EMC Is Required to Initiate Accurate Membrane Protein Topogenesis

Patrick J Chitwood,Szymon Juszkiewicz,Alina Guna,Sichen Shao,Ramanujan S Hegde

doi:10.1016/j.cell.2018.10.009

Patrick J Chitwood, Szymon Juszkiewicz + Show 3 more

Open Access

https://doi.org/10.1016/j.cell.2018.10.009

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Journal: Cell	Publication Date: Nov 1, 2018
Citations: 162	License type: cc-by

Affiliation: MRC Laboratory of Molecular Biology, Harvard University

Abstract

SummaryMammals encode ∼5,000 integral membrane proteins that need to be inserted in a defined topology at the endoplasmic reticulum (ER) membrane by mechanisms that are incompletely understood. Here, we found that efficient biogenesis of β1-adrenergic receptor (β1AR) and other G protein-coupled receptors (GPCRs) requires the conserved ER membrane protein complex (EMC). Reconstitution studies of β1AR biogenesis narrowed the EMC requirement to the co-translational insertion of the first transmembrane domain (TMD). Without EMC, a proportion of TMD1 inserted in an inverted orientation or failed altogether. Purified EMC and SRP receptor were sufficient for correctly oriented TMD1 insertion, while the Sec61 translocon was necessary for insertion of the next TMD. Enforcing TMD1 topology with an N-terminal signal peptide bypassed the EMC requirement for insertion in vitro and restored efficient biogenesis of multiple GPCRs in EMC-knockout cells. Thus, EMC inserts TMDs co-translationally and cooperates with the Sec61 translocon to ensure accurate topogenesis of many membrane proteins.

Highlights

A membrane protein’s topology is determined during its initial biogenesis and is generally maintained throughout the protein’s lifetime (Shao and Hegde, 2011)
ER membrane protein complex (EMC) Is Required for Optimal b1-Adrenergic Receptor Biogenesis in Cells Among the several membrane proteins reported to be impacted by EMC disruption, we chose to analyze G protein-coupled receptors (GPCRs)
The C terminus of a well-characterized b1-adrenergic receptor (b1AR) construct (Warne et al, 2009) was appended with GFP and RFP separated by a viral P2A sequence (Figure 1A)

Summary

Introduction

A membrane protein’s topology is determined during its initial biogenesis and is generally maintained throughout the protein’s lifetime (Shao and Hegde, 2011). The topology of a single-pass membrane protein is defined by its sole first transmembrane domain (TMD). Multi-pass membrane proteins have more than one TMD, it is apparent from inspection of known membrane protein structures that their orientations are strongly interdependent on each other. Fixing the topology of one TMD generally constrains the others, simplifying the topogenesis problem. For most multi-pass membrane proteins, the first TMD is thought to be critical for setting overall topology by essentially defining the ‘‘reading frame’’ for interpretation of downstream TMDs (Blobel, 1980). An understanding of membrane protein topogenesis necessarily requires knowledge of how the first TMD is recognized, oriented, and inserted into the lipid bilayer

Results

Discussion

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