Pleiner, Tino and Pinton Tomaleri, Giovani and Januszyk, Kurt and Inglis, Alison J. and Hazu, Masami and Voorhees, Rebecca M. (2020) Structural basis for membrane insertion by the human ER membrane protein complex. Science . ISSN 0036-8075. (In Press) https://resolver.caltech.edu/CaltechAUTHORS:20200521-122757560
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Abstract
A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit ER membrane protein complex (EMC) is a conserved co- and post-translational insertase at the endoplasmic reticulum. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.
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| Additional Information: | © 2020 American Association for the Advancement of Science. Published online May 21, 2020. We thank Israel Sanchez-Fernandez, Chi-Min Ho, Naima Sharaf, Chengcheng Fan, Gabe Lander, Andrey Malyutin, and Songye Chen for technical assistance; and Bil Clemons and the entire Voorhees lab for thoughtful discussion. Cryo-EM was performed in the Beckman Institute Center for TEM at Caltech, and data was processed using the Caltech High Performance Cluster, supported by a grant from the Gordon and Betty Moore Foundation. This research used resources of the Advanced Light Source, a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. Funding: This work was supported by grants from the Heritage Medical Research Institute, the Kinship Foundation, the Pew-Stewart Foundation, and the NIH’s National Institute of General Medical Sciences (DP2GM137412). T.P. is funded by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. Author contributions: T.P., G.P.T., and K.J. expressed and purified the sample. K.J. and R.M.V. collected and processed the cryo-EM data. K.J. and G.P.T. built and refined the atomic model. T.P., A.J.I., and M.H. performed the functional experiments. R.M.V. wrote the manuscript with input from all authors. The authors declare no competing interests. Data and materials availability: Atomic coordinates and cryo-EM maps have been deposited in the Protein Data Bank under accession code 6WW7 and in the Electron Microscopy Data Bank under accession codes EMD-21929 (overall map), 21930 (lumenal map), and 21931 (cytosolic map). Plasmids for the GFP-nanobody purification strategy are available on Addgene, and requests for materials should be addressed to R.M.V. | ||||||||||||||||
| Group: | Heritage Medical Research Institute | ||||||||||||||||
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| Record Number: | CaltechAUTHORS:20200521-122757560 | ||||||||||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20200521-122757560 | ||||||||||||||||
| Official Citation: | Structural basis for membrane insertion by the human ER membrane protein complex. Tino Pleiner, Giovani Pinton Tomaleri, Kurt Januszyk, Alison J. Inglis, Masami Hazu and Rebecca M. Voorhees. published online May 21, 2020; doi: 10.1126/science.abb5008 | ||||||||||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||
| ID Code: | 103379 | ||||||||||||||||
| Collection: | CaltechAUTHORS | ||||||||||||||||
| Deposited By: | Tony Diaz | ||||||||||||||||
| Deposited On: | 21 May 2020 19:49 | ||||||||||||||||
| Last Modified: | 21 May 2020 20:16 |
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