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Diversity in subcellular targeting of the PP2A B′η subfamily members

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Abstract

Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase comprising a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to be responsible for substrate specificity and localization of the PP2A complex. In plants, three families of B subunits exist, i.e. B (B55), B′, and B′′. Here, we report differential subcellular targeting within the Arabidopsis B′η subfamily, which consists of the close homologs B′η, B′θ, B′γ and B′ζ. Phenotypes of corresponding knockouts were observed, and particularly revealed delayed flowering for the B′η knockout. The B′ subunits were linked to fluorescent tags and transiently expressed in various tissues of onion, tobacco and Arabidopsis. B′η and B′γ targeted the cytosol and nucleus. B′ζ localized to the cytoplasm and partly co-localized with mitochondrial markers when the N-terminus was free. Provided its C-terminus was free, the B′θ subunit targeted peroxisomes. The importance of the C-terminal end for peroxisomal targeting was further confirmed by truncation of the C-terminus. The results revealed that the closely related B′ subunits are targeting different organelles in plants, and exemplify the usage of the peptide serine–serine–leucine as a PTS1 peroxisomal signaling peptide.

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Abbreviations

CDPK:

Calcium-dependent protein kinase

GFP:

Green fluorescent protein

NLS:

Nuclear localization signal

PP2A:

Protein phosphatase 2A

PTS1:

Peroxisomal targeting signal 1

RFP:

Red fluorescent protein

YFP:

Yellow fluorescent protein

References

  • Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WJ, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657

    Article  PubMed  Google Scholar 

  • Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18:298–305

    Article  PubMed  CAS  Google Scholar 

  • Blakeslee JJ, Zhou HW, Heath JT, Skottke KR, Barrios JAR, Liu SY, DeLong A (2008) Specificity of RCN1-mediated protein phosphatase 2A regulation in meristem organization and stress response in roots. Plant Physiol 146:539–553

    Article  PubMed  CAS  Google Scholar 

  • Boudsocq M, Droillard MJ, Barbier-Brygoo H, Lauriere C (2007) Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid. Plant Mol Biol 63:491–503

    Article  PubMed  CAS  Google Scholar 

  • Boutry M, Nagy F, Poulsen C, Aoyagi K, Chua NH (1987) Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. Nature 328:340–342

    Article  PubMed  CAS  Google Scholar 

  • Brocard C, Hartig A (2006) Peroxisome targeting signal 1: is it really a simple tripeptide? Biochim Biophys Acta 1763:1565–1573

    Article  PubMed  CAS  Google Scholar 

  • Camilleri C, Azimzadeh J, Pastuglia M, Bellini C, Grandjean O, Bouchez D (2002) The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton. Plant Cell 14:833–845

    Article  PubMed  CAS  Google Scholar 

  • Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics 4:29

    Article  PubMed  Google Scholar 

  • Cho UC, Xu W (2007) Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445:53–57

    Article  PubMed  CAS  Google Scholar 

  • Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241:779–786

    Article  PubMed  CAS  Google Scholar 

  • Dagda RK, Zaucha JA, Wadzinski BE, Strack S (2003) A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem 278:24976–24985

    Article  PubMed  CAS  Google Scholar 

  • Dagda RK, Barwacz CA, Cribbs JT, Strack S (2005) Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bβ2 regulatory subunit for protein phosphate 2A. J Biol Chem 280:27375–27382

    Article  PubMed  CAS  Google Scholar 

  • Droillard MJ, Thibivilliers S, Cazale AC, Barbier-Brygoo H, Lauriere C (2000) Protein kinases induced by osmotic stresses and elicitor molecules in tobacco cell suspensions: two crossroad MAP kinases and one osmoregulation-specific protein kinase. FEBS Lett 474:217–222

    Article  PubMed  CAS  Google Scholar 

  • Eichhorn PJA, Creyghton MP, Bernards R (2009) Protein phosphatase 2A regulatory subunits and cancer. Biochim Biophys Acta 1795:1–15

    PubMed  CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Elofsson A, von Heijne G, Cristóbal S (2003) In silico prediction of the peroxisomal proteome in fungi, plant and animals. J Mol Biol 330:443–456

    Article  PubMed  CAS  Google Scholar 

  • Eubel H, Meyer EH, Taylor NL, Bussell JD, O’Toole N, Heazlewood JL, Castleden I, Small ID, Smith SM, Millar AH (2008) Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes. Plant Physiol 148:1809–1829

    Article  PubMed  CAS  Google Scholar 

  • Farkas I, Dombradi V, Miskei M, Szabados L, Koncz C (2007) Arabidopsis PPP family of serine/threonine phosphatases. Trends Plant Sci 12:169–176

    Article  PubMed  CAS  Google Scholar 

  • Fujiwara MT, Nakamura A, Itoh R, Shimada Y, Yoshida S, Moller SG (2004) Chloroplast division site placement requires dimerization of the ARC11/AtMinD1 protein in Arabidopsis. J Cell Sci 117:2399–2410

    Article  PubMed  CAS  Google Scholar 

  • Fukao Y, Hayashi M, Nishimura M (2002) Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana. Plant Cell Physiol 43:689–696

    Article  PubMed  CAS  Google Scholar 

  • Gorlich D, Mattaj IW (1996) Nucleocytoplasmic transport. Science 271:1513–1518

    Article  PubMed  CAS  Google Scholar 

  • Heazlewood JL, Tonti-Filippini JS, Gout AM, Day DA, Whelan J, Millar AH (2004) Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. Plant Cell 16:241–256

    Article  PubMed  CAS  Google Scholar 

  • Heazlewood JL, Tonti-Filippini J, Verboom RE, Millar AH (2005) Combining experimental and predicted datasets for determination of the subcellular location of proteins in Arabidopsis. Plant Physiol 139:598–609

    Article  PubMed  CAS  Google Scholar 

  • Hombauer H, Weismann D, Mudrak I, Stanzel C, Fellner T, Lackner DH, Ogris E (2007) Generation of active protein phosphatase 2A is coupled to holoenzyme assembly. PLoS Biol 5:e155

    Article  PubMed  Google Scholar 

  • Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35:W585–W587

    Article  PubMed  Google Scholar 

  • Hua S, Sun Z (2001) Support vector machine approach for protein subcellular localization prediction. Bioinformatics 17:721–728

    Article  PubMed  CAS  Google Scholar 

  • Janssens V, Goris J (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signaling. Biochem J 353:417–439

    Article  PubMed  CAS  Google Scholar 

  • Janssens V, Longin S, Goris J (2008) PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends Biochem Sci 33:113–121

    Article  PubMed  CAS  Google Scholar 

  • Johnson TL, Olsen LJ (2003) Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme A thiolase into glyoxysomes. Plant Physiol 133:1991–1999

    Article  PubMed  CAS  Google Scholar 

  • Koroleva OA, Tomlinson ML, Leader D, Shaw P, Doonan JH (2005) High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J 41:162–174

    Article  PubMed  CAS  Google Scholar 

  • Kost B, Spielhofer P, Chua NH (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393–401

    Article  PubMed  CAS  Google Scholar 

  • Kragler F, Lametschwandtner G, Christmann J, Hartig A, Harada JJ (1998) Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5. Proc Natl Acad Sci USA 95:13336–13341

    Article  PubMed  CAS  Google Scholar 

  • Li X, Virshup DM (2002) Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A. Eur J Biochem 269:546–552

    Article  PubMed  CAS  Google Scholar 

  • Li S, Ehrhardt DW, Rhee SY (2006) Systematic analysis of Arabidopsis organelles and a protein localization database for facilitating fluorescent tagging of full-length Arabidopsis proteins. Plant Physiol 141:527–539

    Article  PubMed  CAS  Google Scholar 

  • Lillo C (2008) Signalling cascades integrating light-enhanced nitrate metabolism. Biochem J 415:11–19

    Article  PubMed  CAS  Google Scholar 

  • Lillo C, Smith LH, Nimmo HG, Wilkins MB (1996) Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts. Planta 200:181–185

    Article  CAS  Google Scholar 

  • Lorkovic ZJ, Hilscher J, Barta A (2004) Use of fluorescent protein tags to study nuclear organization of the spliceosomal machinery in transiently transformed living plant cells. Mol Biol Cell 15:3233–3243

    Article  PubMed  CAS  Google Scholar 

  • MacKintosh C (1998) Regulation of cytosolic enzymes in primary metabolism by reversible protein phosphorylation. Curr Opin Plant Biol 1:224–229

    Article  PubMed  CAS  Google Scholar 

  • MacKintosh C, Coggins J, Cohen P (1991) Plant protein phosphatases. Subcellular distribution, detection of protein phosphatase 2C and identification of protein phosphatase 2A as the major quinate dehydrogenase phosphatase. Biochem J 273:733–738

    PubMed  CAS  Google Scholar 

  • Michniewicz M, Zago MK, Abas L, Weijers D, Schweighofer A, Meskiene I, Heisler MG, Ohno C, Zhang J, Huang F, Schwab R, Weigel D, Meyerowitz EM, Luschnig C, Offringa R, Friml J (2007) Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell 130:1044–1056

    Article  PubMed  CAS  Google Scholar 

  • Millar AH, Sweetlove LJ, Giege P, Leaver CJ (2001) Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol 127:1711–1727

    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 51:1126–1136

    Article  PubMed  CAS  Google Scholar 

  • Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A, Eisenhaber F (2003) Motif refinement of the peroxisomal targeting signal 1 and evaluation of taxon-specific differences. J Mol Biol 328:567–579

    Article  PubMed  CAS  Google Scholar 

  • Reumann S, Babujee L, Ma C, Wienkoop S, Siemsen T, Antonicelli GE, Rasche N, Luder F, Weckwerth W, Jahn O (2007) Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19:3170–3193

    Article  PubMed  CAS  Google Scholar 

  • Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C, Clarke JD, Cotton D, Bullis D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel T, Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 12:2985–2994

    Article  Google Scholar 

  • Siegl G, MacKintosh C, Stitt M (1990) Sucrose-phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves: evidence from the effects of okadaic acid and microcystin. FEBS Lett 270:198–202

    Article  PubMed  CAS  Google Scholar 

  • Sommer JM, Cheng QL, Keller GA, Wang CC (1992) In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal. Mol Biol Cell 3:749–759

    PubMed  CAS  Google Scholar 

  • Sontag E (2001) Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal 13:7–16

    Article  PubMed  CAS  Google Scholar 

  • Terol J, Bargues M, Carrasco P, Perez-Alonso M, Paricio N (2002) Molecular characterization and evolution of the protein phosphatase 2A B′ regulatory subunit family in plants. Plant Physiol 129:808–822

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Chevalier D, Larue C, Cho SK, Walker JC (2007) The protein phosphatases and protein kinases of Arabidopsis thaliana. The Arabidopsis book. American Society of Plant Biologists. doi:10.1199/tab.0106

  • Xu Y, Xing Y, Chen Y, Chao Y, Lin Z, Fan E, Yu JW, Strack S, Jeffrey PD, Shi Y (2006) Structure of the protein phosphatase 2A holoenzyme. Cell 127:1239–1251

    Article  PubMed  CAS  Google Scholar 

  • Xu C, Jing R, Mao X, Jia X, Chang X (2007) A wheat (Triticum aestivum) protein phosphatase 2A catalytic subunit gene provides enhanced drought tolerance in tobacco. Ann Bot (Lond) 99:439–450

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Roger Y. Tsien for providing mRFP cDNA and to Zdravko J Lorkovic (Max F. Perutz Laboratories, Vienna, Austria) and Benedikt Kost (Uppsala, Sweden) for kindly providing materials used in this work. We thank Simon G Møller and Jodi Maple (UiS, Norway) for their help and for providing vectors.

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Correspondence to Cathrine Lillo.

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Matre, P., Meyer, C. & Lillo, C. Diversity in subcellular targeting of the PP2A B′η subfamily members. Planta 230, 935–945 (2009). https://doi.org/10.1007/s00425-009-0998-z

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