Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients.

Lakshmi E Miller-Vedam,Natalia Sevillano,Bastian Bräuning,Nicole T Schirle Oakdale,Jesuraj R Prabu,Matthew J Shurtleff,Charles S Craik,Jessica L Bonnar,Adam Frost,Brenda A Schulman,Robert M Stroud,Jonathan S Weissman,Elizabeth A Boydston,Katerina D Popova

doi:10.7554/elife.62611

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.

Highlights

Integral membrane proteins serve diverse and critical cellular roles, including signal transduction, lipid biosynthesis, adhesion, and transport of molecules across the bilayer
We developed systems to produce robust quantities of pure intact yEMC and human EMC (hEMC) to determine structures for the two organisms in which different facets of ER membrane protein complex (EMC) function have been described in detail (Jonikas et al, 2009; Christianson et al, 2012; Guna et al, 2018; Shurtleff et al, 2018)
Parallel efforts converged on an approach involving FLAG affinity-tagging of the EMC5 C-terminus, which was performed for endogenous yEMC and recombinant hEMC in human embryonic kidney (HEK) cells (Figure 1—figure supplements 1–2, Supplementary file 1)

Summary

Introduction

Integral membrane proteins serve diverse and critical cellular roles, including signal transduction, lipid biosynthesis, adhesion, and transport of molecules across the bilayer. We characterized the phenotypes of three distinct classes of EMC clients associated with a series of structure-based EMC mutants Both yEMC and hEMC structures reveal a path for transmembrane helix insertion from the cytoplasm into the membrane via a conserved cavity. Analysis of human disease mutations in hEMC1 and our structure-informed mutations enabled us to decouple the EMC insertase function from non-insertase functions and reveal a potential role of the EMC in differentially controlling the biogenesis of distinct classes of client proteins. These structure-function studies collectively establish that the EMC adopts a modular architecture enabling its diverse functions in membrane protein biogenesis

Results

Discussion

Materials and methods

Funding Funder