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
-
- 4,510
- views
-
- 791
- 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
- Developmental Biology
Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.
-
- Cancer Biology
- Cell Biology
Understanding the cell cycle at the single-cell level is crucial for cellular biology and cancer research. While current methods using fluorescent markers have improved the study of adherent cells, non-adherent cells remain challenging. In this study, we addressed this gap by combining a specialized surface to enhance cell attachment, the FUCCI(CA)2 sensor, an automated image analysis pipeline, and a custom machine learning algorithm. This approach enabled precise measurement of cell cycle phase durations in non-adherent cells. This method was validated in acute myeloid leukemia cell lines NB4 and Kasumi-1, which have unique cell cycle characteristics, and we tested the impact of cell cycle-modulating drugs on NB4 cells. Our cell cycle analysis system, which is also compatible with adherent cells, is fully automated and freely available, providing detailed insights from hundreds of cells under various conditions. This report presents a valuable tool for advancing cancer research and drug development by enabling comprehensive, automated cell cycle analysis in both adherent and non-adherent cells.
