex.
Figure 2: HRD1A and HRD1B interact with UBC32 and modulate the ubiquitylation and stability of UBC32.
Figure 3: UBC32 acts genetically downstream of the HRD1 complex and UBC32 is a versatile protein in facing different levels of stress.
Figure 4: UBE2J1 is targeted by HRD1 for proteasome system degradation.

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Acknowledgements

This research was supported by grant 973 Program 2011CB915402 from the National Basic Research Program of China and grant NSFC 31030047 from the National Science Foundation of China.

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Authors and Affiliations

  1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Beijing 100101, China

    Qian Chen, Yaorong Wu, Lijing Liu, Pengfei Wang, Ruijun Liu, Feng Cui, Qingliang Li, Xiaoyuan Yang & Qi Xie

  2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China

    Qian Chen, Pengfei Wang, Ruijun Liu, Qingliang Li & Qi Xie

  3. Department of Physiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, 21201, Maryland, USA

    Yongwang Zhong & Shengyun Fang

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Contributions

Q.X. and Q.C. designed the research. Q.C., Y.W.Z., L.J.L., P.F.W., F.C. and Q.L.L. performed the research. Q.X., Q.C., Y.W.Z. and S.Y.F. analysed the data. Q.X. and Q.C. wrote the paper. S.Y.F., Y.R.W. and X.Y.Y. revised the manuscript.

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Correspondence to Qi Xie.

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Supplementary Information

Supplementary Methods, Supplementary Figs 1-8, Supplementary Table 1 (PDF 950 kb)

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Chen, Q., Zhong, Y., Wu, Y. et al. HRD1-mediated ERAD tuning of ER-bound E2 is conserved between plants and mammals. Nature Plants 2, 16094 (2016). https://doi.org/10.1038/nplants.2016.94

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