Discovery of EMRE in fungi resolves the true evolutionary history of the mitochondrial calcium uniporter

Mitochondria rapidly take up and buffer cytosolic Ca²⁺ through a selective inner-membrane channel, the mitochondrial calcium uniporter (MCU), thereby shaping intracellular Ca²⁺ signaling and metabolism. Molecular identification of uniporter components in mammals established MCU as the pore-forming subunit and MICU1 as an EF-hand gatekeeper, with EMRE as an essential regulator enabling channel activity. However, comparative genomics had revealed an evolutionary paradox: many fungi encode MCU homologs but lack MICU and EMRE and show no detectable mitochondrial Ca²⁺ uptake, raising questions about the true evolutionary relationships and functional equivalence of fungal and animal MCUs. This study aims to resolve the evolutionary history of the uniporter complex across eukaryotes, to determine whether fungal MCUs are orthologous and functionally equivalent to animal MCU, to identify the phylogenetic distribution of EMRE, and to test functional requirements for mt-Ca²⁺ uptake using heterologous reconstitution.

Early electrophysiological and biochemical studies established a highly selective mitochondrial Ca²⁺ uptake pathway. Genomic and RNAi-based approaches later identified mammalian MCU and MICU1, followed by discovery of MCU regulatory subunits (e.g., EMRE) and paralogs (MCUB, MICU2/3), and structural advances via X-ray and cryo-EM. Prior phylogenetic surveys suggested EMRE was metazoan-specific and that some protists and fungi possessed MCU without other components, prompting speculation that fungal MCUs might function independently of MICU. Structural studies on Ascomycota MCU homologs were used as models for human MCU, despite reports that these fungi and their MCU homologs lack uniporter activity in heterologous systems. This background highlighted the need for comprehensive phylogenomics and functional assays to clarify orthology, paralogy, and co-evolution among MCU, MICU, and EMRE across eukaryotes.

• Phylogenomic dataset: Surveyed 1,156 fully sequenced eukaryotic genomes (collapsed to 969 species where multiple strains shared patterns). Particular enrichment in fungi (776 species). Genome annotations were retrieved from Ensembl (Metazoa, Plants, Fungi, Protozoa). UniProt used for specific sequences.
• Homology detection: Profile HMM searches (HMMER/hmmsearch) built from multiple sequence alignments; Pfam domains used to annotate families (e.g., MCU Pfam PF08678; EMRE Pfam PF11611). BLAST with low-complexity filtering and stringent e-value thresholds. Iterative HMM searches applied to detect divergent EMRE homologs.
• Sequence processing and domain analysis: Sequences aligned with MAFFT; low-coverage positions trimmed. Additional domain scans with HMMscan. Assessed conserved motifs and residues (e.g., DXE motif in MCU; EMRE β-hairpin, TM, polybasic CAD region). Multi-Harmony used to detect clade-specific conservation shifts.
• Phylogenetic inference: Maximum likelihood trees constructed with IQ-TREE, model selection by BIC, branch supports from 1000 ultrafast bootstrap replicates. Mapped duplications/losses across species tree; assessed co-occurrence and co-evolution patterns among MCU, MICU, and EMRE.
• Functional reconstitution in yeast: Saccharomyces cerevisiae strains expressing mitochondrial aequorin (mt-AEQ) were transformed with combinations of MCU and EMRE homologs from human, chytrid fungi (Allomyces macrogynus, Spizellomyces punctatus), and controls. Subcellular fractionation and immunoblotting confirmed expression and mitochondrial localization. Glucose-induced calcium (GIC) stimulation in presence of 1 mM CaCl₂ used to elicit mt-Ca²⁺ transients; luminescence quantified and converted to [Ca²⁺]mt via calibration models.
• Heterologous expression in mammalian cells: HeLa cells transfected/infected with constructs expressing human or fungal MCUs/EMREs; MCU knockdown or knockout lines generated via shRNA/CRISPR. Mitochondrial Ca²⁺ uptake monitored with mt-AEQ during agonist (e.g., histamine) stimulation; rescue assays tested whether human or fungal MCUs restore uptake in MCU-deficient cells.
• Biochemistry and topology: Isolation of mitochondria from HeLa; protease protection and detergent permeabilization to assess membrane insertion/topology. Protein detection via immunoblotting with specific antibodies.
• Statistics: Data presented as mean ± SEM; normality tested by Shapiro–Wilk; one-way ANOVA with Dunnett’s multiple comparisons; significance thresholds including p < 0.001 and p < 0.0001 as reported in figure legends.

• Comprehensive phylogenomics across 1,156 eukaryotes reveals that animal and fungal MCU proteins segregate into two distinct paralogous clades originating from an ancient duplication near the base of opisthokonts. Most fungi retained a fungal-specific paralog (MCUP), while holozoans retained the bona fide animal-like MCU.
• Contrary to earlier beliefs, bona fide EMRE orthologs were identified outside Holozoa, specifically in early-diverging chytrid fungi (e.g., Allomyces macrogynus, Catenaria/Caterina angustilobata, Spizellomyces punctatus), indicating an ancestral presence of EMRE in opisthokonts.
• Only three chytrid fungi retained both the animal-like MCU along with MICU and EMRE, consistent with an animal-like uniporter present before the animal–fungal split.
• Functional assays: Co-expression of chytrid animal-like MCU with its cognate EMRE restored mitochondrial Ca²⁺ uptake in yeast; expression of MCUP paralogs from fungi did not mediate uptake despite correct expression and localization.
• In HeLa cells, human MCU rescued mt-Ca²⁺ uptake in MCU-deficient cells, whereas fungal MCUs (both animal-like and MCUP) failed to rescue in the absence of appropriate EMRE support, underscoring EMRE dependence of animal-like MCUs.
• Sequence analysis showed MCUPs lack key residues conserved in animal-like MCUs that are critical for EMRE interaction and channel function, despite retaining the DXE motif.
• Fungal EMREs are highly divergent overall but conserve essential MCU-interacting features (GXXGXXG and polybasic regions). An extra C-terminal domain in fungal EMREs appears important: truncation impaired reconstitution with fungal MCUs; adding this domain to human EMRE did not enable function with fungal MCUs, suggesting specialized co-adaptation.
• Strong co-occurrence: When considering true orthologs, MCU and EMRE (and MICU) show highly overlapping distributions; MCU components co-occur in ~1144 of 1156 species (~99%), with a small number of apparent exceptions likely due to detection/annotation issues.
• Findings resolve the paradox of MCU presence in fungi without mt-Ca²⁺ uptake: most fungal MCUs are paralogs (MCUPs) not functioning as classical uniporters under tested conditions.

The results demonstrate that the core uniporter machinery—animal-like MCU, EMRE, and MICU—was present in the last common ancestor of opisthokonts and that differential retention and loss shaped present-day distributions. This clarifies why many fungi harbor MCU-like genes without detectable mt-Ca²⁺ uptake: these are paralogous MCUPs lacking key functional determinants and do not operate as EMRE-dependent Ca²⁺ pores in heterologous systems. Discovery of EMRE in chytrids and successful reconstitution of mt-Ca²⁺ uptake with chytrid MCU+EMRE show conservation of MCU–EMRE coupling predating the animal–fungal divergence. The divergence of fungal EMREs, including an extra C-terminal domain, suggests lineage-specific adaptations modulating MCU activation, while conserved interaction motifs explain cross-compatibility with human MCU in some contexts. These insights have broad implications for interpreting prior structural studies that used fungal MCUs as models for human channels and for understanding the evolution of Ca²⁺ homeostasis mechanisms across eukaryotes.

This study provides the most extensive phylogenomic and functional analysis to date of the mitochondrial Ca²⁺ uniporter system. It shows that animal and fungal MCUs are paralogs from an ancient duplication, identifies non-metazoan EMREs in chytrid fungi, and functionally establishes an animal-like, EMRE-dependent uniporter in early-diverging fungi. These findings resolve an evolutionary paradox regarding fungal MCUs, redefine appropriate model systems for structural and functional studies, and highlight conserved MCU–EMRE interfaces as key targets. Future work should characterize the physiological roles of MCUP paralogs, delineate MICU regulation across lineages, analyze the structural impact of the fungal EMRE C-terminal domain, and investigate notable taxa that appear to lack MCU despite retaining other components.

• EMRE is short and highly divergent, complicating homology detection; some presence/absence calls may be affected by sensitivity limits and annotation errors.
• Functional assays relied on heterologous expression in yeast and mammalian cells, which may not capture native assembly factors or chaperones.
• Only a few chytrid species retaining animal-like uniporter components were available for experimental testing, limiting generalization across basal fungi.
• Some taxonomic anomalies (e.g., nematodes reportedly lacking MCU while retaining other components) require further validation to exclude assembly or detection artifacts.
• The exact physiological roles of MCUP paralogs and potential lineage-specific regulators remain unresolved.