The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes

Background The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. Results Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. Conclusions Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-023-01765-1.


Figure S4. Volcano plot analysis of proteins coIP with ERMES components (baits).
The vertical axis corresponds to the mean value of -log10 p-value and the horizontal axis displays the corresponding t-test difference.Red dots represent baits, each was used in three independent coIP experiments.Black dots represent proteins that were used for interactome construction (Fig. 6).The cut-off curve, assigning significant potential interactors, is based on the false discovery rate (FDR=0.05)and the artificial factor s0 (s=1).

Gloeochaete wittrockiana EP00276_Gloeochaete_wittrockiana_P011349
Leptosphaeria maculans XP_003840150.1  A. Polar contacts were predicted using the Pymol "show_contacs.py"script: good polar interactions (proper atoms, distance, and angle) are shown in yellow and not-ideal contacts are marked in purple.

Figure S5 .
Figure S5.Phylogenetic analysis of beta-barrel proteins to investigate the relationship of Porin2 of Parabasalia and Mdm10.The maximum likelihood tree was constructed using IQ-TREE (Best fit; Q.pfam+F+I+G4 model) with 67 sequences and 237 sites.Porins in blue represent previously analyzed sequences [13].The Tom40 clade in pink was used as an outgroup.Ultrafast bootstrap support (UFB) values, and Standard non-parametric (NP) bootstrap support values were calculated using 1000 replicates, and 100 replicates, respectively.aBayes Posterior probability values were calculated using 100 replicates.The support values are represented in the order of aBayes/UFB/NP.Support values below 0.5 (aBayes) and 50(UFB/NP) are omitted or represented by a dash (-), while nodes with a support value of 100 for both Ultrafast and NP bootstrapping and 1 for posterior probability are represented with black solid triangles.

Figure S8 .
Figure S8.Hydrophobic and polar interaction of the interface of TvMmm1a-TvMdm12 heterodimer.

B.Figure S9 .Figure S10 .
Figure S9.Superposition of T. vaginalis and Z. rouxii Mmm1-Mdm12 heterotetramer.Crystal Structure of Mdm12-Mmm1 complex (PDB ID 5YK7) and ColabFold predicted T. vaginalis heterotetramer (TableS4) were used for the analysis.The red lines indicate axes of each displaced dimer of both tetramers.These axes form the displacement angle of 56.4°.