Perturbation of mitochondrial Ca²⁺ homeostasis activates cross-compartmental proteostatic response in Arabidopsis

Abstract

Cross-compartment communication is critical for maintaining cellular homeostasis, which is essential for cell function and survival under stressful conditions. However, the cellular cues that trigger interorganellar communication remain poorly understood. Mitochondrial Ca²⁺ (_mtCa²⁺) homeostasis is fundamental to mitochondrial function; yet, how mitochondrial Ca²⁺ (_mtCa²⁺) homeostasis modulates nuclear gene expression to establish and maintain cellular homeostasis remains unclear. Here, we first characterize the critical role of the mitochondrial Ca²⁺ uniporter (MCU) in control of _mtCa²⁺ uptake and maintaining _mtCa²⁺ homeostasis in planta. Using gain-of-function and sextuple MCU knockdown mutants, we then analyzed the effects of impaired MCU-controlled _mtCa²⁺ homeostasis (iMUCH). We find that iMUCH elicits an interorganellar transcription program that activates multiple compartment-specific unfolded protein responses (UPRs) and genes critical for mitochondrial and cytosolic proteostasis. Additionally, iMUCH induces a post-transcriptional program that selectively represses the synthesis of ribosomes and RNA modification proteins. Furthermore, eukaryotic initiation factor α (eIFα and its phosphorylation likely serve as a protective mechanism under long-term mitochondrial proteotoxic stress induced by iMUCH. Collectively, the data demonstrate that MCU-controlled _mtCa²⁺ homeostasis plays a pivotal role in sustaining mitochondrial and cytosolic proteostasis through an interconnected organelle quality control system, which ultimately determines cell growth and fitness.

Keywords

Mitochondrial Ca²⁺ homeostasis / Mitochondrial Ca²⁺ uniporter / Mitochondrial retrograde signaling / UPR^mt / Intercompartmental proteostasis

References

[1] Andreasson C, Ott M, Buttner S. Mitochondria orchestrate proteostatic and metabolic stress responses. EMBO Rep, 2019, 20(10): e47865

[2] Antonicka H, Lin ZY, Janer A, Aaltonen MJ, Weraarpachai W, Gingras AC, Shoubridge EA. A high-density human mitochondrial proximity interaction network. Cell Metab, 2020, 32(3): 479-497

[3] Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y, Bao XR, Strittmatter L, Goldberger O, Bogorad RL, et al.. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature, 2011, 476(7360): 341-345

[4] Bernardi P, Rasola A. Calcium and cell death: the mitochondrial connection. Subcell Biochem, 2007, 45: 481-506

[5] Boos F, Kramer L, Groh C, Jung F, Haberkant P, Stein F, Wollweber F, Gackstatter A, Zoller E, van der Laan M, et al.. Mitochondrial protein-induced stress triggers a global adaptive transcriptional programme. Nat Cell Biol, 2019, 21(4): 442-451

[6] Boos F, Labbadia J, Herrmann JM. How the mitoprotein-induced stress response safeguards the cytosol: a unified view. Trends Cell Biol, 2020, 30(3): 241-254

[7] Butow RA, Avadhani NG. Mitochondrial signaling: the retrograde response. Mol Cell, 2004, 14(1): 1-15

[8] Calvo-Rodriguez M, Hou SS, Snyder AC, Kharitonova EK, Russ AN, Das S, Fan Z, Muzikansky A, Garcia-Alloza M, Serrano-Pozo A, et al.. Increased mitochondrial calcium levels associated with neuronal death in a mouse model of Alzheimer's disease. Nat Commun, 2020, 11(1): 2146

[9] Carraretto L, Teardo E, Checchetto V, Finazzi G, Uozumi N, Szabo I. Ion channels in plant bioenergetic organelles, chloroplasts, and mitochondria: from molecular identification to function. Mol Plant, 2016, 9(3): 371-395

[10] Couvillion MT, Soto IC, Shipkovenska G, Churchman LS. Synchronized mitochondrial and cytosolic translation programs. Nature, 2016, 533(7604): 499-503

[11] D'Amico D, Sorrentino V, Auwerx J. Cytosolic proteostasis networks of the mitochondrial stress response. Trends Biochem Sci, 2017, 42(9): 712-725

[12] de Souza A, Wang JZ, Dehesh K. Retrograde signals: Integrators of interorganellar communication and orchestrators of plant development. Annu Rev Plant Biol, 2017, 68(68): 85-108

[13] De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature, 2011, 476(7360): 336-340

[14] Fan M, Zhang J, Tsai CW, Orlando BJ, Rodriguez M, Xu Y, Liao M, Tsai MF, Feng L. Structure and mechanism of the mitochondrial Ca²⁺ uniporter holocomplex. Nature, 2020, 582(7810): 129-133

[15] Fessler E, Eckl EM, Schmitt S, Mancilla IA, Meyer-Bender MF, Hanf M, Philippou-Massier J, Krebs S, Zischka H, Jae LT. A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature, 2020, 579(7799): 433-437

[16] Garbincius JF, Elrod JW. Mitochondrial calcium exchange in physiology and disease. Physiol Rev, 2022, 102(2): 893-992

[17] Gerst JE. Pimp my ribosome: ribosomal protein paralogs specify translational control. Trends Genet, 2018, 34(11): 832-845

[18] Giacomello M, Drago I, Pizzo P, Pozzan T. Mitochondrial Ca²⁺ as a key regulator of cell life and death. Cell Death Differ, 2007, 14(7): 1267-1274

[19] Giorgio V, Guo L, Bassot C, Petronilli V, Bernardi P. Calcium and regulation of the mitochondrial permeability transition. Cell Calcium, 2018, 70: 56-63

[20] Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, Campisi J, Collado M, Evangelou K, Ferbeyre G, et al.. Cellular senescence: defining a path forward. Cell, 2019, 179(4): 813-827

[21] Griffiths EJ, Rutter GA. Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells. Biochim Biophys Acta, 2009, 1787(11): 1324-1333

[22] Guo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT, Wiita AP, Xu K, Correia MA, Kampmann M. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature, 2020, 579(7799): 427-432

[23] Harding HP, Novoa I, Zhang YH, Zeng HQ, Wek R, Schapira M, Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell, 2000, 6(5): 1099-1108

[24] Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature, 2011, 475(7356): 324-332

[25] Hoffman NE, Chandramoorthy HC, Shamugapriya S, Zhang X, Rajan S, Mallilankaraman K, Gandhirajan RK, Vagnozzi RJ, Ferrer LM, Sreekrishnanilayam K, et al.. MICU1 motifs define mitochondrial calcium uniporter binding and activity. Cell Rep, 2013, 5(6): 1576-1588

[26] Horwich AL. Molecular chaperones in cellular protein folding: the birth of a field. Cell, 2014, 157(2): 285-288

[27] Houtkooper RH, Mouchiroud L, Ryu D, Moullan N, Katsyuba E, Knott G, Williams RW, Auwerx J. Mitonuclear protein imbalance as a conserved longevity mechanism. Nature, 2013, 497(7450): 451-457

[28] Huang LS, Cobessi D, Tung EY, Berry EA. Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc(1) complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern. J Mol Biol, 2005, 351(3): 573-597

[29] Ivanova A, Law SR, Narsai R, Duncan O, Lee JH, Zhang BT, Van Aken O, Radomiljac JD, van der Merwe M, Yi KK, et al.. A functional antagonistic relationship between auxin and mitochondrial retrograde signaling regulates alternative oxidase1a expression in Arabidopsis. Plant Physiol, 2014, 165(3): 1233-1254

[30] Kamer KJ, Mootha VK. MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter. EMBO Rep, 2014, 15(3): 299-307

[31] Khan NA, Nikkanen J, Yatsuga S, Jackson C, Wang L, Pradhan S, Kivela R, Pessia A, Velagapudi V, Suomalainen A (2017) mTORC1 regulates mitochondrial integrated stress response and mitochondrial myopathy progression. Cell Metab 26(2):419–428 e415. https://doi.org/10.1016/j.cmet.2017.07.007

[32] Kim HE, Grant AR, Simic MS, Kohnz RA, Nomura DK, Durieux J, Riera CE, Sanchez M, Kapernick E, Wolff S, Dillin A (2016) Lipid biosynthesis coordinates a mitochondrial-to-cytosolic stress response. Cell 166(6):1539–1552 e1516. https://doi.org/10.1016/j.cell.2016.08.027

[33] Knight H, Trewavas AJ, Knight MR. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell, 1996, 8(3): 489-503

[34] Knight H, Trewavas AJ, Knight MR. Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J, 1997, 12(5): 1067-1078

[35] Kuhl I, Miranda M, Atanassov I, Kuznetsova I, Hinze Y, Mourier A, Filipovska A, Larsson NG (2017) Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals. Elife 6:e30952. https://doi.org/10.7554/eLife.30952

[36] Kwasniak M, Majewski P, Skibior R, Adamowicz A, Czarna M, Sliwinska E, Janska H. Silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein alters translation in Arabidopsis mitochondria. Plant Cell, 2013, 25(5): 1855-1867

[37] Kwasniak-Owczarek M, Kazmierczak U, Tomal A, Mackiewicz P, Janska H. Deficiency of mitoribosomal S10 protein affects translation and splicing in Arabidopsis mitochondria. Nucleic Acids Res, 2019, 47(22): 11790-11806

[38] Liu JC, Syder NC, Ghorashi NS, Willingham TB, Parks RJ, Sun J, Fergusson MM, Liu J, Holmström KM, Menazza S, Springer DA, Liu C, Glancy B, Finkel T, Murphy E (2020) EMRE is essential for mitochondrial calcium uniporter activity in a mouse model. JCI Insight 5(4):e134063. https://doi.org/10.1172/jci.insight.134063

[39] Logan DC, Knight MR. Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants. Plant Physiol, 2003, 133(1): 21-24

[40] Lokdarshi A, Guan J, Camacho RAU, Cho SK, Morgan PW, Leonard M, Shimono M, Day B, von Arnim AG. Light activates the translational regulatory kinase GCN2 via reactive oxygen species emanating from the chloroplast. Plant Cell, 2020, 32(4): 1161-1178

[41] Loro G, Drago I, Pozzan T, Schiavo FL, Zottini M, Costa A. Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²⁺ handling relationship in plant cells. Plant J, 2012, 71(1): 1-13

[42] Ma J, Chen T, Wu SF, Yang CY, Bai MZ, Shu KX, Li KL, Zhang GQ, Jin Z, He FC, et al.. iProX: an integrated proteome resource. Nucleic Acids Res, 2019, 47(D1): D1211-D1217

[43] McAinsh MR, Pittman JK. Shaping the calcium signature. New Phytol, 2009, 181(2): 275-294

[44] Mehlmer N, Parvin N, Hurst CH, Knight MR, Teige M, Vothknecht UC. A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana. J Exp Bot, 2012, 63(4): 1751-1761

[45] Merkwirth C, Jovaisaite V, Durieux J, Matilainen O, Jordan SD, Quiros PM, Steffen KK, Williams EG, Mouchiroud L, Tronnes SU, et al.. Two conserved histone demethylases regulate mitochondrial stress-induced longevity. Cell, 2016, 165(5): 1209-1223

[46] Missra A, von Arnim AG. Analysis of mRNA translation states in Arabidopsis over the diurnal cycle by polysome microarray. Methods Mol Biol, 2014, 1158: 157-174

[47] Molenaars M, Janssens GE, Williams EG, Jongejan A, Lan J, Rabot S, Joly F, Moerland PD, Schomakers BV, Lezzerini M, et al.. A conserved mito-cytosolic translational balance links two longevity pathways. Cell Metab, 2020, 31(3): 549-563

[48] Mottis A, Herzig S, Auwerx J. Mitocellular communication: shaping health and disease. Science, 2019, 366(6467): 827-832

[49] Munch C, Harper JW. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature, 2016, 534(7609): 710-713

[50] Mustroph A, Juntawong P, Bailey-Serres J. Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods. Methods Mol Biol, 2009, 553: 109-126

[51] Nandagopal N, Roux PP. Regulation of global and specific mRNA translation by the mTOR signaling pathway. Translation, 2015, 3(1): e983402

[52] Ng S, Ivanova A, Duncan O, Law SR, Van Aken O, De Clercq I, Wang Y, Carrie C, Xu L, Kmiec B, et al.. A membrane-bound NAC transcription factor, ANAC017, mediates mitochondrial retrograde signaling in Arabidopsis. Plant Cell, 2013, 25(9): 3450-3471

[53] Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO Rep, 2016, 17(10): 1374-1395

[54] Quiros PM, Mottis A, Auwerx J. Mitonuclear communication in homeostasis and stress. Nat Rev Mol Cell Biol, 2016, 17(4): 213-226

[55] Quiros PM, Prado MA, Zamboni N, D'Amico D, Williams RW, Finley D, Gygi SP, Auwerx J. Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J Cell Biol, 2017, 216(7): 2027-2045

[56] Resentini F, Ruberti C, Grenzi M, Bonza MC, Costa A. The signatures of organellar calcium. Plant Physiol, 2021, 187(4): 1985-2004

[57] Richter U, Lahtinen T, Marttinen P, Myohanen M, Greco D, Cannino G, Jacobs HT, Lietzen N, Nyman TA, Battersby BJ. A mitochondrial ribosomal and RNA decay pathway blocks cell proliferation. Curr Biol, 2013, 23(6): 535-541

[58] Rizzuto R, De Stefani D, Raffaello A, Mammucari C. Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol, 2012, 13(9): 566-578

[59] Ruberti C, Feitosa-Araujo E, Xu Z, Wagner S, Grenzi M, Darwish E, Lichtenauer S, Fuchs P, Parmagnani AS, Balcerowicz D, Schoenaers S, de la Torre C, Mekkaoui K, Nunes-Nesi A, Wirtz M, Vissenberg K, Van Aken O, Hause B, Costa A, Schwarzländer M (2022) MCU proteins dominate in vivo mitochondrial Ca²⁺ uptake in Arabidopsis roots. Plant Cell 34(11):4428-4452. https://doi.org/10.1093/plcell/koac242

[60] Samluk L, Urbanska M, Kisielewska K, Mohanraj K, Kim MJ, Machnicka K, Liszewska E, Jaworski J, Chacinska A. Cytosolic translational responses differ under conditions of severe short-term and long-term mitochondrial stress. Mol Biol Cell, 2019, 30(15): 1864-1877

[61] Sancak Y, Markhard AL, Kitami T, Kovacs-Bogdan E, Kamer KJ, Udeshi ND, Carr SA, Chaudhuri D, Clapham DE, Li AA, et al.. EMRE is an essential component of the mitochondrial calcium uniporter complex. Science, 2013, 342(6164): 1379-1382

[62] Selles B, Michaud C, Xiong TC, Leblanc O, Ingouff M. Arabidopsis pollen tube germination and growth depend on the mitochondrial calcium uniporter complex. New Phytol, 2018, 219(1): 58-65

[63] Shi Z, Fujii K, Kovary KM, Genuth NR, Rost HL, Teruel MN, Barna M. Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide. Mol Cell, 2017, 67(1): 71-83

[64] Slater EC. The mechanism of action of the respiratory inhibitor, antimycin. Biochim Biophys Acta, 1973, 301(2): 129-154

[65] St-Pierre J, Topisirovic I. Nucleus to mitochondria: lost in transcription, found in translation. Dev Cell, 2016, 37(6): 490-492

[66] Suhm T, Kaimal JM, Dawitz H, Peselj C, Masser AE, Hanzén S, Ambrožič M, Smialowska A, Björck ML, Brzezinski P, Nyström T, Büttner S, Andréasson C, Ott M (2018) Mitochondrial translation efficiency controls cytoplasmic protein homeostasis. Cell Metab 27(6):1309–1322 e1306. https://doi.org/10.1016/j.cmet.2018.04.011

[67] Sun L, Xu Y, Bai S, Bai X, Zhu H, Dong H, Wang W, Zhu X, Hao F, Song CP. Transcriptome-wide analysis of pseudouridylation of mRNA and non-coding RNAs in Arabidopsis. J Exp Bot, 2019, 70(19): 5089-5600

[68] Sun F, Zhang Y-F, Jiang P-P, Li Y, Cao S-K, Xu C-H, Wang Y. Advances and prospects of plant mitochondrial pentatricopeptide repeat proteins in post-transcriptional processing. New Crops, 2024, 2: 100063

[69] Teardo E, Carraretto L, Wagner S, Formentin E, Behera S, De Bortoli S, Larosa V, Fuchs P, Lo Schiavo F, Raffaello A, et al.. Physiological characterization of a plant mitochondrial calcium uniporter in vitro and in vivo. Plant Physiol, 2017, 173(2): 1355-1370

[70] Teardo E, Carraretto L, Moscatiello R, Cortese E, Vicario M, Festa M, Maso L, De Bortoli S, Cali T, Vothknecht UC, et al.. A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis. Nat Plants, 2019, 5(6): 581-588

[71] Topf U, Suppanz I, Samluk L, Wrobel L, Boser A, Sakowska P, Knapp B, Pietrzyk MK, Chacinska A, Warscheid B. Quantitative proteomics identifies redox switches for global translation modulation by mitochondrially produced reactive oxygen species. Nat Commun, 2018, 9: 324

[72] Topf U, Uszczynska-Ratajczak B, Chacinska A (2019) Mitochondrial stress-dependent regulation of cellular protein synthesis. J Cell Sci 132(8):jcs226258. https://doi.org/10.1242/jcs.226258

[73] Van Aken O, Zhang B, Carrie C, Uggalla V, Paynter E, Giraud E, Whelan J. Defining the mitochondrial stress response in Arabidopsis thaliana. Mol Plant, 2009, 2(6): 1310-1324

[74] Wagner S, Behera S, De Bortoli S, Logan DC, Fuchs P, Carraretto L, Teardo E, Cendron L, Nietzel T, Fussl M, et al.. The EF-Hand Ca²⁺ binding protein MICU choreographs mitochondrial Ca²⁺ dynamics in Arabidopsis. Plant Cell, 2015, 27(11): 3190-3212

[75] Waltz F, Nguyen TT, Arrive M, Bochler A, Chicher J, Hammann P, Kuhn L, Quadrado M, Mireau H, Hashem Y, et al.. Small is big in Arabidopsis mitochondrial ribosome. Nat Plants, 2019, 5(1): 106-117

[76] Wang X, Chen XJ. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature, 2015, 524(7566): 481-484

[77] Wang LJ, Li HH, Zhao CZ, Li SF, Kong LY, Wu WW, Kong WS, Liu Y, Wei YY, Zhu JK, et al.. The inhibition of protein translation mediated by AtGCN1 is essential for cold tolerance in Arabidopsis thaliana. Plant, Cell Environ, 2017, 40(1): 56-68

[78] Wang X, Auwerx J (2017) Systems phytohormone responses to mitochondrial proteotoxic stress. Mol Cell 68(3):540–551 e545. https://doi.org/10.1016/j.molcel.2017.10.006

[79] Wang Y, Nguyen NX, She J, Zeng W, Yang Y, Bai XC, Jiang Y (2019) Structural mechanism of EMRE-dependent gating of the human mitochondrial calcium uniporter. Cell 177(5):1252–1261 e1213. https://doi.org/10.1016/j.cell.2019.03.050

[80] Wikstrom MK, Berden JA. Oxidoreduction of cytochrome b in the presence of antimycin. Biochim Biophys Acta, 1972, 283(3): 403-420

[81] Wrobel L, Topf U, Bragoszewski P, Wiese S, Sztolsztener ME, Oeljeklaus S, Varabyova A, Lirski M, Chroscicki P, Mroczek S, et al.. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature, 2015, 524(7566): 485-488

[82] Zhao Y, Wang X, Lei Q, Zhang X, Wang Y, Ji H, Ma C, Wang P, Song CP, Zhu X. The SnRK1-JMJ15-CRF6 module integrates energy and mitochondrial signaling to balance growth and the oxidative stress response in Arabidopsis. New Phytol, 2025, 246(1): 158-175

[83] Zhu JK. Abiotic stress signaling and responses in plants. Cell, 2016, 167(2): 313-324

[84] Zhu X, Feng Y, Liang G, Liu N, Zhu JK. Aequorin-based luminescence imaging reveals stimulus- and tissue-specific Ca²⁺ dynamics in Arabidopsis plants. Mol Plant, 2013, 6(2): 444-455

Funding

National Natural Science Foundation of China(32188102)

Guangdong S&T Program

National Key R&D Program of China

Shenzhen Science and Technology Program

RIGHTS & PERMISSIONS

The Author(s)