3.A.27.  The Endoplasmic Reticulum Membrane Protein Insertion Complex (EMC) Family

Guna et al. 2018 found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic C-terminal transmembrane domains. These proteins are instead shielded in the cytosol by calmodulin. Dynamic release from calmodulin allowed sampling of the endoplasmic reticulum (ER), where the conserved ER membrane protein complex (EMC) was shown to be essential for efficient insertion in vitro and in cells. Purified EMC in synthetic liposomes catalyzed the insertion of its substrates in a reconstituted system. Thus, EMC is a transmembrane domain insertase, a function that may explain its widely pleiotropic membrane-associated phenotypes across organisms (Guna et al. 2018). This family can be considered to be the second C-terminal Tail-Anchored Membrane Protein Biogenesis/Insertion Complex Family, the first having TC# 3.A.21. This system has been reviewed (Volkmar and Christianson 2020). Cell-free synthesis strategies to probe co-translational folding of proteins within lipid membranes has been reviewed (Harris et al. 2022). Several cryo-EM structures of the endoplasmic reticulum membrane complex are available (Bai and Li 2022). The mechanism(s) of TMD insertion by EMC are beginning to reveal the range of EMC's membrane protein substrates (Hegde 2022).

The ten known proteins of the EMC are EMC1 - EMC10.  These proteins have sizes between 110 and 993 aas, seven of them having sizes between 180 and 300 aas in humans (see 3.A.27.1.1).  These proteins have between ) and 3 TMSs each.  None of them are clearly homologous to any of the proteins in TCDB based on sequence similarity.  However, Guna et al. 2018 reported that EMC3 is distantly related to the yeast Get1 protein and the mammalian TRC40 protein, subunits of the insertase for the TRC pathway (Wang et al. 2014) (see TC#s 3.A.19 and 3.A.21). Get1 and EMC3 may have evolved from the ancestral prokaryotic insertase of the YidC family as claimed by Guna et al., 2018,  The EMC has been genetically implicated in several membrane-associated processes such as quality control, trafficking, protein maturation, and lipid homeostasis (Jonikas et al. 2009; Christianson et al. 2011). 

High-throughput genetic interaction analyses have revealed the EMC, and its disruption has since been found to affect wide-ranging processes, including protein trafficking, organelle communication, ER stress, viral maturation, lipid homeostasis, integral membrane insertion and folding, and others (Chitwood and Hegde 2019).  Biochemical reconstitution experiments showed that EMC can directly mediate the insertion of TMSs into the lipid bilayer. A central role for EMC as a TMD insertion factor explains its high abundance, wide conservation, and pleiotropic phenotypes. Tian et al. 2019 identified 36 EMC-dependent membrane proteins and 171 EMC- independent membrane proteins, and found that a common feature among EMC-dependent proteins is the occurance of TMSs with polar and/or charged residues; the EMC may be involved in handling TMDs with residues challenging for membrane integration.

Hiramatsu et al. 2019 investigated the EMC function using the Drosophila photoreceptor as a model system. EMC was necessary only for the biogenesis of a subset of multi-pass membrane proteins such as rhodopsin (Rh1), TRP, TRPL, Csat, Cni, SERCA, and Na+K+ATPase alpha, but not for that of secretory or single-pass membrane proteins. In EMC-deficient cells, Rh1 was translated to its C-terminus but degraded independently from ER-associated degradation. Thus, EMC exerts its effect after translation but during the membrane integration of TMDs. EMC is not required for the stable expression of the first three TMSs of Rh1 but is required for that of the fourth and fifth TMSs. EMC is required for the ER membrane insertion of succeeding TMDs of multi-pass membrane proteins (Hiramatsu et al. 2019). 

O'Donnell et al. 2020 defined the composition and architecture of human EMC. EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's TMS. The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed, intramembrane groove large enough to accommodate a single substrate TMS. A gap between the cytosolic vestibule and the intramembrane groove provides a potential path for substrate egress from EMC. Thus, EMC facilitates energy-independent membrane insertion of TMSs, explain why only short lumenal domains are translocated by EMC, and constrains models of EMC's proposed chaperone function (O'Donnell et al. 2020).

The nine- or ten-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. Pleiner et al. 2020 determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-EM, permitting building of a nearly complete atomic model. They used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. The EMC may use local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer (Pleiner et al. 2020).

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), O'Keefe et al. 2021 showed that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can access the membrane insertase activity of the EMC via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients (O'Keefe et al. 2021).

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Leznicki et al. 2022 discovered an unexpected complexity to LD membrane protein biogenesis, identifying a role for the EMC during co-translational insertion into the ER. TA proteins contain a single C-terminal transmembrane domain that must be post-translationally recognized, guided to, and ultimately inserted into the correct cellular compartment. The majority of TA proteins begin their biogenesis in the ER and utilize two parallel strategies for targeting and insertion: the guided-entry of tail-anchored proteins (GET) and ER-membrane protein complex (EMC) pathways. Guna et al. 2022 described how these two sets of machinery target, transfer, and insert TAs into the lipid bilayer in close collaboration with quality control machinery. They highlighted the unifying features of the insertion process as revealed by structures of the GET and EMC membrane protein complexes. The core of the GET insertase is conserved within structures of the ER membrane proteincomplex (EMC), which acts in parallel to insert a different subset of TA proteins. Structures of the dislo-cases, Spf1 and Msp1, show how they remove mislocalised TA proteins from the ER and outer mitochondrial membranes, respectively (Sinning and McDowell 2022).

The mechanism of signal-anchor triage during early steps of membrane protein insertion has been examined (Wu and Hegde 2023). Most membrane proteins use their first TMS, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology has been poorly understood. Using site-specific crosslinking, Wu and Hegde 2023 detected a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. Thus, EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis. 

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. However, Wu et al. 2023 found that TMDs near the carboxyl terminus of a mammalian multipass proteins are inserted post-translationally by the EMC. Site-specific crosslinking showed that the EMC's cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome-translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of pentameric ion channels that are crucial for neurotransmission (Wu et al. 2023).

The reaction belived to be catalyzed by the EMC complex is:

C-terminal tail anchored protein with a moderately hydrophobic TMS (associated with calmodulin in the cytoplasm) or type III TMP → C-terminal tail anchored protein with a moderately hydrophobic TMS (anchored to the ER membrane) or type III TMP.

 

 



This family belongs to the Guided Entry of Tail-anchored Protein (GET) Superfamily.

 

References:

Bai, L. and H. Li. (2022). Cryo-EM structures of the endoplasmic reticulum membrane complex. FEBS J. 289: 102-112.

Bai, L., Q. You, X. Feng, A. Kovach, and H. Li. (2020). Structure of the ER membrane complex, a transmembrane-domain insertase. Nature. [Epub: Ahead of Print]

Chitwood, P.J. and R.S. Hegde. (2019). The Role of EMC during Membrane Protein Biogenesis. Trends Cell Biol. 29: 371-384.

Chitwood, P.J., S. Juszkiewicz, A. Guna, S. Shao, and R.S. Hegde. (2018). EMC Is Required to Initiate Accurate Membrane Protein Topogenesis. Cell 175: 1507-1519.e16.

Christianson, J.C., J.A. Olzmann, T.A. Shaler, M.E. Sowa, E.J. Bennett, C.M. Richter, R.E. Tyler, E.J. Greenblatt, J.W. Harper, and R.R. Kopito. (2011). Defining human ERAD networks through an integrative mapping strategy. Nat. Cell Biol. 14: 93-105.

Coukos, R., D. Yao, M.I. Sanchez, E.T. Strand, M.E. Olive, N.D. Udeshi, J.S. Weissman, S.A. Carr, M.C. Bassik, and A.Y. Ting. (2021). An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens. Elife 10:.

Guna, A., M. Hazu, G. Pinton Tomaleri, and R.M. Voorhees. (2022). A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum. Cold Spring Harb Perspect Biol. [Epub: Ahead of Print]

Guna, A., N. Volkmar, J.C. Christianson, and R.S. Hegde. (2018). The ER membrane protein complex is a transmembrane domain insertase. Science 359: 470-473.

Harris, N.J., E. Reading, and P.J. Booth. (2022). Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes. Methods Mol Biol 2433: 273-292.

Hegde, R.S. (2022). The Function, Structure, and Origins of the ER Membrane Protein Complex. Annu. Rev. Biochem. 91: 651-678.

Hiramatsu, N., T. Tago, T. Satoh, and A.K. Satoh. (2019). ER membrane protein complex is required for the insertions of late-synthesized transmembrane helices of Rh1 in photoreceptors. Mol. Biol. Cell mbcE19080434. [Epub: Ahead of Print]

Jonikas, M.C., S.R. Collins, V. Denic, E. Oh, E.M. Quan, V. Schmid, J. Weibezahn, B. Schwappach, P. Walter, J.S. Weissman, and M. Schuldiner. (2009). Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323: 1693-1697.

Leznicki, P., H.O. Schneider, J.V. Harvey, W.Q. Shi, and S. High. (2022). Co-translational biogenesis of lipid droplet integral membrane proteins. J Cell Sci 135:.

Li, Y.P., R.J. Shen, Y.M. Cheng, Q. Zhao, K. Jin, Z.B. Jin, and S. Zhang. (2023). Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3. Heliyon 9: e20146.

Marquez, J., J. Criscione, R.M. Charney, M.S. Prasad, W.Y. Hwang, E.K. Mis, M.I. García-Castro, and M.K. Khokha. (2020). Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects. J Clin Invest. [Epub: Ahead of Print]

O''Donnell, J.P., B.P. Phillips, Y. Yagita, S. Juszkiewicz, A. Wagner, D. Malinverni, R.J. Keenan, E.A. Miller, and R.S. Hegde. (2020). The architecture of EMC reveals a path for membrane protein insertion. Elife 9:.

O''Keefe, S., G. Zong, K.B. Duah, L.E. Andrews, W.Q. Shi, and S. High. (2021). An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum. Commun Biol 4: 828.

Pleiner, T., G.P. Tomaleri, K. Januszyk, A.J. Inglis, M. Hazu, and R.M. Voorhees. (2020). Structural basis for membrane insertion by the human ER membrane protein complex. Science 369: 433-436.

Sinning, I. and M.A. McDowell. (2022). Cryo-EM insights into tail-anchored membrane protein biogenesis in eukaryotes. Curr. Opin. Struct. Biol. 75: 102428.

Tian, S., Q. Wu, B. Zhou, M.Y. Choi, B. Ding, W. Yang, and M. Dong. (2019). Proteomic Analysis Identifies Membrane Proteins Dependent on the ER Membrane Protein Complex. Cell Rep 28: 2517-2526.e5.

Volkmar, N. and J.C. Christianson. (2020). Squaring the EMC - how promoting membrane protein biogenesis impacts cellular functions and organismal homeostasis. J Cell Sci 133:.

Wang, F., C. Chan, N.R. Weir, and V. Denic. (2014). The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase. Nature 512: 441-444.

Wu, H. and R.S. Hegde. (2023). Mechanism of signal-anchor triage during early steps of membrane protein insertion. Mol. Cell. [Epub: Ahead of Print]

Wu, H., L. Smalinskaitė, and R.S. Hegde. (2023). EMC rectifies the topology of multipass membrane proteins. Nat Struct Mol Biol. [Epub: Ahead of Print]

Examples:

TC#NameOrganismal TypeExample
3.A.27.1.1

ER membrane protein insertase complex of ten recognized proteins, EMC1 - 10 (Guna et al. 2018).  Inserts C-terminal moderately hydrophobic TMSs of tail anchored (TA) proteins into the endoplasmic reticular membrane (Guna et al. 2018). EMC also inserts the first N-terminal TMS of G-proteins and some receptors co-translationally and cooperates with the Sec61 translocon to ensure accurate topogenesis of many membrane proteins (Chitwood et al. 2018). Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects (Marquez et al. 2020). The high resolution 3-d structure of this EMC complex has been determined (O'Donnell et al. 2020). EMC10 plays a regulatory role in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex (Coukos et al. 2021). Inherited retinal dystrophies (IRDs) are a heterogeneous group of visual disorders caused by different pathogenic mutations in genes and regulatory sequences. The endoplasmic reticulum (ER) membrane protein complex (EMC) subunit 3 (EMC3) is the core unit of the EMC insertase that integrates transmembrane peptides into lipid bilayers (Li et al. 2023).  The C-terminus of EMC3 is essential for EMC functions, and EMC3 may be the gene for retinal degenerative diseases (Li et al. 2023).

EMC1 - 10 of Homo sapiens
EMC1, Q8N766, 993 aas and 1 C-terminal TMS
EMC2, Q15006, 297 aas and 0 TMSs
EMC3, Q9P0I2, 261 aas and 3 TMSs; may be a member of the YidC family (see EMC family description)
EMC4, Q5J8M3, 183 aas and 2 TMSs
EMC5 (MMGT1; TMEM32), Q8N4V1, 131 aas and 2 TMSs.  This protein is a member of the MMgT family (TC# 1.A.67)
EMC6 (TMEM93), Q9BV81, 110 aas and 2 TMSs
EMC7, Q9NPA0, 242 aas and 2 TMSs, one N-terminal and one near the C-terminus
EMC8, O43402, 210 aas and 0 - 2 TMSs; homologous to EMC9
EMC9, Q9Y3B6, 208 aas and 0 - 2 TMSs; homologous to EMC8
EMC10, Q5UCC4, 262 aas and 2 TMSs, one N-terminal and one C-terminal

 
3.A.27.1.2

ER membrane protein insertase complex of eight recognized proteins, EMC1 - 7 + EMC10 (Bai et al. 2020). These authors have determined the high resolution structure of the complex.  It co-translationally inserts TMSs of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMSs of some tail-anchored proteins. Bai et al. 2020 reported theCryoEM structure. The Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. They identified a five-TMS fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. The flexibility of Emc4 and the hydrophilicity of the pocket are required for EMC function. The structure reveals notable evolutionary conservation with prokaryotic insertases, suggesting that eukaryotic TMS insertion involves a similar mechanism (Bai et al. 2020).

EMC of Saccharomyces cerevisiae
EMC1, 760 aas and 2 TMSs, N- and C-terminal (P25574)
EMC2, 292 aas and 0 TMSs (P47133)
EMC3, 253 aas and 3 TMSs in a 1 + 2 TMS arrangement. (P36039). It resembles CLAC Ca2+ channels (see TC# 1.A.106.2.1).
EMC4, 190 aas and 2 TMSs in the C-terminal half of the protein. (P53073) It resembles MMgT Mg2+ channels and is identical to the channel protein with TC# 1.A.67.1.7.
EMC5, 141 aas and 2 TMSs in the N-terminal half of the protein. (P40540)
MEC6, 108 aas and 3 TMSs in a 2 + 1 TMS arrangement (Q12431)
MEC7 or Sop4 of 234 aas and 2 TMSs, N- and C-terminal (NP_012343.1)
MEC10, 205 aas and 1 N-terminal TMS (Q12025)