Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients
- Whitehead Institute for Biomedical Research, United States
- Max Plank Institute for Biochemistry, Germany
- University of California, San Francisco, United States
- Max Planck Institute of Biochemistry, Germany
- FairJourney Biologics, Portugal
- Stanford University, United States
- St Jude Children's Research Hospital, United States
Abstract
Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.
Data availability
All data generated or analyzed during this study are included in the manuscript or will have been made available in public repositories. Flow cytometry data and analysis code is available at Github (https://github.com/katerinadpopova/emcstructurefunction). Electron microscopy maps are available at the EMDB (unsharpened, sharpened, half maps, FSC file) (accession codes EMDB - 11732, 11733, 23003, 23033), models at the PDB (accession codes PDB - 7ADO, 7ADP, 7KRA, 7KTX), and raw cryo-EM data at EMPIAR. Key Resource Table is included as an appendix to the main article and is referenced throughout the Methods section with relevant reagents used or generated during the course of the study allowing for replication of these or request of specific cell lines and reagents. Supplementary file 1 contains raw mass spectrometry data. Supplementary file 4 contains un-cropped western blots. Supplementary file 5 contains plasmid sequences for mutant constructs generated for this study.
Article and author information
Author details
Lakshmi E Miller-Vedam
Elizabeth A Boydston
Matthew J Shurtleff
Jonathan S Weissman
Funding
Deutsche Forschungsgemeinschaft
- Brenda A Schulman
Chan Zuckerberg Initiative
- Adam Frost
Max Planck Institute for Dynamics of Complex Technical Systems Magdeburg
- Brenda A Schulman
National Institutes of Health (P50AI150476,1P41CA196276-01)
- Natalia Sevillano
- Charles S Craik
Helen Hay Whitney Foundation
- Matthew J Shurtleff
Peter und Traudl Engelhorn Stiftung
- Bastian Bräuning
Jane Coffin Childs Memorial Fund for Medical Research
- Nicole T Schirle Oakdale
National Institutes of Health (1DP2OD017690-01)
- Adam Frost
National Institutes of Health (GM24485)
- Robert M Stroud
Howard Hughes Medical Institute
- Jonathan S Weissman
The funders had no role in study design, data collection, data interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Miller-Vedam et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,462
- views
-
- 787
- downloads
-
- 73
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients
eLife 9:e62611.
https://doi.org/10.7554/eLife.62611
Further reading
-
- Cell Biology
Aging is associated with a number of physiologic changes including perturbed circadian rhythms; however, mechanisms by which rhythms are altered remain unknown. To test the idea that circulating factors mediate age-dependent changes in peripheral rhythms, we compared the ability of human serum from young and old individuals to synchronize circadian rhythms in culture. We collected blood from apparently healthy young (age 25–30) and old (age 70–76) individuals at 14:00 and used the serum to synchronize cultured fibroblasts. We found that young and old sera are equally competent at initiating robust ~24 hr oscillations of a luciferase reporter driven by clock gene promoter. However, cyclic gene expression is affected, such that young and old sera promote cycling of different sets of genes. Genes that lose rhythmicity with old serum entrainment are associated with oxidative phosphorylation and Alzheimer’s Disease as identified by STRING and IPA analyses. Conversely, the expression of cycling genes associated with cholesterol biosynthesis increased in the cells entrained with old serum. Genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions. We did not observe a global difference in the distribution of phase between groups, but found that peak expression of several clock-controlled genes (PER3, NR1D1, NR1D2, CRY1, CRY2, and TEF) lagged in the cells synchronized ex vivo with old serum. Taken together, these findings demonstrate that age-dependent blood-borne factors affect circadian rhythms in peripheral cells and have the potential to impact health and disease via maintaining or disrupting rhythms respectively.
-
- Biochemistry and Chemical Biology
- Cell Biology
Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.
