Abstract
A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways1. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis2,3. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone4. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex ‘clients’ are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.
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Acknowledgements
We are grateful to E. Whiteman and X. Li for carrying out the initial experiments for parts of this project, S.W. Kang, S. Shao, and Z. Zhang for discussions, P. Sengupta, J. Magadán, and C. Ott for constructs, J. Taunton and J. Garrison for cotransin, S. Shao for comments on the manuscript, and Y. Ye for discussions and sharing results before publication. This work was supported by the Intramural Research Program of the National Institutes of Health (R.S.H.) and a postdoctoral fellowship from The Wenner-Gren Foundations (T.H.).
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T.H. performed most of the experiments, with contributions from A.S. (ubiquitination assays in modified lysates), M.M. (defining the substrate specificity of BAG6), H.D.E. (characterizing the Fr-RRL system), E.G. (BAG6 crosslinking analysis) and R.S.H. (in vivo studies). R.S.H. conceived the project, guided the experiments and wrote the paper with input from all of the authors.
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The file contains Supplementary Technical Notes relating to each of the main figures and Supplementary Figures 1-23 with legends. (PDF 9380 kb)
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Hessa, T., Sharma, A., Mariappan, M. et al. Protein targeting and degradation are coupled for elimination of mislocalized proteins. Nature 475, 394–397 (2011). https://doi.org/10.1038/nature10181
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DOI: https://doi.org/10.1038/nature10181
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