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.
Reviewing Editor
- Volker Dötsch, Goethe University, Germany
Version history
- Received: August 30, 2020
- Accepted: November 17, 2020
- Accepted Manuscript published: November 25, 2020 (version 1)
- Version of Record published: January 5, 2021 (version 2)
- Version of Record updated: January 6, 2021 (version 3)
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
-
- 4,268
- views
-
- 762
- downloads
-
- 64
- 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
Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.
-
- Cancer Biology
- Cell Biology
Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.
