Structural basis for membrane insertion by the human ER membrane protein complex
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 endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms 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.
Additional Information© 2020 American Association for the Advancement of Science. Received 2 March 2020; accepted 12 May 2020. Published online 21 May 2020. We thank I. Sanchez-Fernandez, C.-M. Ho, N. Sharaf, C. Fan, G. Lander, A. Malyutin, and S. Chen for technical assistance and B. Clemons, M. Schmeing, 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 Department of Energy 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.
Accepted Version - nihms-1634020.pdf
Supplemental Material - abb5008_MDAR_Reproducibility_Checklist.pdf
Supplemental Material - abb5008_Pleiner_SM.pdf
Supplemental Material - abb5008s1.mp4
Supplemental Material - abb5008s2.mp4
Supplemental Material - abb5008s3.mp4