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

Mitochondrial calcium (mt-Ca²⁺) uptake, a crucial process regulating various cellular functions, is mediated by the mitochondrial calcium uniporter (MCU). The mammalian uniporter involves multiple components: MCU (pore-forming subunit), MICU1 (a negative sensor and activator), and EMRE (essential for channel function). Previous comparative genomic analyses, primarily focusing on a limited number of eukaryotic species, identified MCU and MICU1, revealing a correlated evolutionary history. However, EMRE appeared to be an animal-specific innovation, leading to a paradox regarding the functional role of MCU in fungi, where other uniporter components were seemingly absent. This study aimed to resolve this evolutionary puzzle by performing a comprehensive phylogenomic analysis of the mt-Ca²⁺ uniporter across a vastly expanded dataset of eukaryotic species. The objective was to trace the evolutionary history of the uniporter components and determine their functional relationships to clarify the mechanisms and significance of mt-Ca²⁺ uptake in different eukaryotic lineages. This research is crucial for understanding the evolution of cellular calcium regulation and provides a basis for future comparative studies of uniporter structure and function across diverse organisms. The presence of MCU homologs in fungal species devoid of other uniporter components and evidence of mt-Ca²⁺ uptake represents a significant evolutionary question. Addressing this gap in knowledge requires a detailed examination of the evolutionary relationships between MCU, EMRE, and MICU across a broad range of eukaryotic species, potentially leading to a deeper understanding of the uniporter's structure and function.

Previous studies have identified key components of the mammalian mitochondrial calcium uniporter (MCU), including the pore-forming subunit MCU itself, the regulatory subunit MICU1, and the essential component EMRE. Research has shown a strong co-evolutionary relationship between MCU and MICU1 across various eukaryotic lineages, suggesting a functional dependence. However, the absence of EMRE homologs outside metazoans raised questions about the evolutionary history and functional diversity of the uniporter. Prior work on fungal MCU homologs revealed conflicting results regarding their ability to mediate mt-Ca²⁺ uptake, prompting further investigation into the role of MCU and the possibility of alternative uniporter mechanisms in fungi. The evolutionary relationships among uniporter components were not fully understood, particularly in fungi. The existing literature suggested an animal-specific innovation for EMRE, leaving the function and evolutionary history of fungal MCUs ambiguous. This created the need for a broad phylogenomic analysis to resolve these inconsistencies and gain a complete picture of the uniporter's evolution across eukaryotes.

This study employed a multi-faceted approach combining comprehensive phylogenomic analysis with functional reconstitution experiments. The phylogenomic analysis utilized a vast dataset of 1,156 fully sequenced eukaryotic genomes to trace the evolutionary history of MCU, EMRE, and MICU. Profile-based sequence searches, protein domain composition assessments, and phylogenetic reconstructions were employed to determine the evolutionary relationships among these proteins across diverse eukaryotic lineages. The analysis included the reconstruction of molecular phylogenies for MCU and MICU, investigating gene duplication and loss events, to better understand the evolutionary pathways of these proteins. Functional reconstitution experiments involved the heterologous expression of MCU and EMRE from various species (including chytrid fungi) in yeast (Saccharomyces cerevisiae) and HeLa cells to assess their ability to mediate mt-Ca²⁺ uptake. This allowed the researchers to directly test the hypothesis that animal-like MCU complexes containing both MCU and EMRE were necessary and sufficient for functional mt-Ca²⁺ uniporter activity. The experiments also involved genetic manipulation, such as MCU knock-down in HeLa cells, to control for endogenous uniporter activity and assess the rescue effect of different MCU homologs. A detailed description of cloning methods, yeast strains, and other relevant experimental details were provided. Statistical analyses were performed to assess the significance of experimental findings. The proteomic analysis was supplemented with molecular phylogenetic analysis, using various phylogenetic algorithms to reconstruct the evolutionary history of MCU, MICU, and EMRE homologs. Maximum-likelihood trees were constructed based on multiple sequence alignments using software such as MAFFT and IQ-TREE. These phylogenetic trees then provided insights into the evolutionary relationships and the timing of key duplication and loss events that shaped the uniporter's evolution.

This research revealed several key findings: First, the study demonstrated that animal and fungal MCUs represent distinct paralogous subfamilies resulting from an ancestral gene duplication event at the base of opisthokonts. Phylogenetic analysis revealed distinct evolutionary trajectories for these paralogs, indicating functional divergence. Second, the researchers identified EMRE orthologs outside the Holozoa clade, specifically in chytrid fungi (Allomyces macrogynus, Catenaria anguillulae, and Spizellomyces punctatus). This contradicted the previous notion of EMRE as an animal-specific innovation and expanded our understanding of the evolution of the uniporter. Third, functional reconstitution experiments showed that co-expression of chytrid fungal MCU and EMRE homologs reconstituted mt-Ca²⁺ uptake in yeast mitochondria. This proved that the animal-like uniporter complex was indeed functional in these early-diverging fungi and was not a recent animal-specific development. Furthermore, studies using HeLa cells revealed that fungal MCU homologs that lacked EMRE were unable to rescue mt-Ca²⁺ uptake in MCU knock-down cells, while animal-like MCUs from chytrid fungi did restore mt-Ca²⁺ uptake activity upon co-expression with EMRE. Notably, the researchers identified key amino acid residues in MCU that are conserved in animal-like MCUs but absent in fungal-specific paralogs, suggesting a structural basis for the functional differences between these paralogs. Sequence analysis of EMRE also revealed conserved domains essential for MCU interaction, emphasizing a key role of the EMRE-MCU interaction in the evolution of the uniporter.

The findings of this study address the long-standing evolutionary paradox concerning the presence of MCU in fungi without other uniporter components and functional mt-Ca²⁺ uptake. The discovery of EMRE homologs in chytrid fungi demonstrates that the EMRE-dependent animal-like uniporter complex existed in the common ancestor of animals and fungi. The loss of EMRE in most fungal lineages, together with the inability of fungal-specific MCU paralogs to mediate mt-Ca²⁺ uptake, suggests a functional diversification of these MCU paralogs, potentially with non-uniporter-related roles. This implies that the complex MCU-EMRE-MICU machinery evolved earlier than previously assumed, potentially predating the divergence of animals and fungi. The strong co-evolutionary pattern observed between MCU, EMRE, and MICU supports the functional interdependence of these components in animal-like uniporters, highlighting the regulatory coupling between uniporter gating and EMRE regulation. This study also reveals a significant amount of functional variation in MCU proteins across different eukaryotic lineages, highlighting a need for future studies to investigate the functional specificity of MCU in various organisms. Future research should focus on examining the functional roles of fungal-specific MCU paralogs, which might have evolved distinct functions unrelated to mt-Ca²⁺ uptake. A better understanding of the evolutionary pressures leading to the retention or loss of uniporter components in different lineages is also critical.

This comprehensive phylogenomic and functional study resolved the evolutionary paradox surrounding the mitochondrial calcium uniporter. The findings demonstrate that the core uniporter complex, comprising MCU, EMRE, and MICU, predates the divergence of animals and fungi. The identification of EMRE in chytrid fungi and the functional reconstitution of mt-Ca²⁺ uptake in these fungi highlight the ancestral role of EMRE and the conservation of the animal-like uniporter mechanism. The functional divergence between animal and fungal MCUs underscores the importance of comparative studies to elucidate the structure-function relationships within the uniporter. Future studies could focus on the structural and functional characterization of the uniporter in other eukaryotic groups, particularly those with unique variations in uniporter composition.

While this study significantly advances our understanding of uniporter evolution, several limitations exist. The phylogenetic analysis relies on available genome sequences, which may not fully represent the diversity of eukaryotic lineages. Functional reconstitution experiments were primarily conducted in yeast and HeLa cells, which might not perfectly reflect the native cellular context of other species. Further studies are needed to investigate the diversity and functional roles of the uniporter in a broader range of eukaryotic organisms. The study primarily focused on MCU, EMRE, and MICU and did not fully explore the roles of other uniporter regulators that might exist. Deeper functional analyses in different organisms might help clarify how the uniporter operates in diverse cellular contexts.