The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins

Shurtleff, Matthew J.; Itzhak, Daniel N.; Hussmann, Jeffrey A.; Oakdale, Nicole T. Schirle; Boydston, Elizabeth A.; Jonikas, Martin C.; Weibezahn, Jimena; Popova, Katerina D.; Jan, Calvin H.; Sinitcyn, Pavel; Vembar, Shruthi S.; Hernández, Hilda; Cox, Jürgen; Burlingame, Alma L.; Brodsky, Jeffrey L.; Frost, Adam; Borner, Georg H. H.; Weissman, Jonathan S.

doi:10.7554/elife.37018

Cited by 178 publications

(282 citation statements)

References 54 publications

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“…In the uninfected condition, we did not observe dramatic changes in TE for cellular mRNAs in EMC4 KO vs WT cells (Figure 3A and Figure 3 - Source Data 1). This is in line with previously reported results using CRISPRi knockdown against EMC subunits suggesting that deletion of EMC minimally impacts the synthesis rates of its client proteins (Shurtleff et al, 2018). Similarly, we did not find any substantial differences in viral TE in EMC4 KO cells relative to WT cells at 18 (0.43x) and 44hpi (2.17x) (Figures 3B and 3C and Figure 3 - Source Data 1).…”

Section: Resultssupporting

confidence: 93%

The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

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Shurtleff²,

Popova³

et al. 2019

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11Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic 12 reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is 13 indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential 14 for accurate folding and post-translational stability rather than translation efficiency. Specifically, 15we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of 16 EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-17 synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we 18 showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts 19 an aberrant topology in the absence of the EMC leading to degradation. Together, our data 20 highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve 21 optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing 22 challenging transmembrane proteins during synthesis.Subsequently, the polyprotein is cleaved by the viral NS2B-NS3 protease and the cellular signal 27 peptidase into the individual structural (C, prM, E) and non-structural (NS1-5) proteins required 28 for replication and assembly into new virions (Neufeldt et al., 2018). Similar to cellular 29 transmembrane proteins, the viral polyprotein relies on the host cell machinery for targeting to the 30 ER, translocation across the membrane and insertion of transmembrane domains (TMDs) into 31 the lipid bilayer during its translation. These processes are facilitated by the signal recognition 32 particle (SRP), the translocon-associated protein (TRAP) complex (also known as signal-33 sequence receptor complex), the Sec61 translocon and the signal peptidase complex. These cell 34 components were all identified as essential host factors for flavivirus infection in genetic screens 35 underscoring their importance for virus replication (Krishnan et al., 2008; Marceau et al., 2016; 36 Sessions et al., 2009; Zhang et al., 2016). For most transmembrane proteins, recognition of a 37 hydrophobic TMD or signal sequence by the SRP followed by transfer to the Sec61 translocation 38 channel are sufficient for membrane targeting and accurate topogenesis (Shao and Hegde, 2011). 39Recently, it was shown that the ER membrane protein complex (EMC) plays a role in the insertion 40 and/or stabilization of certain transmembrane proteins (Chitwood et al., 2018; Guna et al., 2018; 41 Shurtleff et al., 2018). Intriguingly, several genome-wide CRISPR knockout (KO) screens for 42 flavivirus dependency factors showed strong enrichment of the EMC (Ma et al., 2015; Marceau 43 et al., 2016; Savidis et al., 2016; Zhang et al., 2016). However, its role in the virus life cycle is not 44 yet understood. 45The EMC was originally discovered in a genetic screen for yeast mutants modulating the unfolded 46 protein response (UPR) (Jonikas et al., 2009). Genetic interac...

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Section: Resultssupporting

confidence: 93%

The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

Ngo

Shurtleff²,

Popova³

et al. 2019

Preprint

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“…Although the intramembrane groove is well suited to serve as a chaperone for a single TMD, such a function seems unlikely to occur at the Sec61 translocon as proposed on the basis of ribosome profiling experiments (Shurtleff et al, 2018). Co-translational engagement of EMC by TMDs emerging from the ribosome-Sec61 complex could only occur once a TMD can diffuse at least 100 Å away.…”

Section: Discussionmentioning

confidence: 99%

“…The "guided entry of TA proteins" (GET) pathway (Chio et al, 2017; and the relationship of this function to its insertase activity, is not known. Consistent with either function, many membrane proteins are partially or strongly impacted in their biogenesis by the loss of EMC in numerous organisms including yeast, worms, flies, and mammals (Bircham et al, 2011;Chitwood et al, 2018;Lakshminarayan et al, 2020;Louie et al, 2012;Richard et al, 2013;Satoh et al, 2015;Shurtleff et al, 2018;Talbot et al, 2019;Taylor et al, 2005;Volkmar et al, 2019). Thus, EMC is a highly abundant component of the ER needed for cellular and organism homeostasis.…”

Section: Introductionmentioning

confidence: 87%

The architecture of EMC reveals a path for membrane protein insertion

O’Donnell

Phillips

Yagita

et al. 2020

Preprint

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Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results show that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that binds a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.

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“…Cue1 is an essential component of the ERAD pathway [27]. Emc4 is a member of the conserved ER transmembrane complex (EMC) and is required for efficient folding of proteins in the ER [21, 28]. The EMC is also proposed to facilitate the transfer of phosphatidylserine from the ER to mitochondria [29].…”

Section: Resultsmentioning

confidence: 99%

A subset of UPR-induced transmembrane proteins are prematurely degraded during lipid perturbation

Shyu

et al. 2017

Preprint

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BackgroundPhospholipid homeostasis in biological membranes is essential to maintain functions of organelles such as the endoplasmic reticulum. Phospholipid perturbation has been associated to non-alcoholic fatty liver disease, obesity and other metabolic disorders. However, in most cases, the biological significance of lipid disequilibrium remains unclear. Previously, we reported that Saccharomyces cerevisiae adapts to lipid disequilibrium by upregulating several protein quality control pathways such as the endoplasmic reticulum-associated degradation (ERAD) pathway and the unfolded protein response (UPR).ResultsSurprisingly, we observed certain ER-resident transmembrane proteins (TPs), which form part of the UPR programme, to be destabilised under lipid perturbation (LP). Among these, Sbh1 was prematurely degraded by fatty acid remodelling and membrane stiffening of the ER. Moreover, the protein translocon subunit Sbh1 is targeted for degradation through its transmembrane domain in an unconventional Doa10-dependent manner.ConclusionPremature removal of key ER-resident TPs might be an underlying cause of chronic ER stress in metabolic disorders.

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The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins

Cited by 178 publications

References 54 publications

The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

The architecture of EMC reveals a path for membrane protein insertion

A subset of UPR-induced transmembrane proteins are prematurely degraded during lipid perturbation

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