ER-mitochondria Encounter Structure Research Articles

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

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

BackgroundThe 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.ResultsHomology 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.ConclusionsOur investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

TORC1 mediated regulation of mitochondrial integrity and calcium ion homeostasis by Wat1/mLst8 in S. pombe

The mTOR complexes play a fundamental role in mitochondrial biogenesis and cellular homeostasis. Wat1, an ortholog of mammalian Lst8 is an important component of TOR complex and is essential for the regulation of downstream signaling. Earlier we reported the role of Wat1 in oxidative stress response. Here, we have shown that the abrogation of wat1 causes respiratory defects and mitochondrial depolarization that leads to a decrease in ATP production. The confocal and electron microscopy in wat1Δ cells revealed the fragmented mitochondrial morphology implying its role in mitochondrial fission. Furthermore, we also showed its role in autophagy and the maintenance of calcium ion homeostasis. Additionally, tor2–287 mutant cells also exhibit defects in mitochondrial integrity indicating the TORC1-dependent involvement of Wat1 in the maintenance of mitochondrial homeostasis. The interaction studies of Wat1 and Tor2 with Por1 and Mmm1 proteins revealed a plausible cross-talk between mitochondria and endoplasmic reticulum through the Mitochondria-associated membranes (MAM) and endoplasmic reticulum-mitochondria encounter structure (ERMES) complex, involving TORC1. Taken together, this study demonstrates the involvement of Wat1/mLst8 in harmonizing various mitochondrial functions, redox status, and Ca2+ homeostasis.

In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

H2O2 Induces Calcium and ERMES Complex-Dependent Mitochondrial Constriction and Division as Well as Mitochondrial Outer Membrane Remodeling in Aspergillus nidulans

The dynamin-like protein DnmA and its receptor FisA are essential for H2O2-induced mitochondrial division in Aspergillus nidulans. Here, we show that in the absence of DnmA or FisA, mitochondria show few spontaneous transient constrictions, the frequency of which is extensively increased by H2O2 or the carbonyl cyanide m-chlorophenyl hydrazone (CCCP). While H2O2-induced constrictions are transient, CCCP induces a drastic and irreversible alteration of mitochondrial filaments. H2O2 induces a gradual mitochondrial depolarization, while CCCP-induced depolarization is abrupt. The calcium chelator BAPTA-AM prevents the formation of mitochondrial constrictions induced by either H2O2 or CCCP. H2O2 also induces major rearrangements of the mitochondrial outer membrane, which remain after constrictions dissipate, as well as changes in endoplasmic reticulum (ER) and nuclear morphology. Similar mitochondrial constriction, ER and nuclear morphology changes are detected during the early stages of asexual development. ER and ER-Mitochondria encounter structure (ERMES) complex—composed of proteins Mdm10, Mmm1, Mdm43 and Mdm12—are important for mitochondrial division in Saccharomyces cerevisiae. As the Mdm10 ortholog MdmB was found to be essential in A. nidulans, we evaluated its functions in ΔmdmB terminal mutants and ΔmdmB heterokaryons. ΔmdmB conidia produce a short germ tube that fails to grow further, in which inherited mitochondria become gigantic and round shaped, lacking clear contacts with the ER. In slow-growing ΔmdmB heterokaryotic mycelia, multiple hyphae contain very long mitochondria with high ROS levels, as occur in ΔdnmA and ΔfisA mutants. In this hyphae, H2O2 fails to induce mitochondrial constrictions but not outer mitochondrial membrane reshaping, indicating that these are two separate effects of H2O2. Our results indicate that H2O2 induces a generalized mitochondrial constriction response, prior to actual division, involving gradual depolarization; they also indicate that Ca2+ and the ERMES complex are critical for both mitochondrial constriction and division. This supports a view of mitochondrial dynamics as the result of a cascade of signaling events that can be initiated in vivo by H2O2.

Respiratory defects caused by mutations affecting the Endoplasmic Reticulum‐Mitochondria Encounter Structure (ERMES) can be rescued by the deletion of COQ11

Coenzyme Q (CoQ) is an essential redox‐active lipid that plays a major role in the electron transport chain, driving mitochondrial ATP synthesis. Deficiency of CoQ causes a wide range of clinical deficiencies, highlighting the need to study the biosynthesis of this lipid to design therapeutics to treat these symptoms. In Saccharomyces cerevisiae, CoQ biosynthesis takes place exclusively in the mitochondrial matrix using a multi‐subunit protein‐lipid complex, the CoQ Synthome, that includes the polypeptides Coq3‐Coq9 and Coq11. A recently identified regulator of CoQ Synthome assembly and CoQ production is the ER‐mitochondria encounter structure (ERMES). ERMES is a tethering complex that bridges the ER and mitochondria, and the CoQ Synthome resides in specific membrane niches or domains directly adjacent to this complex. Loss of ERMES results in transcriptionally upregulated expression of COQ genes, yet inefficient synthesis of CoQ due to a destabilized CoQ Synthome. In this work, ERMESΔcoq11Δ mutants have been generated in an effort to correct this defect. Deletion of COQ11 has been shown to promote mitochondrial CoQ content, enhance CoQ Synthome stability, and rescue the respiratory deficiency of the coq10Δ mutant. We seek to investigate the functional roles of Coq11 and ERMES to better understand the regulation of CoQ biosynthesis and aid in the development of more effective therapeutics for diseases linked to CoQ deficiencies.

Vacuole and mitochondria patch (vCLAMP) and ER-mitochondria encounter structure (ERMES) maintain cell survival by protecting mitochondrial functions in Candida albicans

Candida albicans is an important opportunistic fungus in the clinic. In recent years, with the widespread use of antibiotics, drug-resistant strains have been isolated in the clinic, so finding new drug targets has become an urgent problem to be solved. The vacuole and mitochondria patch (vCLAMP) and the ER-mitochondria encounter structure (ERMES) are new types of inner membrane junction systems in Saccharomyces cerevisiae. However, the functions in maintaining cell survival of the two structures have not yet been elucidated in C. albicans. In this study, VAM6 and MDM34 knockout mutants (vam6Δ/Δmet-MDM34) were constructed using an induction system regulated by the MET3 promoter. PI-positive assays showed that deletion of vCLAMP and ERMES led to abnormal growth of C. albicans. Furthermore, the vam6Δ/Δmet-MDM34 mutant exhibited obvious mitochondrial fragmentation, mtDNA damage, reduced ATP levels, and abnormal mitochondrial membrane potential, indicating its important role in maintaining the structures and functions of mitochondria. Moreover, deletion of vCLAMP and ERMES inhibited filamentous growth. Overall This study shows that vCLAMP and ERMES play important roles in maintaining the survival of C. albicans cells.

ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function

Proteostasis is a challenge for cellular organisms, as all known protein synthesis machineries are error-prone. Here we show by cell fractionation and microscopy studies that misfolded proteins formed in the endoplasmic reticulum can become associated with and partly transported into mitochondria, resulting in impaired mitochondrial function. Blocking the endoplasmic reticulum-mitochondria encounter structure (ERMES), but not the mitochondrial sorting and assembly machinery (SAM) or the mitochondrial surveillance pathway components Msp1 and Vms1, abrogated mitochondrial sequestration of ER-misfolded proteins. We term this mitochondria-associated proteostatic mechanism for ER-misfolded proteins ERAMS (ER-associated mitochondrial sequestration). We testify to the relevance of this pathway by using mutant α-1-antitrypsin as an example of a human disease-related misfolded ER protein, and we hypothesize that ERAMS plays a role in pathological features such as mitochondrial dysfunction.

The structural organization and functions of the ERMES complex in fungi

<p indent=0mm>In eukaryotic cells, mitochondria, the double-membrane bound organelle, form different networks by undergoing constant fission and fusion to meet the changing energy demands. The proper function of mitochondria and the regulation of mitochondrial dynamics depend on the proper contact of mitochondria with other subcellular organelles and the materials exchanged between the two contacting organelles. Mitochondria form contact sites with many organelles including lipid droplets, peroxisomes, vacuoles, and the endoplasmic reticulum (ER). The ER-mitochondria encounter structure (ERMES) mediates the contact between the ER and mitochondria and plays important roles in regulating mitochondrial morphology and functions. Malfunctions of the ERMES complex affect mitochondrial dynamics, calcium signalling, lipid transport, drug resistance and tolerance in fungi, and the virulence of pathogenic fungi. In this review, we discuss the structural organization, functions, and the regulation of the ERMES complex.

TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2–TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER–mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2–TOM20 interaction is thus an additional player in the control of apoptosis.

Emr1 regulates the number of foci of the endoplasmic reticulum-mitochondria encounter structure complex

The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex creates contact sites between the endoplasmic reticulum and mitochondria, playing crucial roles in interorganelle communication, mitochondrial fission, mtDNA inheritance, lipid transfer, and autophagy. The mechanism regulating the number of ERMES foci within the cell remains unclear. Here, we demonstrate that the mitochondrial membrane protein Emr1 contributes to regulating the number of ERMES foci. We show that the absence of Emr1 significantly decreases the number of ERMES foci. Moreover, we find that Emr1 interacts with the ERMES core component Mdm12 and colocalizes with Mdm12 on mitochondria. Similar to ERMES mutant cells, cells lacking Emr1 display defective mitochondrial morphology and impaired mitochondrial segregation, which can be rescued by an artificial tether capable of linking the endoplasmic reticulum and mitochondria. We further demonstrate that the cytoplasmic region of Emr1 is required for regulating the number of ERMES foci. This work thus reveals a crucial regulatory protein necessary for ERMES functions and provides mechanistic insights into understanding the dynamic regulation of endoplasmic reticulum-mitochondria communication.

The Endoplasmic Reticulum-Mitochondria Encounter Structure and its Regulatory Proteins.

In fungi, the endoplasmic reticulum-mitochondria encounter structure (ERMES) is present between the endoplasmic reticulon (ER) and mitochondria to promote the formation of the ER-mitochondria contact sites. Four constitutive components (Mmm1, Mdm12, Mdm34, and Mdm10) assemble to form the ERMES complex while regulator proteins are required for regulating the organization and function of the ERMES complex. Multiple regulator proteins, including Gem1, Lam6, Tom7, and Emr1, of the ERMES complex, have been identified recently. In this review, we discuss the organization of the ERMES complex and the roles of the regulator proteins of the ERMES complex.

Leri: A web-server for identifying protein functional networks from evolutionary couplings.

Information on the co-evolution of amino acid pairs in a protein can be used for endeavors such as protein engineering, mutation design, and structure prediction. Here we report a method that captures significant determinants of proteins using estimated co-evolution information to identify networks of residues, termed "residue communities", relevant to protein function. On the benchmark dataset (67 proteins with both catalytic and allosteric residues), the Pearson's correlation between the identified residues in the communities at functional sites is 0.53, and it is higher than 0.8 by taking account of conserved residues derived from the method. On the endoplasmic reticulum-mitochondria encounter structure complex, the results indicate three distinguishable residue communities that are relevant to functional roles in the protein family, suggesting that the residue communities could be general evolutionary signatures in proteins. Based on the method, we provide a webserver for the scientific community to explore the signatures in protein families, which establishes a powerful tool to analyze residue-level profiling for the discovery of functional sites and biological pathway identification. This web-server is freely available for non-commercial users at https://kornmann.bioch.ox.ac.uk/leri/services/ecs.html, neither login nor e-mail required.

ER-mitochondria contacts promote mitochondrial-derived compartment biogenesis.

Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid overabundance stress. How cells form MDCs is unclear. Here, we show that MDCs are dynamic structures that form and stably persist at sites of contact between the ER and mitochondria. MDC biogenesis requires the ER-mitochondria encounter structure (ERMES) and the conserved GTPase Gem1, factors previously implicated in lipid exchange and membrane tethering at ER-mitochondria contacts. Interestingly, common genetic suppressors of abnormalities displayed by ERMES mutants exhibit distinct abilities to rescue MDC formation in ERMES-depleted strains and are incapable of rescuing MDC formation in cells lacking Gem1. Thus, the function of ERMES and Gem1 in MDC biogenesis may extend beyond their conventional role in maintaining mitochondrial phospholipid homeostasis. Overall, this study identifies an important function for ER-mitochondria contacts in the biogenesis of MDCs.

The endoplasmic reticulum-mitochondria encounter structure: coordinating lipid metabolism across membranes.

Endosymbiosis, the beginning of a collaboration between an archaeon and a bacterium and a founding step in the evolution of eukaryotes, owes its success to the establishment of communication routes between the host and the symbiont to allow the exchange of metabolites. As far as lipids are concerned, it is the host that has learnt the symbiont's language, as eukaryote lipids appear to have been borrowed from the bacterial symbiont. Mitochondria exchange lipids with the rest of the cell at membrane contact sites. In fungi, the endoplasmic reticulum-mitochondria encounter structure (ERMES) is one of the best understood membrane tethering complexes. Its discovery has yielded crucial insight into the mechanisms of intracellular lipid trafficking. Despite a wealth of data, our understanding of ERMES formation and its exact role(s) remains incomplete. Here, I endeavour to summarise our knowledge on the ERMES complex and to identify lingering gaps.

The mitochondrial intermembrane space-facing proteins Mcp2 and Tgl2 are involved in yeast lipid metabolism.

Mitochondria are unique organelles harboring two distinct membranes, the mitochondrial inner and outer membrane (MIM and MOM, respectively). Mitochondria comprise only a subset of metabolic pathways for the synthesis of membrane lipids; therefore most lipid species and their precursors have to be imported from other cellular compartments. One such import process is mediated by the ER mitochondria encounter structure (ERMES) complex. Both mitochondrial membranes surround the hydrophilic intermembrane space (IMS). Therefore, additional systems are required that shuttle lipids between the MIM and MOM. Recently, we identified the IMS protein Mcp2 as a high-copy suppressor for cells that lack a functional ERMES complex. To understand better how mitochondria facilitate transport and biogenesis of lipids, we searched for genetic interactions of this suppressor. We found that MCP2 has a negative genetic interaction with the gene TGL2 encoding a neutral lipid hydrolase. We show that this lipase is located in the intermembrane space of the mitochondrion and is imported via the Mia40 disulfide relay system. Furthermore, we show a positive genetic interaction of double deletion of MCP2 and PSD1, the gene encoding the enzyme that synthesizes the major amount of cellular phosphatidylethanolamine. Finally, we demonstrate that the nucleotide-binding motifs of the predicted atypical kinase Mcp2 are required for its proper function. Taken together, our data suggest that Mcp2 is involved in mitochondrial lipid metabolism and an increase of this involvement by overexpression suppresses loss of ERMES.

Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins.

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.

Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release.

The distribution of the pro-apoptotic protein Bax in the outer mi-tochondrial membrane (OMM) is a central point of regulation of apoptosis. It is now widely recognized that parts of the endoplasmic reticulum (ER) are closely associated to the OMM, and are actively involved in different signaling processes. We addressed a possible role of these domains, called Mitochon-dria-Associated Membranes (MAMs) in Bax localization and function, by ex-pressing the human protein in a yeast mutant deleted of MDM34, a ERMES (ER-Mitochondria Encounter Structure) component. By affecting MAMs stabil-ity, the deletion of MDM34 altered Bax mitochondrial localization, and de-creased its capacity to release cytochrome c. Furthermore, the deletion of MDM34 decreased the size of an incompletely released, MAMs-associated pool of cytochrome c.

The Endoplasmic Reticulum-Mitochondria Encounter Structure Complex Coordinates Coenzyme Q Biosynthesis.

Loss of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) complex that resides in contact sites between the yeast ER and mitochondria leads to impaired respiration; however, the reason for that is not clear. We find that in ERMES null mutants, there is an increase in the level of mRNAs encoding for biosynthetic enzymes of coenzyme Q6 (CoQ6), an essential electron carrier of the mitochondrial respiratory chain. We show that the mega complexes involved in CoQ6 biosynthesis (CoQ synthomes) are destabilized in ERMES mutants. This, in turn, affects the level and distribution of CoQ6 within the cell, resulting in reduced mitochondrial CoQ6. We suggest that these outcomes contribute to the reduced respiration observed in ERMES mutants. Fluorescence microscopy experiments demonstrate close proximity between the CoQ synthome and ERMES, suggesting a spatial coordination. The involvement of the ER-mitochondria contact site in regulation of CoQ6 biogenesis highlights an additional level of communication between these two organelles.