The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?

The EMC is an ancient and highly conserved protein complex

Using homology-searching algorithms EMC candidate proteins were identified in the vast majority of sequenced genomes representing the complete diversity of eukaryotes (Figure 1). Emc8 and Emc9 are homologues but only a single homologue could be detected in most genomes. A subset of opisthokont Emc8/9 sequences were subjected to a phylogenetic analysis demonstrating that vertebrate Emc8 and Emc9 are the result of a vertebrate-specific duplication of Emc8 (Figure S1; see legend for methods). Based on this knowledge, I suggest that the vertebrate Emc8 and Emc9 be renamed Emc8a and Emc8b, respectively.

Figure 1. Coulson plot showing distribution of EMC components across eukaryotes.

Coloured pies indicate presence of a particular subunit. Plot was generated using the Coulson plot generator (Field et al., 2013). Asterisks indicate presence of orthologue in a different member of the genus but absent in the indicated species (see Table S1). Abbreviations: Vertebrates: Hsap, Homo sapiens; Mdom, Monodelphis domesticus; Drer, Danio rerio; Xtro, Xenopus tropicalis; Ggal, Gallus gallus; Mmus, Mus musculus; Invertebrates: Cele, Caenorhabditis elegans; Dmel, Drosophila melanogaster; Bflo, Branchiostoma floridae; Nvec, Nematostella vectensis; Tadh, Trichoplax adhaerens; Unicellular Holozoa: Mbre, Monosiga brevicollis; Cowc, Capsaspora owczarzaki; Sarc, Sphaeroforma arctica; Sros, Salpingoeca rosetta; Fungi: Spom, Schizosaccharomyces pombe; Scer, Saccharomyces cerevisiae; Ncra, Neurospora crassa; Cneo, Cryptococcus neoformans; Umay, Ustilago maydis; Bden, Batrachochytrium dendrobatidis; Ncer, Nosema ceranae; Ecun, Encephalitozoon cuniculi; Pir, Piromyces sp.; Spun, Spizellomyces punctatus; Rirr, Rhizophagus irregularis; Crev, Coemansia reversa; Ccor, Conidiobolus coronatus; Cang, Catenaria anguillulae; Rall, Rozella allomyces; Apusozoa: Ttra, Thecamonas trahens; Fonticulids: Fonticula alba; Amoebozoa: Acas, Acanthamoeba castellanii; Ddis, Dictyostelium discoideum; Ehis, Entamoeba histolytica; Ppal, Polysphondylium pallidum; Excavata: Ngru, Naegleria gruberi; Gint, Giardia intesinalis; Tvag, Trichomonas vaginalis; Bsal, Bodo saltans; Lmaj, Leishmania major; Tbru, Trypanosoma brucei; Stramenopiles: Bhom, Blastocystis hominis; Alim, Aurantiochytrium limacinum; Aana, Aureococcus anophagefferens; Tpse, Thalassiosira pseudonana; Ptri, Phaeodactylum tricornutum; Psoj, Phytophthora sojae; Esil, Ectocarpus siliculosus; Ngad, Nannochloropsis gaditana; Alveolates: Ptet, Paramecium tetraurelia; Tthe, Tetrahymena thermophila; Otri, Oxytricha trifallax; Tpar, Theileria parva; Smin, Symbiodinium minutum; Tgon, Toxoplasma gondii; Cpar, Cryptosporidium parvum; Pfal, Plasmodium falciparum; Rhizaria: Bnat, Bigelowiella natans; Rfil, Reticulomyxa filosa; Archaeplastida: Crei, Chlamydomonas reinhardtii; Cmer, Cyanidioschyzon merolae; Cyp, Cyanophora paradoxa; Atha, Arabidopsis thaliana; Ppat, Physcomitrella patens; Otau, Ostreococcus tauri; Gsul, Galdieria sulphuraria; Mpus, Micromonas pusilla; Ccri, Chondrus crispus; CCTH: Ehux, Emiliania huxleyi; Gthe, Guillardia theta.

A complete EMC (Emc1-8, 10) was found in at least one representative from each major lineage including animals, fungi, excavates, amoebozoa, green algae, plants, stramenopiles, alveolates, rhizaria, and haptophytes (Figure 1). The relative sequence conservation of EMC components across diverse taxa suggests that the EMC has an ancient and critical role in cellular function.

Yeast Sop4 and YDR056C are Emc7 and Emc10, respectively

Although previous reports suggest S. cerevisiae EMC comprises only six subunits, I identified Sop4 and YDR056C as orthologues of Emc7 and Emc10, respectively. Supporting this, Jonikas et al. (2009), the original discoverers of the EMC, show by co-immunoprecipitation analyses that Sop4 and YDR056C are interacting partners of FLAG-tagged Emc3. This experiment not only confirms my bioinformatic classification but also puts into perspective a previous study on Sop4’s role in membrane protein quality control (Luo et al., 2002). Furthermore, tracing the evolutionary history of the EMC in fungi demonstrates that Emc8 was lost only in Ascomycetes and a few basally diverging fungi whereas most fungi retain Emc8 (as well as Emc7 and 10).

The EMC has been independently lost in several lineages

Although the EMC was identified in representative taxa from every major eukaryote supergroup, I was unable to identify even a single EMC member in the genomes of the microsporidians Nosema ceranae and Encephalitozoon cuniculi, the metamonad Giardia intestinalis, the stramenopile Blastocystis hominis, the alveolate Theileria parva, and the red alga Cyanidioschyzon merolae (Figure 1 and Figure 2). Trichomonas vaginalis, another metamonad retains only a rather divergent Emc2, that passed the test for orthology, but only weakly, suggesting that this protein is under relaxed selection, perhaps repurposed, or in the process of being lost. All other genomes from the remaining 65 species investigated contained clear representatives of EMC homologues (Figure 1).

Figure 2. Evolutionary history of the EMC.

EMC 1-8 and 10 evolved prior to the divergence of the major eukaryote lineages. Green and red dashes represent gains and losses of EMC components, respectively. Coloured pies are schematic representations of which EMC components were present at different points over the course of evolution.

These disparate organisms that lack the EMC prompted the question: What cellular or biochemical features tie these diverse organisms together? The microsporidians, metamonads and B. hominis all contain reduced anaerobic mitochondria-related organelles (MROs) and also lack the EMC. However, the amoebozoan Entamoeba histolytica retains Emc1-4, 7 and 10, the apicomplexan Cryptosporidium parvum retains Emc1-4, and 8, and the fungus Piromyces sp. retains Emc1-4, 6, 7, and 10, but all three organisms also contain extremely reduced MROs. T. parva and C. merolae contain relatively normal mitochondria but completely lack the EMC. Thus, it seems that further insight into the cell biology of these organisms is required to understand why only these few species from unrelated lineages have lost the EMC. At this point, of the proposed functions of the EMC, its involvement in multipass membrane protein assembly is the best candidate for generalization to other eukaryotes. It explains the connection to ERAD as a secondary effect of misassembled multipass proteins and explains why an organism with extremely reduced mitochondria (E. histolytica) might retain the EMC. Finally, although EMC involvement as an ER-mitochondria tether is attractive, the distribution of the only known MOM-localized interactor of EMC (Tom5) has not been identified in organisms other than animals and fungi (Maćasev et al., 2004). Thus, until an ancient interaction partner is identified, the role of EMC as an ancient tether remains speculative.