Skip to main content
SpringerLink
Log in

The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively

Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price includes VAT (France)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Anderson JM, Charbonneau H, Jones HP, McCann RO, Cormier MJ (1980) Characterization of the plant nicotinamide adenine dinucleotide kinase activator protein and its identification as calmodulin. Biochemistry 19(13):3113–3120

    Article  PubMed  CAS  Google Scholar 

  • Azimzadeh J, Nacry P, Christodoulidou A, Drevensek S, Camilleri C, Amiour N, Parcy F, Pastuglia M, Bouchez D (2008) Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin. Plant Cell 20(8):2146–2159. doi:10.1105/tpc.107.056812

    Article  PubMed  CAS  Google Scholar 

  • Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1(1):11–21. doi:10.1038/3503603535036035

    Article  PubMed  CAS  Google Scholar 

  • Biro RL, Daye S, Serlin BS, Terry ME, Datta N, Sopory SK, Roux SJ (1984) Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution. Plant Physiol 75(2):382–386

    Article  PubMed  CAS  Google Scholar 

  • Boavida LC, Borges F, Becker JD, Feijo JA (2011) Whole genome analysis of gene expression reveals coordinated activation of signaling and metabolic pathways during pollen-pistil interactions in Arabidopsis. Plant Physiol 155(4):2066–2080. doi:10.1104/pp.110.169813

  • Bouche N, Yellin A, Snedden WA, Fromm H (2005) Plant-specific calmodulin-binding proteins. Annu Rev Plant Biol 56:435–466. doi:10.1146/annurev.arplant.56.032604.144224

    Article  PubMed  CAS  Google Scholar 

  • Bussemer J, Chigri F, Vothknecht UC (2009) Arabidopsis ATPase family gene 1-like protein 1 is a calmodulin-binding AAA-ATPase with a dual localization in chloroplasts and mitochondria. FEBS J 276(14):3870–3880. doi:10.1111/j.1742-4658.2009.07102.x

    Article  PubMed  CAS  Google Scholar 

  • Chiasson D, Ekengren SK, Martin GB, Dobney SL, Snedden WA (2005) Calmodulin-like proteins from Arabidopsis and tomato are involved in host defense against Pseudomonas syringae pv. tomato. Plant Mol Biol 58(6):887–897. doi:10.1007/s11103-005-8395-x

  • Chigri F, Soll J, Vothknecht UC (2005) Calcium regulation of chloroplast protein import. Plant J 42(6):821–831. doi:10.1111/j.1365-313X.2005.02414.x

    Article  PubMed  CAS  Google Scholar 

  • Chigri F, Hormann F, Stamp A, Stammers DK, Bolter B, Soll J, Vothknecht UC (2006) Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32. Proc Natl Acad Sci USA 103(43):16051–16056. doi:10.1073/pnas.0607150103

    Article  PubMed  CAS  Google Scholar 

  • Clapham DE (2007) Calcium signaling. Cell 131(6):1047–1058. doi:10.1016/j.cell.2007.11.028

    Article  PubMed  CAS  Google Scholar 

  • Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122(2):383–384

    Article  PubMed  CAS  Google Scholar 

  • DeFalco TA, Bender KW, Snedden WA (2010) Breaking the code: Ca2+ sensors in plant signalling. Biochem J 425:27–40. doi:10.1042/Bj20091147

    Article  CAS  Google Scholar 

  • Dobney S, Chiasson D, Lam P, Smith SP, Snedden WA (2009) The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J Biol Chem 284(46):31647–31657. doi:10.1074/jbc.M109.056770

    Article  PubMed  CAS  Google Scholar 

  • Dohmann EMN, Levesque MP, De Veylder L, Reichardt I, Jürgens G, Schmid M, Schwechheimer C (2008a) The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability. Development 135(11):2013–2022. doi:10.1242/dev.020743

    Google Scholar 

  • Dohmann EMN, Levesque MP, Isono E, Schmid M, Schwechheimer C (2008b) Auxin responses in mutants of the Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome. Plant Physiol 147(3):1369–1379. doi:10.1104/pp.108.121061

    Google Scholar 

  • Foreman J, Demidchik V, Bothwell JH, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JD, Davies JM, Dolan L (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422(6930):442–446. doi:10.1038/nature01485

    Article  PubMed  CAS  Google Scholar 

  • Garrigos M, Deschamps S, Viel A, Lund S, Champeil P, Moller JV, Lemaire M (1991) Detection of Ca2+ -binding proteins by electrophoretic migration in the presence of Ca2+ combined with Ca-45(2+) overlay of protein blots. Anal Biochem 194(1):82–88

    Article  PubMed  CAS  Google Scholar 

  • Girzalsky W, Saffian D, Erdmann R (2010) Peroxisomal protein translocation. Bba-Mol Cell Res 1803(6):724–731. doi:10.1016/j.bbamcr.2010.01.002

    CAS  Google Scholar 

  • Gopalakrishna R, Anderson WB (1982) Ca2+ -induced hydrophobic site on calmodulin—application for purification of calmodulin by phenyl-sepharose affinity-chromatography. Biochem Biophys Res Commun 104(2):830–836

    Article  PubMed  CAS  Google Scholar 

  • Hanada K, Kuromori T, Myouga F, Toyoda T, Li WH, Shinozaki K (2009) Evolutionary persistence of functional compensation by duplicate genes in Arabidopsis. Genome Biol Evol 1:409–414. doi:10.1093/gbe/evp043

    Article  PubMed  Google Scholar 

  • Huang SB, Taylor NL, Whelan J, Millar AH (2009) Refining the definition of plant mitochondrial presequences through analysis of sorting signals, n-terminal modifications, and cleavage motifs. Plant Physiol 150(3):1272–1285. doi:10.1104/pp.109.137885

    Article  PubMed  CAS  Google Scholar 

  • Jarrett HW, Brown CJ, Black CC, Cormier MJ (1982) Evidence that calmodulin is in the chloroplast of peas and serves a regulatory role in photosynthesis. J Biol Chem 257(22):13795–13804

    PubMed  CAS  Google Scholar 

  • Koop HU, Steinmuller K, Wagner H, Rossler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199(2):193–201

    Article  PubMed  CAS  Google Scholar 

  • Kretsinger RH, Nockolds CE (1973) Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem 248(9):3313–3326

    PubMed  CAS  Google Scholar 

  • Kuhn S, Bussemer J, Chigri F, Vothknecht UC (2009) Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria. Plant J 58(4):694–705. doi:10.1111/j.1365-313X.2009.03810.x

    Article  PubMed  CAS  Google Scholar 

  • Lewit-Bentley A, Rety S (2000) EF-hand calcium-binding proteins. Curr Opin Struct Biol 10(6):637–643. doi:S0959-440X(00)00142-1

    Article  PubMed  CAS  Google Scholar 

  • Liang L, Flury S, Kalck V, Hohn B, Molinier J (2006) CENTRIN2 interacts with the Arabidopsis homolog of the human XPC protein (AtRAD4) and contributes to efficient synthesis-dependent repair of bulky DNA lesions. Plant Mol Biol 61(1–2):345–356. doi:10.1007/s11103-006-0016-9

    Article  PubMed  CAS  Google Scholar 

  • Ma W, Smigel A, Tsai YC, Braam J, Berkowitz GA (2008) Innate immunity signaling: cytosolic Ca2+ elevation is linked to downstream nitric oxide generation through the action of calmodulin or a calmodulin-like protein. Plant Physiol 148(2):818–828. doi:10.1104/pp.108.125104

    Article  PubMed  CAS  Google Scholar 

  • Magnan F, Ranty B, Charpenteau M, Sotta B, Galaud JP, Aldon D (2008) Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thaliana, alter plant responses to abiotic stress and abscisic acid. Plant J 56(4):575–589. doi:10.1111/j.1365-313X.2008.03622.x

    Article  PubMed  CAS  Google Scholar 

  • Maune JF, Klee CB, Beckingham K (1992) Ca2+ binding and conformational change in 2 series of point mutations to the individual Ca2+ -binding sites of calmodulin. J Biol Chem 267(8):5286–5295

    PubMed  CAS  Google Scholar 

  • McCormack E, Braam J (2003) Calmodulins and related potential calcium sensors of Arabidopsis. New Phytol 159(3):585–598. doi:10.1046/j.1469-8137.2003.00845.x

    Article  CAS  Google Scholar 

  • Miernyk JA, Fang TK, Randall DD (1987) Calmodulin antagonists inhibit the mitochondrial pyruvate dehydrogenase complex. J Biol Chem 262(32):15338–15340

    PubMed  CAS  Google Scholar 

  • Mokranjac D, Neupert W (2009) Thirty years of protein translocation into mitochondria: unexpectedly complex and still puzzling. Bba-Mol Cell Res 1793(1):33–41. doi:10.1016/j.bbamcr.2008.06.021

    CAS  Google Scholar 

  • Molinier J, Ramos C, Fritsch O, Hohn B (2004) CENTRIN2 modulates homologous recombination and nucleotide excision repair in Arabidopsis. Plant Cell 16(6):1633–1643. doi:10.1105/Tpc.021378

    Article  PubMed  CAS  Google Scholar 

  • Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51(6):1126–1136. doi:10.1111/j.1365-313X.2007.03212.x

    Article  PubMed  CAS  Google Scholar 

  • Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A, Eisenhaber F (2003) Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J Mol Biol 328(3):581–592. doi:S002228360300319X

    Article  PubMed  CAS  Google Scholar 

  • Park H, Park C, Koo S, Cheong M, Kim K, Kim M, Lim C, Lee S, Yun D-J, Chung W (2010) AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana. Plant Cell Rep 29(11):1297–1304. doi:10.1007/s00299-010-0916-7

    Article  PubMed  CAS  Google Scholar 

  • Potocky M, Jones MA, Bezvoda R, Smirnoff N, Zarsky V (2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol 174(4):742–751. doi:10.1111/j.1469-8137.2007.02042.x

    Article  PubMed  CAS  Google Scholar 

  • Pou De Crescenzo MA, Gallais S, Leon A, Laval-Martin DL (2001) Tween-20 activates and solubilizes the mitochondrial membrane-bound, calmodulin dependent NAD+ finase of Avena sativa L. J Membr Biol 182(2):135–146

    Article  PubMed  CAS  Google Scholar 

  • Reddy VS, Ali GS, Reddy AS (2002) Genes encoding calmodulin-binding proteins in the Arabidopsis genome. J Biol Chem 277(12):9840–9852. doi:10.1074/jbc.M111626200M111626200

    Article  PubMed  CAS  Google Scholar 

  • Reumann S, Ma C, Lemke S, Babujee L (2004) AraPerox. A database of putative Arabidopsis proteins from plant peroxisomes. Plant Physiol 136(1):2587–2608. doi:10.1104/pp.104.043695pp.104.043695

    Article  PubMed  CAS  Google Scholar 

  • Sauer A, Robinson DG (1985) Calmodulin dependent NAD-kinase is associated with both the outer and inner mitochondrial membranes in maize roots. Planta 166(2):227–233

    Article  CAS  Google Scholar 

  • Strimmer K, von Haeseler A (1996) Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol Biol Evol 13(7):964–969

    CAS  Google Scholar 

  • Strynadka NC, James MN (1989) Crystal structures of the helix-loop-helix calcium-binding proteins. Annu Rev Biochem 58:951–998. doi:10.1146/annurev.bi.58.070189.004511

    Article  PubMed  CAS  Google Scholar 

  • Thomas CL, Schmidt D, Bayer EM, Dreos R, Maule AJ (2008) Arabidopsis plant homeodomain finger proteins operate downstream of auxin accumulation in specifying the vasculature and primary root meristem. Plant J 59(3):426–436. doi:10.1111/j.1365-313X.2009.03874.x

    Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882

    Article  PubMed  CAS  Google Scholar 

  • Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE, Jones JDG (1998) Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91(phox)). Plant J 14(3):365–370

    Article  PubMed  CAS  Google Scholar 

  • Vanderbeld B, Snedden WA (2007) Developmental and stimulus-induced expression patterns of Arabidopsis calmodulin-like genes CML37, CML38 and CML39. Plant Mol Biol 64(6):683–697. doi:10.1007/s11103-007-9189-0

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Zhang WZ, Song LF, Zou JJ, Su Z, Wu WH (2008) Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis. Plant Physiol 148(3):1201–1211. doi:10.1104/pp.108.126375

    Article  PubMed  CAS  Google Scholar 

  • Whelan J, Hugosson M, Glaser E, Day DA (1995) Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria. Plant Mol Biol 27(4):769–778

    Article  PubMed  CAS  Google Scholar 

  • White PJ, Broadley MR (2003) Calcium in plants. Ann Bot (Lond) 92(4):487–511. doi:10.1093/aob/mcg164mcg164

    Article  CAS  Google Scholar 

  • Yang T, Poovaiah BW (2002) Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin. Proc Natl Acad Sci USA 99(6):4097–4102. doi:10.1073/pnas.052564899

    Article  PubMed  CAS  Google Scholar 

  • Yang T, Poovaiah BW (2003) Calcium/calmodulin-mediated signal network in plants. Trends Plant Sci 8(10):505–512. doi:S1360138503002188

    Article  PubMed  CAS  Google Scholar 

  • Zielinski RE (1998) Calmodulin and calmodulin-binding proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 49:697–725. doi:10.1146/annurev.arplant.49.1.697

    Article  PubMed  CAS  Google Scholar 

  • Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136(1):2621–2632. doi:10.1104/pp.104.046367

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. J. Whelan (University of Western Australia) for the pAOX clone and Dr. I. Finkemeier (LMU Munich) for help with the elucidation of micro array data. Technical assistance of Laura Kleinknecht and Claudia Sippel is also gratefully acknowledged. This work was supported by a grant from ERA-NETs “Plant Genomics” (ERAPG 08-044) to UV and by grants from the Deutsche Forschungsgemeinschaft to FC (CH 966/1-1) and CG (GI 154/13-1 and GI 154/13-2).

Author information

Authors and Affiliations

  1. Department of Biology of the LMU Munich, Center for Integrated Protein Science (Munich), 82152, Martinsried, Germany

    Fatima Chigri & Ute C. Vothknecht

  2. Department of Biology I, LMU Munich, 82152, Martinsried, Germany

    Sandra Flosdorff, Sahra Pilz, Eva Kölle & Ute C. Vothknecht

  3. Lehrstuhl für Botanik, TU Munich, Wissenschaftszentrum Weihenstephan, 85350, Freising, Germany

    Esther Dolze & Christine Gietl

Authors
  1. Fatima Chigri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandra Flosdorff
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sahra Pilz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eva Kölle
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Esther Dolze
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christine Gietl
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ute C. Vothknecht
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ute C. Vothknecht.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 590 kb)

Supplementary material 2 (PDF 262 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chigri, F., Flosdorff, S., Pilz, S. et al. The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively. Plant Mol Biol 78, 211–222 (2012). https://doi.org/10.1007/s11103-011-9856-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-011-9856-z

Keywords

search

Navigation