Introduction
About 20-30% of the genes that make up the human genome encode for integral membrane proteins (1). In line with their remarkable abundance, membrane proteins are essential for cellular function with key roles in a myriad of biological processes that include small molecule transport, signal transduction, intracellular trafficking and organelle biosynthesis. Functionally, 40-50% of the membrane proteins in Saccharomyces cerevisiae are involved in small molecule transport, while, in humans, about 15% of the membrane proteins are G-protein-coupled receptors (GPCRs) that mediate signal transduction (1). In addition to their crucial biological role, dysfunctional membrane proteins are etiologic agents of many human diseases such as neurological and cardiac disorders, cystic fibrosis as well as cancer (2). It is therefore not surprising that human membrane proteins are of major pharmaceutical importance. For example, more than half of the known targets for small-molecule drugs are membrane proteins with GPCRs accounting for 30%, ion channels 7%, transporters 4% and other receptors and cell-surface proteins for 5% (3). Despite the biophysical complexity of cellular membranes, the structure of membrane proteins is relatively simple containing most often segments of 20-25 hydrophobic residues. These adopt an α-helical conformation and span the lipid bilayer, thereby effectively anchoring the protein into the membrane. The lipid-exposed part of helical transmembrane domains (TMDs) mainly comprises leucine, isoleucine, alanine, valine and phenyl alanine, which mediate the contact with membrane lipids. In addition to these apolar residues, TMDs also contain functionally important polar and charged residues that are located at the inside of the helix, while aromatic residues, tryptophan and tyrosine, are prominent at the ends of TMDs that are in contact with the lipid headgroups (1). Helical membrane proteins are prominent in all cellular membranes and display a large variety in size and shape as shown in figure 1 (adapted from 4).
Some membrane proteins have a single TMD (bitopic) with the termini on opposite sides of the membrane, whereas other have multiple TMDs (polytopic). Based on topology – the number and way TMDs are oriented into the membrane – membrane proteins are categorized into four distinct topological classes (type I-IV). Type I, II and III represent bitopic membrane proteins with the N-terminus exposed to either the ER lumen (type I and III) or cytosol (type II), while the C-terminus is localized to the cytosol (type I and III) or ER lumen (type II). Type I proteins are, moreover, initially synthesized with a signal sequence that is proteolytically removed after membrane translocation. Type II and III proteins don’t contain a cleavable signal sequence (4). Polytopic membrane proteins (type IV) can contain up to 20 TMDs often forming a tightly packed bundle in the membrane. For most polytopic membrane proteins, the first TMD probably defines the overall topology by establishing the orientation of the subsequent TMDs. Nearly all human membrane proteins are synthesized and assembled at the endoplasmic reticulum (ER) (5). They remain, moreover, embedded in the membrane as they are shipped towards their final destinations along the secretory pathway. The initial membrane protein topology is maintained as membrane proteins are transported intracellularly, indicating that the final topology is established during their biosynthesis at the ER (4). The endoplasmic membrane houses different molecular factors that facilitate membrane protein insertion and thereby determine its topology (5). Of these, the Sec61 translocon represents the best characterized one and its role in the insertion of most membrane proteins into the ER membrane is well established. In addition, the ER membrane protein complex (EMC) represents a newly identified membrane protein insertase that directly mediates the insertion of TMDs into the lipid bilayer (6). The detailed structure of the human EMC was presented recently, providing mechanistic insight of membrane protein insertion at the ER (7). Here, I will discuss this structure as well as its current functional understanding.
How are membrane proteins made?
Within their primary structure, membrane proteins contain several important topological determinants. These are: hydrophobic TMDs that anchor the protein into the lipid bilayer as well as a strong presence of positively charged residues (lysine and arginine) in the loops between the TMDs that face the cytoplasm, while the corresponding loops at the extracytoplasmic side of the membrane are reduced in these residues. This asymmetric distribution of positively charged residues is known as the positive-inside rule and holds true for nearly all α-helical membrane proteins from all organisms and membrane systems (1). It is believed that the electrostatic interaction of these positively charged residues with the Sec61 translocon prevents their translocation. The Sec61 translocon in the ER membrane is the principal site where membrane proteins are inserted into the lipid bilayer, final topology is established and folded into their biological active conformation. The Sec61 complex is a heterotrimer made of three membrane-embedded subunits: Sec61α, Sec61β and Sec61γ. It is now well established that the α subunit forms the protein-conducting channel and enables TMDs to exit laterally into the lipid bilayer (8). Similar to soluble proteins, membrane proteins are synthesized by cytosolic ribosomes but owing to their hydrophobic nature newly synthesized membrane proteins would readily aggregate in the aqueous cytoplasm, thereby forming toxic aggregates. To avoid this, the biosynthesis of membrane proteins occurs in two distinct steps: recognition/targeting to the ER localized translocon and insertion/translocation into the ER membrane (8). These steps are assisted by various molecular factors that shield the nascent membrane protein from the aqueous cytoplasm to prevent its aggregation. The vast majority of eukaryotic membrane proteins are inserted cotranslationally into the ER membrane via the ubiquitous SRP/Sec61 pathway (8). Moreover, this pathway is also used for the transport of secretory proteins. In bacteria, however, all membrane proteins are inserted cotranslationally as well as a subset of secretory proteins.
A schematic representation of eukaryotic cotranslational membrane protein insertion is shown in figure 2 (adapted from 8). The targeting step is initiated when a hydrophobic TMD that emerges from the ribosomal exit tunnel is recognized by the signal recognition particle (SRP) on the ribosome. The eukaryotic SRP is made up of a 300-nucleotide 7S RNA backbone and six associated protein subunits. One of these (SRP54), possesses a deep groove that is used for binding of hydrophobic segments and displays GTPase activity. SRP54 is located near the ribosomal exit tunnel and is therefore ideally positioned to capture a TMD of a nascent membrane protein as it emerges from the ribosome (8). The substrate-binding groove of SRP54 is able to accommodate a variety of hydrophobic segments, explaining its promiscuous nature that ensures successful targeting of many membrane proteins. Following the binding of SRP to the first TMD, the translation of the nascent membrane protein is transiently slowed down. This increases the time window for membrane targeting and thereby prevents that TMDs are exposed to the cytosol before associating with the Sec61 translocon. The SRP-ribosome-nascent chain complex (RNC) is guided to the SRP receptor (SR) located at Sec61 translocon in the ER membrane. Eukaryotic SR is a heterodimer composed of α and β subunits that display both GTPase activity. SRP54 binds to SRα and it is believed that following their interaction conformational rearrangements within the ribosome expose a part of the exit tunnel to Sec61. Subsequently, Sec61 binds to this exposed site and the nascent chain is transferred from SRP to Sec61. This, in turn, triggers disassembly of the SRP-SR complex and relieves translational arrest. These final events are coordinated by the GTPase activity of SRP54, SRα and SRβ but are not fully understood. Nevertheless, the net result of these final events is that the ribosomal exit tunnel is now directly above the Sec61 translocon, which opens up to allow the growing nascent chain to move across the membrane via its central channel. If a TMD emerges, the Sec61 translocon opens a lateral gate in its channel wall with access to the lipid bilayer through which the TMD integrates sideways from the channel into the membrane (8). Although the Sec61 translocon is the primary site for protein translocation and membrane insertion, the ER contains additional insertion factors that operate in parallel and are jointly responsible for integrating the entire pool of structurally diverse membrane proteins. The ER membrane complex represents one of these additional insertion factors and is discussed below in more detail.
Biological features of human EMC
Biochemical properties of human EMC
EMC is a widely conserved hetero oligomeric complex embedded in the endoplasmic membrane that is in humans made up of nine subunits (EMC1-10) and six in yeast. The predicted topology of each subunit is shown in the left panel of figure 3 (adapted from 6), revealing that EMC2 and EMC8/9 are cytoplasmic proteins that are associated with the other seven subunits that are firmly anchored into the ER membrane through their 12 joint TMDs. EMC1 is the largest subunit with weight of 112 kDa, while EMC6 is, with a weight of 12 kDa, the smallest subunit. Only EMC1, EMC7 and EMC10 have sizeable domains exposed to the ER lumen (6). The six genes of the yeast EMC were first described in 2009 employing a high-throughput genetic screen to identify genes required for protein folding in the ER (9). Subsequently, the yeast EMC subunits were also copurified as a complex (6). The function of the EMC remained enigmatic for a long time mainly because sequence analysis of the EMC subunits did not reveal a clear catalytic or other biochemical activity (6). However, data obtained of EMC mutant cells from different eukaryotes points towards a role in biogenesis, quality control and trafficking of many membrane proteins. Specifically, disruption of EMC subunits seems to predominantly affect polytopic membrane proteins (15). A clear involvement of EMC in membrane protein biogenesis was first shown for tail anchor (TA) proteins (10). These are membrane proteins with a single TMD that is about 50-70 residues from the C-terminus and a large soluble N-terminal domain (right panel of figure 3 adapted from 6). These proteins are typically inserted posttranslationally into the ER membrane without involvement of the Sec61 translocon. In mammals, for example, TA protein insertion requires the TMD recognition complex (TRC), although this pathway does not mediate insertion of all TA proteins into the ER membrane (11).
Recently, it was shown that EMC functions as an insertase for a subset of TA proteins with moderately hydrophobic TMDs, while other higher hydrophobicity TAs require TRC for membrane insertion (10,11). Specifically, in vitro reconstitution of EMC-catalyzed membrane insertion showed that purified EMC is sufficient for insertion of a TRC-independent TA into liposomes. A role for EMC as an independent membrane protein insertase is reminiscent of the insertase activity of YidC and its homologs Oxa1 and Alb3. These proteins facilitate membrane protein insertion in the bacterial plasma membrane, mitochondrial inner membrane and thylakoid membrane of chloroplasts, respectively (12). The ubiquitous presence of YidC homologs in bacteria and archaea raises the question whether the endoplasmic membrane also contains such a homolog. It appeared, however, for a long time that the ER membrane lacked any protein related to the Oxa1/Alb3/YidC family, although a recent bioinformatics study identified multiple proteins embedded in the ER membrane as YidC homologs including EMC3 (13). In addition to facilitating the insertion of TA proteins, EMC is required for efficient in vivo biogenesis of many polytopic membrane proteins, including GPCRs (14). To study this reaction in more detail, EMC-mediated insertion of a model GPCR was reconstituted in vitro by employing a cell free translation system in combination with ER-derived microsomes from wild-type cells or cells lacking EMC6. This showed that the model substrate failed to attain its correct topology in the absence of EMC. More specifically, purified EMC is sufficient for insertion of the first TMD into proteoliposomes (14). Furthermore, the same experimental system was used to define the role of the Sec61 translocon. To this end, proteoliposomes with or without the Sec61 complex were used, revealing that the Sec61 translocon is required for the integration of downstream TMDs. To identify targeting factors involved in the biogenesis of the model substrate, RNCs programmed with radiolabeled stalled translation intermediates of the model GPCR were analyzed by immunoblotting, revealing an interaction with SRP54. Thus, EMC inserts N-terminal TMDs in a cotranslational fashion, whereas subsequent TMDs are integrated into the lipid bilayer Sec-dependently (14). These findings raise intriguing questions about the potential interaction of EMC and the Sec61 complex and this possibly reflects the established cooperation of YidC with the SecYEG machinery during membrane protein insertion in bacteria. With regards to this it is also interesting to note that the amount of EMC in the ER membrane is roughly comparable to the of the Sec61 translocon, while, in bacteria, YidC is in excess over the SecYEG complex (12).
Structural properties of human EMC
Clearly, detailed structural information is needed to provide an answer on how EMC facilitates insertion of membrane proteins into the ER membrane. A structure of human EMC at an overall resolution of 3.4 Å was presented recently (7). To this end, human EMC was affinity purified from a cell line stably expressing a tagged EMC variant after which the purified complex was reconstituted into lipid nanodiscs and subjected to cryo-EM. The structure of human EMC is shown in figure 4 in surface (left and center panel) and ribbon representation (right panel). This reveals that is made up of nine subunits that are organized in three general regions, namely: (i) the membrane spanning region that is composed of 12 TMDs of which nine form the central core, (ii) a basket-shaped cytosolic region that is located between EMC2, 8, 3 and (iii) an L-shaped lumenal region made up of EMC1, 4,7 and 10. The structure, moreover, also shows that EMC6 contains three TMDs of which the first is poorly hydrophobic that is only inserted following assembly with EMC5. In addition, the structure also reveals that EMC4, 7 and 10 collectively possess three TMDs. EMC3 and 6 with three TMDs each comprise the center of the membrane spanning region, while the TMDs of EMC5 and 3 are located at the side and serve as attachment site for the cytosolic and lumenal regions (7).
Furthermore, one helix of EMC1 and one of EMC3 are located on the lumenal side of the ER membrane and may play an important role in substrate insertion. EMC2 is localized to the cytosol and probably serves as structural scaffold for EMC8 and the cytosolic regions of EMC3, 5 and 1. This scaffolding role of EMC2 is in agreement with its importance for stability of the entire complex. The lumenal region is formed by the N-termini of EMC1, 7, 10 and the tail of EMC4. EMC1 is made up of two 8-bladed β-propellers (right panel). This subunit moreover stabilizes the entire complex through contacting most subunits. EMC 7 and 10 are folded into a β-sandwich that are anchored to EMC1 (7).
The structure of the membrane-embedded core region is presented in figure 5 in surface and ribbon representation, respectively, with EMC3 in green, EMC6 in grey and EMC5 in orange. The transmembrane core contains two intramembrane sites that could be involved in the membrane insertion of substrate proteins. One of these represents a lipid-exposed cavity (indicated by an asterisks) that is made up of EMC3 and 6, which is also partially surrounded by the TMDs of EMC4, 7 and 10. This cavity contains a cluster of positively charged residues surrounded by hydrophobic ones (shown as red sticks in right panel of figure 5) and is at the top covered by the lumenal helix of EMC3. YidC, the bacterial homolog of EMC3 relies on a stretch of positively charged residues for membrane insertion (12). It is therefore conceivable that this cavity of EMC3 confers insertase activity. Replacing these positively charged residues in the cavity of EMC3 with alanine strongly impaired co and posttranslational membrane insertion, which confirms that the hydrophilic cavity of EMC3 is required for co and posttranslational insertion of substrate proteins (7). Additionally, members of the Oxa1 superfamily contain methionine-rich cytosolic loops that are believed to capture substrate proteins analogously to SRP54. The primary sequence of human EMC3 is shown in the bottom panel of figure 5 with TMDs in green. In EMC3, these methionine residues (in yellow) are located next to TMD1 and 2 and reside within the cytosol near the insertase cavity. Mutagenesis of these residues strongly affected co and posttranslational insertion (7). Based on these results it is likely that these residues transiently interact with substrate proteins, thereby directing them into the insertase cavity.
Membrane protein insertion by EMC
Based on the data presented above, the following model for EMC-mediated membrane insertion was proposed (figure 6 adapted from 7). First, substrate proteins are either post or cotranslationally targeted to ER membrane. Subsequently, they are released from either chaperones or the ribosome and captured and directed towards the insertase cavity by the methionine-rich cytosolic loops of EMC3 and 6. At this stage, however, substrate proteins are unable to access the cavity because it is axially sealed by the lumenal helix of EMC3 and laterally by the flexible TMDs of EMC4 and 7. To enable insertion, the EMC reduces the energy barrier for membrane insertion via two ways, namely: (i) inducing a local thinning of the lipid bilayer reduces the distance a lumenal domain must be translocated, and (ii) the polar and positively charged residues of the EMC within the membrane enforce the positive inside rule and thereby stabilizing hydrophilic residues present in the TMDs of substrate proteins. Within the membrane, the hydrophobic core of the inserted substrate could interact with the hydrophobic surface of EMC3. The short TMDs of EMC3 and 6 are unable to stably bind substrate proteins, thereby promoting their release into the membrane and subsequent dissociation from EMC. The β-propellers of EMC1 might serve as docking site for lumenal chaperones that assist the folding of newly inserted substrates (15). Thus, members of the Oxa1 superfamily of insertases probably all employ a similar mechanism for membrane protein insertion.
Summarizing conclusion
About 20-30% of the proteins that make up the human proteome are integral membrane proteins. Owing to their crucial biological roles, that range from small molecule transport to signal transduction, membrane proteins are essential for proper cell functioning. It is therefore not surprising that their dysfunction is closely associated with numerous progressive human diseases such as neurological disorders and cancer (1,2). Membrane proteins come in all shapes and sizes but despite their large structural variation, their basic architecture is relatively simple. Most membrane proteins typically contain segments of 20-25 hydrophobic residues. These adopt an α-helical conformation and span the lipid bilayer, thereby effectively anchoring the protein into the membrane (1). Most human membrane proteins are synthesized and assembled at the endoplasmic reticulum (5). They remain embedded in the membrane as they are shipped towards their final destinations along the secretory pathway. Membrane proteins are synthesized by cytosolic ribosomes but owing to their hydrophobic nature newly synthesized membrane proteins would readily aggregate in the aqueous cytoplasm, thereby forming toxic aggregates. To avoid this, their biosynthesis typically occurs cotranslationally with first a targeting step in which the RNC is delivered at the ER membrane by the SRP. Subsequently, the nascent chain is inserted into the membrane which is usually facilitated by the Sec61 translocon (8). In addition to the Sec61 translocon, the ER membrane contains several other insertion factors that mediate insertion of a subset of membrane proteins such as EMC. This is a conserved oligomeric complex that is in humans made up of nine subunits (EMC1-10). Several lines of evidence point towards a role of EMC in the biogenesis many membrane proteins (15). Firstly, it was shown that EMC is sufficient for insertion of TA proteins in vitro (11) and secondly it was also reported that under similar experimental conditions EMC mediates insertion of a model GPCR into ER-derived microsomes (14). Interestingly, one EMC subunit (EMC3) is a member of the Oxa1 superfamily of which the members facilitate membrane protein insertion in the bacterial plasma membrane (YidC), mitochondrial inner membrane (Oxa1) and thylakoid membrane of chloroplasts (Alb3), respectively (12,13). The recently determined structure of human EMC unveils the molecular details of how EMC3 processes substrate proteins (7), which is probably similar for all members of the Oxa1 superfamily. In fact, these proteins are probably derived of an ancient insertase module that was only able to insert relatively small and simple membrane proteins. During evolution, however, this module was decorated with additional subunits and acquired more specialized functions such as chaperone activity (6). This shows how an ancient structural scaffold diversified over time to facilitate the insertion, folding and assembly of membrane proteins into different cellular membranes throughout all kingdoms of life.
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