1.A.77 The Mg2+/Ca2+ Uniporter (MCU) Family Mitochondrial Ca²⁺ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival. Mitochondrial Ca²⁺ uptake occurs via the ruthenium red sensitive Ca²⁺ uniporter (MCU), and efflux occurs via a Na⁺/Ca²⁺ exchanger (mNCX). De Stefani et al. (2011) and Baughman et al. (2011) simultaneously reported the identity of a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a uniporter regulator that is present in organisms in which mitochondrial Ca²⁺ uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU reduced mitochondrial Ca²⁺ uptake, and MCU overproduction increased the matrix Ca²⁺ concentration evoked by inositol 1,4,5-trisphosphate-generating agonists. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivities of the uniporter 90. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca²⁺ concentration transients when overexpressed. Thus, MCU is the channel responsible for ruthenium-red-sensitive mitochondrial Ca²⁺ uptake. Distant prokaryotic homologues have been identified (A. Lee and MH Saier, unpublished results). A small (10 KDa) protein with 1 TMS, EMRE, is required for interaction of MCU with MICU1 and 2 (see figure4F in Sancak et al. 2013). The entire complex includes MICU1, MICU2, EMRE and MCU, and has a molecular size of 480,000 Daltons. The MICU family has TC# 8.A.44 and functions in regulation. The EMRE family has TC# 8.A.45 and funtions to interconnect MUC and MICU in the complex (Sancak et al. 2013). Mitochondrial calcium uniporter, MCU, forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1 (1.A.76.1.1), and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca²⁺ uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MICU1 (TC# 8.A.44.1.1) is an essential component of the MCU system, and serves as the gatekeeper of MCU-mediated Ca²⁺ uptake that is essnetial to prevent Ca²⁺ overload and associated stress (Mallilankaraman et al., 2012a; Mallilankaraman et al., 2012b). The oligomeric channel can incorporate an inhibitory subunit, MCUb, that exerts a dominant-negative effect on channel formation (Raffaello et al., 2013). Also essential is the EMRE subunit (TC# 8.A.45.1.1) which binds MICU1 via transmembrane helices to control Ca²⁺ transport activity (Tsai et al. 2016). MCUs interact with Miro1 (Q8IXI2), a mitochondrial Rho GTPase that seems to regulate MCU activities (Niescier et al. 2018). Carboxylate-Capped Analogues of Ru265 are prodrugs that inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders (Bigham et al. 2022) MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retained function but confered resistance to Ru360, the most potent inhibitor of the uniporter (Baughman et al., 2011). MCU homologues and their cytoplasmic regulatory protein partners with two EF-hand motifs, MICU, are both present in many but not all eukaryotes having MCU (Bick et al., 2012). The phylogenies and domain orders of MCU Ca²⁺ channel homologues have been reported (Bick et al., 2012). MCU is involved in the apoptosis in PC12 cells induced by 1-methyl-4-phenylpyridinium ions (MPP) (Wang et al. 2018). Diplacone, isolated from Paulownia tomentosa mature fruit, induces ferroptosis-mediated cell death through mitochondrial Ca²⁺ influx and the mitochondrial permeability transition (see TC# 1.A.129) (Oh et al. 2023). Ca²⁺ flux across the inner mitochondrial membrane (IMM) regulates cellular bioenergetics, intra-cellular cytoplasmic Ca²⁺ signals, and various cell death pathways. Ca²⁺ entry into the mitochondria occurs due to the highly negative membrane potential (ΔΨ_m) through a selective inward rectifying MCU channel (Nemani et al. 2018). In addition to being regulated by various mitochondrial matrix resident proteins such as MICUs, MCUb, MCUR1 and EMRE, the channel is transcriptionally regulated by an upstream Ca²⁺ cascade, post transnational modification and divalent cations. The mode of regulation either inhibits or enhances MCU channel activity and thus regulates mitochondrial metabolism and cell fate (Nemani et al. 2018). MCU inhibition protects against intestinal ischemia‒reperfusion by inhibiting Drp1-dependent mitochondrial fission (Kadier et al. 2024). Mitochondrial calcium uniporter knockdown in hippocampal neurons alleviates anxious and depressive behavior in the 3XTG Alzheimer's disease mouse model (Wang et al. 2024). The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein with 2 TMSs. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI (Hicks et al., 2003). Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2 for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium(MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg²⁺ -sensitive ⁴⁵Ca²⁺ uptake by vesicles of an Escherichia coli mutant that is defective in Ca²⁺ and Na⁺ efflux. Hicks et al. (2003) hypothesized that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg²⁺ transporters, Ca²⁺ transporters, probable Mg²⁺/Ca²⁺ channel proteins. Such proteins could provide Mg²⁺ , which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction e.g., during cytoplasmic pH regulation. AtpZ and AtpI have 2 and 4 TMSs respectively. The Na⁺ F₁F_O ATP synthase of the anaerobic acetogenic bacterium Acetobacterium woodii has a unique hybrid rotor that contains nine copies of a F_O-like c subunit with 1 ion binding site in the 2TMS protein, and one copy of a V_O-like c(1) subunit with one ion binding site in four transmembrane helices. Brandt et al. (2013) cloned and expressed its Na⁺ F₁F_O ATP synthase operon in E. coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_Ohybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli did not substitute for AtpI from A. woodii. Thus, the native AtpI is required for assembly of the hybrid rotor. The uniporter is membrane potential dependent and sensitive to ruthenium red and its derivative Ru360. It has high conductance and selectivity. Ca²⁺ entry into mitochondria activates the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. MCU is the pore-forming subunit of the uniporter holocomplex. Oxenoid et al. 2016 reported the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore. The channel assembly displays a unique ion channel architecture and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; these rings appear to form the selectivity filter (Oxenoid et al. 2016). Ca²⁺ uptake into mitochondria regulates bioenergetics, apoptosis, and Ca²⁺ signaling. The primary pathway for mitochondrial Ca²⁺ uptake is MCU. Mitochondrial Ca²⁺ uptake is tightly regulated to maintain low matrix [Ca²⁺] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. Vais et al. 2020 defined a regulatory mechanism in which cytoplasmic Ca²⁺ regulation of intermembrane space-localized MICU1/2 is controlled by Ca²⁺-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca²⁺ that permeates through the channel pore regulates Ca²⁺ affinities of coupled inhibitory and activating sensors in the matrix. Ca²⁺ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca²⁺ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca²⁺ regulation of channel activity. These results demonstrate how Ca²⁺ influx into mitochondria is tuned by coupled Ca²⁺-regulatory mechanisms on both sides of the inner mitochondrial membrane (Vais et al. 2020). As noted above, MCU controls mitochondrial bioenergetics, and its activity varies greatly between tissues. Xue et al. 2023 highlighted a recently identified MCU-EMRE-UCP1 complex, named thermoporter, in the adaptive thermogenesis of brown adipose tissue (BAT). The thermoporter enhances MCU activity to promote thermogenic metabolism, demonstrating a BAT-specific regulation for MCU activity. The generalized reaction believed to be catalyzed by (AtpZ)_n, (AtpI)_n and (AtpZ)_n-x AtpIx is: Mg²⁺ or Ca²⁺ (out) ⇌ Mg²⁺ or Ca²⁺ (in)
References:
Balderas, E., S.H.J. Lee, N.K. Rai, D.M. Mollinedo, H.E. Duron, and D. Chaudhuri. (2024). Mitochondrial Calcium Regulation of Cardiac Metabolism in Health and Disease. Physiology (Bethesda) 39: 0.
Baughman, J.M., F. Perocchi, H.S. Girgis, M. Plovanich, C.A. Belcher-Timme, Y. Sancak, X.R. Bao, L. Strittmatter, O. Goldberger, R.L. Bogorad, V. Koteliansky, and V.K. Mootha. (2011). Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476: 341-345.
Bick, A.G., S.E. Calvo, and V.K. Mootha. (2012). Evolutionary diversity of the mitochondrial calcium uniporter. Science 336: 886.
Bigham, N.P., Z. Huang, J. Spivey, J.J. Woods, S.N. MacMillan, and J.J. Wilson. (2022). Carboxylate-Capped Analogues of Ru265 Are MCU Inhibitor Prodrugs. Inorg Chem 61: 17299-17312.
Brandt, K., D.B. Müller, J. Hoffmann, C. Hübert, B. Brutschy, G. Deckers-Hebestreit, and V. Müller. (2013). Functional production of the Na⁺ F1F(O) ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI. J. Bioenerg. Biomembr. 45: 15-23.
De Stefani, D., A. Raffaello, E. Teardo, I. Szabò, and R. Rizzuto. (2011). A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476: 336-340.
Docampo R., Moreno SN. and Plattner H. (2014). Intracellular calcium channels in protozoa. Eur J Pharmacol. 739:4-18.
Drago, I., P. Pizzo, and T. Pozzan. (2011). After half a century mitochondrial calcium in- and efflux machineries reveal themselves. EMBO. J. 30: 4119-4125.
Dubinin, M.V., E.Y. Talanov, K.S. Tenkov, V.S. Starinets, I.B. Mikheeva, M.G. Sharapov, and K.N. Belosludtsev. (2020). Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition. Biochim. Biophys. Acta. Mol Basis Dis 1866: 165674.
Hicks, D.B., Z. Wang, Y. Wei, R. Kent, A.A. Guffanti, H. Banciu, D.H. Bechhofer, and T.A. Krulwich. (2003). A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg²⁺ uptake. Proc. Natl. Acad. Sci. USA 100: 10213-10218.
Huang, G. and R. Docampo. (2018). The Mitochondrial Ca Uniporter Complex (MCUC) of Trypanosoma brucei Is a Hetero-oligomer That Contains Novel Subunits Essential for Ca Uptake. MBio 9:.
Huang, G. and R. Docampo. (2020). The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans. mBio 11:.
Huang, Z. and J.J. Wilson. (2023). Structure-Activity Relationships of Metal-Based Inhibitors of the Mitochondrial Calcium Uniporter. ChemMedChem e202300106. [Epub: Ahead of Print]
Islam, M.N., G.A. Gusarova, S.R. Das, L. Li, E. Monma, M. Anjaneyulu, L. Mthunzi, S.K. Quadri, E. Owusu-Ansah, S. Bhattacharya, and J. Bhattacharya. (2022). The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury. Nat Commun 13: 5837.
Kadier, T., Y.G. Zhang, Y.X. Jing, Z.Y. Weng, S.S. Liao, J. Luo, K. Ding, C. Cao, R. Chen, and Q.T. Meng. (2024). MCU inhibition protects against intestinal ischemia‒reperfusion by inhibiting Drp1-dependent mitochondrial fission. Free Radic Biol Med 221: 111-124.
Kholmukhamedov, A., R. Janecke, H.J. Choo, and S.M. Jobe. (2018). The mitochondrial calcium uniporter regulates procoagulant platelet formation. J Thromb Haemost. [Epub: Ahead of Print]
MacEwen, M.J., A.L. Markhard, M. Bozbeyoglu, F. Bradford, O. Goldberger, V.K. Mootha, and Y. Sancak. (2020). Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU. Life Sci Alliance 3:.
Mallilankaraman, K., C. Cárdenas, P.J. Doonan, H.C. Chandramoorthy, K.M. Irrinki, T. Golenár, G. Csordás, P. Madireddi, J. Yang, M. Müller, R. Miller, J.E. Kolesar, J. Molgó, B. Kaufman, G. Hajnóczky, J.K. Foskett, and M. Madesh. (2012). MCUR1 is an essential component of mitochondrial Ca²⁺ uptake that regulates cellular metabolism. Nat. Cell Biol. 14: 1336-1343.
Mallilankaraman, K., P. Doonan, C. Cárdenas, H.C. Chandramoorthy, M. Müller, R. Miller, N.E. Hoffman, R.K. Gandhirajan, J. Molgó, M.J. Birnbaum, B.S. Rothberg, D.O. Mak, J.K. Foskett, and M. Madesh. (2012). MICU1 Is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca²⁺ Uptake that Regulates Cell Survival. Cell 151: 630-644.
Morales-Rios, E., I.N. Watt, Q. Zhang, S. Ding, I.M. Fearnley, M.G. Montgomery, M.J. Wakelam, and J.E. Walker. (2015). Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans. Open Biol 5:.
Nemani, N., S. Shanmughapriya, and M. Madesh. (2018). Molecular regulation of MCU: Implications in physiology and disease. Cell Calcium 74: 86-93.
Nguyen, N.X., J.P. Armache, C. Lee, Y. Yang, W. Zeng, V.K. Mootha, Y. Cheng, X.C. Bai, and Y. Jiang. (2018). Cryo-EM structure of a fungal mitochondrial calcium uniporter. Nature. [Epub: Ahead of Print]
Niescier, R.F., K. Hong, D. Park, and K.T. Min. (2018). MCU Interacts with Miro1 to Modulate Mitochondrial Functions in Neuron.s. J. Neurosci. 38: 4666-4677.
O'Brien, J.E. and M.H. Meisler. (2013). Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability. Front Genet 4: 213.
Oh, E.S., H.W. Ryu, Y.N. Song, M.J. Kang, Y.H. Huh, J.Y. Park, S.M. Oh, S.Y. Lee, Y.J. Park, D.Y. Kim, H. Ro, S.T. Hong, S.U. Lee, D.O. Moon, and M.O. Kim. (2023). Diplacone Isolated from Mature Fruit Induces Ferroptosis-Mediated Cell Death through Mitochondrial Ca Influx and Mitochondrial Permeability Transition. Int J Mol Sci 24:.
Oxenoid, K., Y. Dong, C. Cao, T. Cui, Y. Sancak, A.L. Markhard, Z. Grabarek, L. Kong, Z. Liu, B. Ouyang, Y. Cong, V.K. Mootha, and J.J. Chou. (2016). Architecture of the mitochondrial calcium uniporter. Nature 533: 269-273.
Ozkurede, U., S. Pakkiriswami, and J.C. Liu. (2024). MICUs protect the heart by regulating mitochondrial calcium. Trends Pharmacol Sci 45: 950-952.
Quan, X., T.T. Nguyen, S.K. Choi, S. Xu, R. Das, S.K. Cha, N. Kim, J. Han, A. Wiederkehr, C.B. Wollheim, and K.S. Park. (2015). Essential role of mitochondrial Ca²⁺ uniporter in the generation of mitochondrial pH gradient and metabolism-secretion coupling in insulin-releasing cells. J. Biol. Chem. 290: 4086-4096.
Raffaello, A., D. De Stefani, D. Sabbadin, E. Teardo, G. Merli, A. Picard, V. Checchetto, S. Moro, I. Szabò, and R. Rizzuto. (2013). The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. EMBO. J. 32: 2362-2376.
Sancak, Y., A.L. Markhard, T. Kitami, E. Kovács-Bogdán, K.J. Kamer, N.D. Udeshi, S.A. Carr, D. Chaudhuri, D.E. Clapham, A.A. Li, S.E. Calvo, O. Goldberger, and V.K. Mootha. (2013). EMRE is an essential component of the mitochondrial calcium uniporter complex. Science 342: 1379-1382.
Soontharapirakkul, K., W. Promden, N. Yamada, H. Kageyama, A. Incharoensakdi, A. Iwamoto-Kihara, and T. Takabe. (2011). Halotolerant cyanobacterium Aphanothece halophytica contains an Na⁺-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance. J. Biol. Chem. 286: 10169-10176.
Tsai, M.F., C.B. Phillips, M. Ranaghan, C.W. Tsai, Y. Wu, C. Willliams, and C. Miller. (2016). Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. Elife 5:.
Vais, H., R. Payne, U. Paudel, C. Li, and J.K. Foskett. (2020). Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca uptake. Proc. Natl. Acad. Sci. USA 117: 21731-21739.
Wang, C., R. Baradaran, and S.B. Long. (2020). Structure and Reconstitution of an MCU-EMRE Mitochondrial Ca Uniporter Complex. J. Mol. Biol. 432: 5632-5648.
Wang, H., M. Zhao, J. Chen, Y. Ren, G. Wang, W. Li, and F. Zou. (2018). Mitochondrial calcium uniporter-mediated inhibition of 1-methyl-4-phenylpyridinium ions neurotoxicity in PC12 cells. Neuroreport 29: 570-576.
Wang, X., H. Yang, W. Zhou, J. Liu, and N. Xu. (2019). Deletion of Affects ATP Synthase Function and Enhances Production of L-Valine in. J Microbiol Biotechnol 29: 1288-1298.
Wang, Y., L.H. Wu, F. Hou, Z.J. Wang, M.N. Wu, C. Hölscher, and H.Y. Cai. (2024). Mitochondrial calcium uniporter knockdown in hippocampal neurons alleviates anxious and depressive behavior in the 3XTG Alzheimer''s disease mouse model. Brain Res 149060. [Epub: Ahead of Print]
Xiao, R. and X.Z. Xu. (2009). Function and regulation of TRP family channels in C. elegans. Pflugers Arch 458: 851-860.
Xue, K., D. Wu, and Y. Qiu. (2023). Thermoporter: a new regulatory mechanism for mitochondrial calcium uniporter activity. Trends Cell Biol. 33: 182-184.
Yoo, J., M. Wu, Y. Yin, M.A. Herzik, Jr, G.C. Lander, and S.Y. Lee. (2018). Cryo-EM structure of a mitochondrial calcium uniporter. Science 361: 506-511.
Yuan, Y., C. Cao, M. Wen, M. Li, Y. Dong, L. Wu, J. Wu, T. Cui, D. Li, J.J. Chou, and B. OuYang. (2020). Structural Characterization of the N-Terminal Domain of the Mitochondrial Calcium Uniporter. ACS Omega 5: 6452-6460.
Zhang, G., L. Han, Z. Li, Y. Chen, Q. Li, S. Wang, and H. Shi. (2023). Screening of immunogenic proteins and evaluation of vaccine candidates against Mycoplasma synoviae. NPJ Vaccines 8: 121.
Examples:
TC#	Name	Organismal Type	Example
1.A.77.1.1	Inner membrane 40KD mitochondrial Ca²⁺channel-forming uniporter, MCU or MICU2 (DUF607; 350 aas; coiled coil domain protein 109 A) (De Stefani et al., 2011; Drago et al., 2011). It functions with MICU1, an essential component of the system, as well as the gatekeeper for Ca²⁺uptake (Mallilankaraman et al. 2012a; Mallilankaraman et al. 2012b). It contributes to metabolism-insulin secretion coupling in clonal pancreatic beta-cells (Alam et al. 2012). MCU-mediated Ca²⁺ uptake in beta cells is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells (Quan et al. 2015). The mitochondrial calcium uniporter of pulmonary type 2 cells determines the severity of acute lung injury (Islam et al. 2022). Regulation of mitochondrial calcium uptake by the mitochondrial calcium uniporter complex is crucial for heart function. It has been demonstrated that mitochondrial calcium uptake (MICU)1 and MICU2, regulatory subunits of the complex, help maintain calcium homeostasis in cardiac mitochondria, provide potential targets for therapies aimed at improving mitochondrial function in heart disease (Ozkurede et al. 2024).	Animals	MCU of Mus musculus (Q3UMR5)

1.A.77.1.10	MCU homologue (355 aas; 2 TMSs)	Bacteria	*MCU homologue of Chlorobium phaeobacteroides* (A1BIL6)**

1.A.77.1.11	Mitochondrial calcium uniporter, MCU, of 362 aas	Alveolata (Ciliates)	MCU of Tetrahymena thermophila

1.A.77.1.12	The mitochondrial calcium uniporter regulatory subunit MCUb of 336 aas; part of the MCU complex (Sancak et al. 2013). MCU regulates procoagulant platelet formation (Kholmukhamedov et al. 2018) and interacts with the c-subunit of the mitochondrial ATPase (Huang and Docampo 2020). It functions in animals (but not in fungi or protozoans) with another protein, EMRE or SMDT1 of 107 aas (TC# 8.A.45.1.1) that interacts with and renders the channel functional (MacEwen et al. 2020). The mitochondrial calcium uniporter of pulmonary type 2 cells determines the severity of acute lung injury (Islam et al. 2022). Metal coordination complexes, particularly multinuclear ruthenium complexes, are the most widely investigated MCU inhibitors due to both their potent inhibitory activities as well as their longstanding use for this application (Huang and Wilson 2023).	Animals	MCUb of Homo sapiens

1.A.77.1.13	*Mitochondrial calcium uniporter of 658 aas (Docampo et al.* 2013).**	Choanoflagellates	MCU of Monosiga brevicollis

1.A.77.1.14	Mitochondrial calcium uniporter of 297 aas.	Euglenozoa	MCU of Leishmania donovani

1.A.77.1.15	MCU of 488 aas and 2 TMSs. The 3.8 Å cryoEM structure has been solved (Nguyen et al. 2018). The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a structure similar to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn (Nguyen et al. 2018).		MCU of the fungus, Neosartorya fischeri

1.A.77.1.16	Mitochondrial calcium uniporter protein, MCU, of 302 aas and 2 TMSs. It interacts with subunit c of the ATP synthase (Huang and Docampo 2020).		MCU of Trypanosoma cruzi

1.A.77.1.17	CMU of 248 aas and 2 TMSs. It functions together with the EMRE regulatory protein (TC#8.A.45.1.6). MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans. Wang et al. 2020 functionally reconstituted an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and determined a cryo-EM structure of the complex at 3.5 Å resolution. They demonstrated Ca²⁺ uptake into proteoliposomes containing the purified complex. Uptake depended on EMRE as well as cardiolipin. The structure revealed a tetrameric channel with a single ion pore. EMRE was located at the periphery of the transmembrane domain and associates primarily with the first TMS of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels (Wang et al. 2020).		Mcu of Tribolium castaneum

1.A.77.1.2	MCU homologue of 338 aas and 2 TMSs.	Plants	*MCU homologue of Arabidopsis thaliana* (Q1PE15)**

1.A.77.1.3	Algal MCU homologue (300 aas; 2 TMSs)	Algae	*MCU homologue of Chlamydomonas reinhardtii* (A8J6W0)**

1.A.77.1.4	Slime mold MCU homologue of 275 aas and 2 TMSs. The structure of the N-terminal domain (NTD) has been solved at 1.7 A resolution (Yuan et al. 2020). The oligomeric DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. This domain may regulate channel activity (Yuan et al. 2020).	Slime molds	*MCU homologue of Dictyostellium discoideum* (Q54LT0)**

1.A.77.1.5	Fungal MCU homologue of 493 aas and 2 TMS. The cryo-electron microscopy structure of the full-length MCU to an overall resolution of ~3.7 Å has been determined (Yoo et al. 2018). The structure reveals a tetrameric architecture, with the soluble and transmembrane domains adopting different symmetric arrangements within the channel. The conserved W-D-Phi-Phi-E-P-V-T-Y sequence motif of the MCU pore forms a selectivity filter comprising two acidic rings separated by one helical turn along the central axis of the channel pore (Yoo et al. 2018).	Fungi	*MCU homologue of Neurospora crassa* (Q7S4I4)**

1.A.77.1.6	*MCU homologue of 355 aas; 4 TMSs (2+2) (Docampo et al.* 2013).**	Euglenozoa	*MCU homologue of Trypanosoma cruzi* (E7KWU4)**

1.A.77.1.7	Mitochondrial Ca²⁺ Uniporter, a channel complex. MCU is a putative 5TMS protein (307 aas) with homology to MCU Ca²⁺/Mg²⁺ channels in the C-terminal 2TMS domain. The N-terminal domain is found only in Trypanosoma and Leishmania species. The TbMCU complex possesses four subunits, MCU (307 aas), MCUb (214 aas), MCUc (254 aas) and MCUd (249 aas)), present only in trypanosomatids and required for Ca²⁺ transport. These four subunits interact through their transmembrane helices to form hetero-oligomers in a ~380 KDa complex (Huang and Docampo 2018).	Euglenozoa	Channel homologues of Trypanosoma brucei MCU MCUb MCUc MCUd

1.A.77.1.8	Ciliate MCU homologue 362 aas; 2 TMSs	Alveolata (Ciliates)	*MCU homologue of Paramecium tetraurelia* (A0E7U6)**

1.A.77.1.9	MCU homologue (766 aas; 2 TMSs)	Bacteria	*MCU homologue of Cytophaga hutchinsonii* (Q11Z39)**

Examples:
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TC#	Name	Organismal Type	Example
1.A.77.2.1	Putative Mg²⁺ transporter, AtpZ	Proteobacteria	*AtpZ of Helicobacter pylori* (Q1CUJ6)**

1.A.77.2.10	AtpZ homologue of 87 aas.	Proteobacteria	AtpZ of Hippea maritima

1.A.77.2.11	AtpZ homologue of 90 aas	Proteobacteria	AtpZ of Campylobacter curvus

1.A.77.2.12	AtpZ homologue of 60 aas	Euryarchaeota	AtpZ homologue of Methanothermococcus okinawensis

1.A.77.2.13	AtpZ homologue of 80 aas	Proteobacteria	AtpZ of Geobacter metallireducens

1.A.77.2.14	AtpZ homologue of 105 aas	Proteobacteria	AtpZ of Acidophilium multivorum

1.A.77.2.15	AtpZ homologue of 96 aas	Proteobacteria	AtpZ of Tistrella mobilis

1.A.77.2.16	*Putative Mg²⁺ channel of 113 aas and 2 TMSs. Part of the F-type ATPase (Morales-Rios et al.* 2015).**		Magnesium channel of Paracoccus denitrificans

1.A.77.2.17	*Putative Mg²⁺ channel, AtpI, that functions with a Na⁺-transporting F-type ATPase (Soontharapirakkul et al.* 2011).**		AtpI of Aphanothece halophytica

1.A.77.2.2	AtpZ homologue (125 aas; 2 TMSs)	Proteobacteria	*AtpZ homologue of Anaeromyxobacter* sp. Fw109-5 (A7HIX1)**

1.A.77.2.3	AtpZ of 92 aas	Bacteria	*AtpZ of Desulfovibrio vulgaris* (A1VF64)**

1.A.77.2.4	AtpZ of 106 aas	Bacteria	*AtpZ of Chlorobium tepidum* (Q8KGE5)**

1.A.77.2.5	AtpZ of 105 aas	Bacteria	*AtpZ of Rhodomicrobium vannielii* (E3I7U2)**

1.A.77.2.6	AtpZ of 108 aas	α-Proteobacteria	*AtpZ of Maricaulis maris* (Q0AMJ5)**

1.A.77.2.7	ATP synthase protein Z of 114 aas	Bacteria	AtpZ of Rhodobacter capsulatus

1.A.77.2.8	The Mg²⁺ uptake channel, AtpZ. Postulated to form homo- and/or hetero oligomers [(AtpZ)n-x (AtpI)x] (Hicks et al., 2003). The AtpI homologue (P22475) is in subfamily 1.A.77.3 and has TC# 1.A.77.3.1.	Bacteria	The AtpZI Mg²⁺/Ca²⁺ channel of Bacillus pseudofirmus AtpZ (Q9EXJ9)

1.A.77.2.9	AtpZ of 112 aas.	Archaea	*AtpI of Methanosarcina acetivorans* (Q8TN54)**

Examples:
TC#	Name	Organismal Type	Example
1.A.77.3.1	AtpI of 133 aas, This protein is a part of a two component channel and as such is also listed with TC# 1.A.77.2.8.	Firmicutes	AtpI of Bacillus pseudofirmus

1.A.77.3.10	AtpI homologue of 126 aas	Proteobacteria	AtpI of Ferrimonas balearica

1.A.77.3.11	AtpI of 126 aas	Proteobacteria	AtpI of E. coli

1.A.77.3.12	AtpI homologue of 185 aas	Proteobacteria	AtpI of Ralstonia solanacearum

1.A.77.3.13	ATP synthase protein I	Bacteria	AtpI of Mycoplasma gallisepticum )

1.A.77.3.14	ATP synthase I, AtpI	Bacteria	AtpI of Acetohalobium arabaticum

1.A.77.3.15	ATP synthase subunit I	Bacteria	AtpI of Geobacter uraniireducens

1.A.77.3.16	ATP synthase I	Bacteria	AtpI of Fervidobacterium pennivorans

1.A.77.3.17	*AtpI of the Na⁺ ATPase. Essential for assembly of the c-ring of the rotor (Brandt et al.* 2013).**	Firmicutes	AtpI of Acetobacterium woodii

1.A.77.3.18	AtpI homologue of 122 aas and 4 TMSs	Firmicutes	AtpI homologue of Clostridium sticklandii

1.A.77.3.19	AtpI homologue of 109 aas	Thermatogae	AtpI of Thermatoga thermarum

1.A.77.3.2	ATP synthase protein I	Bacilli	AtpI of Bacillus subtilis

1.A.77.3.20	ATP synthase, subunit I of 117 aas and 4 TMSs		AtpI of Staphylococcus aureus

1.A.77.3.21		Cyanobacteria	AtpI of Synechococcus sp.

1.A.77.3.22	ATP snthase subunit I, AtpI of 147 aas	Bacteria	AtpI of Halothermothrix orenii

1.A.77.3.23	ATP synthase, subunit I, AtpI of 153 aas	Bacteria	AtpI of Mycobacterium leprae

1.A.77.3.24	AtpI of 255 aas and 4 TM	Red Algae	AtpI of Galdieria sulfuraria

1.A.77.3.25	Putative AtpI of 122 aas and 4 TMSs	Deferribacteres	AtpI of Denitrovibrio acetophilus

1.A.77.3.26	Uncharacterized protein of 189 aas and 5 TMSs	Proteobacteria	UP of Anaeromyxobacter dehalogenans

1.A.77.3.27	AtpI homologue of 133 aas	Fusobacteria	AtpI of Leptotrichia buccalis

1.A.77.3.28	AtpI homologue of 126 aas	Fusobacteria	AtpI of Ilyobacter polytrophus

1.A.77.3.29	AtpI homologue of 127 aas	Fusobacteria	AtpI of Propionigenium modestum

1.A.77.3.3	ATP synthase subunit I	Bacteria	AtpI of Desulfococcus oleovorans

1.A.77.3.30	AtpI homologue of 135 aas	Fusobacteria	AtpI of Sebaldella termitidis

1.A.77.3.31	AtpI homologue of 164aas and 4 TMSs	Tenericutes	AtpI of Mycoplasma fermentans

1.A.77.3.32	AtpI homologue of 150 aas	Tenericutes	AtpI of Mycoplasma arthritidis

1.A.77.3.33	*AtpI homologue of 161 aas. Immunogenic proteins have been evaluated as vaccine candidates against Mycoplasma synoviae* (Zhang et al. 2023).**	Tenericutes	AtpI of Mycoplasma synoviae

1.A.77.3.34	AtpI of 140 aas and 4 TMSs	Proteobacteria	AtpI of Desulfotalea psychrophila

1.A.77.3.35	Putative AtpI of 129 aas and 4 TMSs	Firmicutes	AtpI of Heliobacterium modesticaldum

1.A.77.3.36	Putative AtpI of 139 aas and 4 TMSs	Acidobacteria	AtpI of Candidatus Koribacter versatilis

1.A.77.3.37	Putative AtpI of 133 aas and 4 TMSs	Proteobacteria	AtpI of Desulfobacula toluolica

1.A.77.3.38	Putative AtpI of 140 aas and 4 TMSs	Proteobacteria	AtpI of Syntrophus aciditrophicus

1.A.77.3.39	Putative AtpI of 256 aas and 4 or 5 TMSs. The N-terminus may include a single TMS plus a hydrophilic domain before the C-terminal AtpI domain.	Rhodophyta (Eukaryote)	AtpI of Chondrus crispus

1.A.77.3.4	ATP synthase protein I	Gram-positive bacteria	AtpI of Bacillus megaterium

1.A.77.3.40	AtpI of 156 aas and 4 TMSs	Rhizaria (Eukaryote)	AtpI of Paulinella chromatophora

1.A.77.3.41	Putative AtpI of 119 aas and 4 TMSs	Deferribacteres	AtpI of Deferribacter desulfuricans

1.A.77.3.42	Putative AtpI of 138 aas and 4 TMSs	Acidobacteria	AtpI of Granulicella tundricola

1.A.77.3.43	Putative AtpI of 121 aas and 4 TMSs	Nitrospirae	AtpI of Thermodesulfovibrio yellowstonii

1.A.77.3.44	Putative AtpI of 160 aas and 4 TMSs	Tenericutes	AtpI of Mycoplasma mobile

1.A.77.3.45	ATP synthase I-like protein, AtpI, of 385 aas amd 3 - 4 TMSs.		AtpI of Chlamydomonas reinhardtii (Chlamydomonas smithii)

1.A.77.3.5	AtpI homologue	Firmicutes	AtpI homologue of Coprococcus catus

1.A.77.3.6	AtpI homologue of 122aas and 4 TMSs	Firmicutes	AtpI homologue of Paenibacillus mucilaginosus

1.A.77.3.7	ATP synthase protein I	Bacteria	AtpI of Vibrio cholerae serotype O1

1.A.77.3.8	AtpI homologue of 150 aas	Proteobacteria	AtpI of Klebsiella pneumoniae

1.A.77.3.9	AtpI homologue of 135 aas	Proteobacteria	AtpI of Pseudomonas putida

Examples:
TC#	Name	Organismal Type	Example
1.A.77.4.1	Fusion protein with N-terminal 4 TMS AtpI domain and large soluble C-terminal α/β-hydrolase domain.	Algae	Fusion protein of Galdieria sulphuraria

1.A.77.4.2	Fusion protein with N-terminal 4 TMS AtpI domain and large soluble C-terminal α/β-hydrolase domain.	Slime molds	Fusion protein of Dictyostelium discoideum

1.A.77.4.3	Fusion protein with N-terminal 4 TMS AtpI domain and large soluble C-terminal α/β-hydrolase domain.	Amoeba	Fusion protein of Entamoeba histolytica

Examples:
TC#	Name	Organismal Type	Example
1.A.77.5.1	AtpI homologue of 147 aas and 4 TMSs. Deletion of the gene encoding the ortholog, cg1360, affects ATP synthase function and enhances production of L-Valine in Corynebacterium glutamicum (Wang et al. 2019).	Actinobacteria	AtpI of Corynebacterium diphtheriae

1.A.77.5.2	AtpI homologue of 137 aas	Actinobacteria	AtpI of Streptomyces avermitilis

1.A.77.5.3	AtpI homologue of 145 aas	Actinobacteria	AtpI of Frankia alni

1.A.77.5.4	Putative AtpI of 177 aas and 4 TMSs		AtpI of Saccharomonospora cyanea

1.A.77.5.5	Putative ATP synthase protein I2 of 161 aas and 4 TMSs		Putative reductase of Actinokineospora spheciospongiae

1.A.77.5.6	Uncharacterized protein of 157 aas and 3-4 TMSs		UP of Cellulomonas fimi

Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example