Abstract
The Gβγ heterodimer is an important signal transducer. Gβ, however, is prone to misfolding due to its requirement for Gγ and chaperones for proper folding. How cells dispose of misfolded Gβ (mfGβ) is not clear. Here, we showed that mfGβ was able to be polyubiquitinated and subsequently degraded by the proteasome. It was sequestered in aggresomes after the inhibition of the proteasome activity with MG132. Sustained activation of Gβγ signaling further elevated cellular levels of the ubiquitinated Gβ. Moreover, Nudel, a regulator of cytoplasmic dynein, the microtubule minus end-directed motor, directly interacted with both the unubiquitinated and ubiquitinated mfGβ. Increasing the levels of both mfGβ and Nudel promoted the association of Gβ with both Nudel and dynein, resulting in robust aggresome formation in a dynein-dependent manner. Depletion of Nudel by RNAi reduced the dynein-associated mfGβ, impaired the MG132-induced aggresome formation, and markedly prolonged the half-life of nascent Gβ. Therefore, cytosolic mfGβ is recruited to dynein by Nudel and transported to the centrosome for rapid sequestration and degradation. Such a process not only eliminates mfGβ efficiently for the control of protein quality, but may also help to terminate the Gβγ signaling.
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Acknowledgements
We thank Dr Yixian Zheng (Carnegie Institution for Science, USA) and Dr Ronggui Hu (Institute of Biochemistry and Cell Biology (IBCB), Chinese Academy of Sciences (CAS)) for helpful suggestions and discussions, Qiongping Huang, Wei Yu and Yan Li for technical assistance. We also thank Drs Narasimhan Gautam (Washington University at St. Louis, USA) for Gβ cDNAs, Phillip B Wedegaertner (Thomas Jefferson University, USA) for pcDNA3-Gγ2, Sami Bahri (Institute of Molecular and Cell Biology, Singapore) for pXJ40-Flag-LGN, Gordon Chan (University of Alberta, Canada) for the ninein antibody, Gang Pei (IBCB, CAS) for pcDNA3-HA-β2AR, and Shuo Yang (ICBC, CAS) for pFlag-Tctex1. This work was supported by the National Basic Research Program of China (2010CB912102 and 2012CB945003), the National Natural Science Foundation of China (30830060 and 31010103910), Chinese Academy of Sciences (XDA01010107), and Science and Technology Commission of Shanghai Municipality (09JC1416200).
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Supplementary information
Supplementary information, Figure S1
The detection of endogenous Gγ. (PDF 88 kb)
Supplementary information, Figure S2
The structural requirements of the Nudel-Gβ2 interaction. (PDF 147 kb)
Supplementary information, Figure S3
The centrosomal localization of Gβ. (PDF 79 kb)
Supplementary information, Figure S4
The aggresome formation upon the overexpression of different Gβ isoforms with Nudel. (PDF 72 kb)
Supplementary information, Figure S5
Inactivation of dynein does not affect the protein levels of Nudel and Gβ. (PDF 85 kb)
Supplementary information, Figure S6
Validation of the use of the RIPA buffer in this study. (PDF 87 kb)
Supplementary information, Figure S7
The Gβ isoforms are prone to polyubiquitination upon overexpression. (PDF 94 kb)
Supplementary information, Figure S8
The comparison of different approaches to the isolation of the dynein motor complex. (PDF 98 kb)
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Wan, Y., Yang, Z., Guo, J. et al. Misfolded Gβ is recruited to cytoplasmic dynein by Nudel for efficient clearance. Cell Res 22, 1140–1154 (2012). https://doi.org/10.1038/cr.2012.41
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DOI: https://doi.org/10.1038/cr.2012.41
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