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Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria

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Abstract

The sub-cellular location of enzymes of fatty acid β-oxidation in plants is controversial. In the current debate the role and location of particular thiolases in fatty acid degradation, fatty acid synthesis and isoleucine degradation are important. The aim of this research was to determine the sub-cellular location and hence provide information about possible functions of all the putative 3-ketoacyl-CoA thiolases (KAT) and acetoacetyl-CoA thiolases (ACAT) in Arabidopsis. Arabidopsis has three genes predicted to encode KATs, one of which encodes two polypeptides that differ at the N-terminal end. Expression in Arabidopsis cells of cDNAs encoding each of these KATs fused to green fluorescent protein (GFP) at their C-termini showed that three are targeted to peroxisomes while the fourth is apparently cytosolic. The four KATs are also predicted to have mitochondrial targeting sequences, but purified mitochondria were unable to import any of the proteins in vitro. Arabidopsis also has two genes encoding a total of five different putative ACATs. One isoform is targeted to peroxisomes as a fusion with GFP, while the others display no targeting in vivo as GFP fusions, or import into isolated mitochondria. Analysis of gene co-expression clusters in Arabidopsis suggests a role for peroxisomal KAT2 in β-oxidation, while KAT5 co-expresses with genes of the flavonoid biosynthesis pathway and cytosolic ACAT2 clearly co-expresses with genes of the cytosolic mevalonate biosynthesis pathway. We conclude that KATs and ACATs are present in the cytosol and peroxisome, but are not found in mitochondria. The implications for fatty acid β-oxidation and for isoleucine degradation in mitochondria are discussed.

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Abbreviations

AOX:

Alternative oxidase

ACAT:

Acetoacetyl-CoA thiolase

KAT:

3-Ketoacyl-CoA thiolase

Rubisco SSU:

Small subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase

References

  • Baker A, Sparkes IA (2005) Peroxisome protein import: some answers, more questions. Curr Opin Plant Biol 8:640–647

    Article  CAS  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  CAS  PubMed  Google Scholar 

  • Bathgate B, Baker A, Leaver CJ (1989) Two genes encode the adenine nucleotide translocator of maize mitochondria. Isolation, characterisation and expression of the structural genes. Eur J Biochem 183:303–310

    Article  CAS  PubMed  Google Scholar 

  • Brugiere S, Kowalski S, Ferro M, Seigneurin-Berny D, Miras S, Salvi D, Ravanel S, d’Herin P, Garin J, Bourguignon J, Joyard J, Rolland N (2004) The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions. Phytochemistry 65:1693–1707

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  • Chew O, Whelan J (2004) Just read the message: a model for sorting of proteins between mitochondria and chloroplasts. Trends Plant Sci 9:318–319

    Article  CAS  PubMed  Google Scholar 

  • Chew O, Rudhe C, Glaser E, Whelan J (2003) Characterization of the targeting signal of dual-targeted pea glutathione reductase. Plant Mol Biol 53:341–356

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97:3718–3723

    Article  CAS  PubMed  Google Scholar 

  • Danpure CJ, Lumb MJ, Birdsey GM, Zhang X (2003) Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease. Biochim Biophys Acta 1647:70–75

    CAS  PubMed  Google Scholar 

  • Derrick S, Large PJ (1993) Activities of the enzymes of the Ehrlich pathway and formation of branched-chain alcohols in Saccharomyces cerevisiae and Candida utilis grown in continuous culture on valine or ammonium as sole nitrogen source. J Gen Microbiol 139:2783–2792

    CAS  PubMed  Google Scholar 

  • Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95:14863–14868

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Footitt S, Slocombe SP, Larner V, Kurup S, Wu Y, Larson T, Graham I, Baker A, Holdsworth M (2002) Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP. EMBO J 21:2912–2922

    Article  CAS  PubMed  Google Scholar 

  • Fujiki Y, Sato T, Ito M, Watanabe A (2000) Isolation and characterization of cDNA clones for the e1beta and E2 subunits of the branched-chain alpha-ketoacid dehydrogenase complex in Arabidopsis. J Biol Chem 275:6007–6013

    Article  CAS  PubMed  Google Scholar 

  • Fukao T, Scriver CR, Kondo N (2001) The clinical phenotype and outcome of mitochondrial acetoacetyl-CoA thiolase deficiency (β-ketothiolase or T2 deficiency) in 26 enzymatically proved and mutation-defined patients. Mol Genet Metab 72:109–114

    Article  CAS  PubMed  Google Scholar 

  • Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N, Carde JP, Bryce JH, Graham IA, Smith SM (2001) Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid β-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant J 28:1–12

    Article  CAS  PubMed  Google Scholar 

  • Graham IA, Eastmond PJ (2002) Pathways of straight and branched chain fatty acid catabolism in higher plants. Prog Lipid Res 41:156–181

    Article  CAS  PubMed  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  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Hooks MA (2002) Molecular biology, enzymology, and physiology of ß-oxidation. In: Baker A, Graham I (eds) Plant peroxisomes. Kluwer Academic Publishers, London, pp 19–55

    Google Scholar 

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

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Kleffmann T, Russenberger D, von Zychlinski A, Christopher W, Sjolander K, Gruissem W, Baginsky S (2004) The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions. Curr Biol 14:354–362

    Article  CAS  PubMed  Google Scholar 

  • Koh S, Andre A, Edwards H, Ehrhardt D, Somerville S (2005) Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections. Plant J 44:516–529

    Article  CAS  PubMed  Google Scholar 

  • Kruft V, Eubel H, Jansch L, Werhahn W, Braun HP (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol 127:1694–1710

    Article  CAS  PubMed  Google Scholar 

  • Kumar M, Verma R, Raghava GP (2006) Prediction of mitochondrial proteins using support vector machine and hidden markov model. J Biol Chem 281:5357–5363

    Article  CAS  PubMed  Google Scholar 

  • Lange PR, Eastmond PJ, Madagan K, Graham IA (2004) An Arabidopsis mutant disrupted in valine catabolism is also compromised in peroxisomal fatty acid β-oxidation. FEBS Lett 571:147–153

    Article  CAS  PubMed  Google Scholar 

  • Lee MN, Whelan J (2004) Identification of signals required for import of the soybean F(A)d subunit of ATP synthase into mitochondria. Plant Mol Biol 54:193–203

    Article  CAS  PubMed  Google Scholar 

  • Lister R, Chew O, Lee MN, Heazlewood JL, Clifton R, Parker KL, Millar AH, Whelan J (2004) A transcriptomic and proteomic characterization of the Arabidopsis mitochondrial protein import apparatus and its response to mitochondrial dysfunction. Plant Physiol 134:777–789

    Article  CAS  PubMed  Google Scholar 

  • Masterson C, Wood C (2001) Mitochondrial and peroxisomal β-oxidation capacities of organs from a non-oilseed plant. Proc Biol Sci 268:1949–1953

    Article  CAS  PubMed  Google Scholar 

  • Mockler TC, Chan S, Sundaresan A, Chen H, Jacobsen SE, Ecker JR (2005) Applications of DNA tiling arrays for whole-genome analysis. Genomics 85:1–15

    Article  CAS  PubMed  Google Scholar 

  • Murcha MW, Elhafez D, Millar AH, Whelan J (2004) The N-terminal extension of plant mitochondrial carrier proteins is removed by two-step processing: the first cleavage is by the mitochondrial processing peptidase. J Mol Biol 344:443–454

    Article  CAS  PubMed  Google Scholar 

  • Murcha MW, Millar AH, Whelan J (2005) The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane. J Mol Biol 351:16–25

    Article  CAS  PubMed  Google Scholar 

  • Pendle AF, Clark GP, Boon R, Lewandowska D, Lam YW, Andersen J, Mann M, Lamond AI, Brown JW, Shaw PJ (2005) Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol Biol Cell 16:260–269

    Article  CAS  PubMed  Google Scholar 

  • Pereto J, Lopez-Garcia P, Moreira D (2005) Phylogenetic analysis of eukaryotic thiolases suggests multiple proteobacterial origins. J Mol Evol 61:65–74

    Article  CAS  PubMed  Google Scholar 

  • Picault N, Hodges M, Palmieri L, Palmieri F (2004) The growing family of mitochondrial carriers in Arabidopsis. Trends Plant Sci 9:138–146

    Article  CAS  PubMed  Google Scholar 

  • Pohlmeyer K, Soll J, Steinkamp T, Hinnah S, Wagner R (1997) Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane. Proc Natl Acad Sci USA 94:9504–9509

    Article  CAS  PubMed  Google Scholar 

  • Pracharoenwattana I, Cornah JE, Smith SM (2005) Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell 17:2037–2048

    Article  CAS  PubMed  Google Scholar 

  • Rassow J, Dekker PJ, van Wilpe S, Meijer M, Soll J (1999) The preprotein translocase of the mitochondrial inner membrane: function and evolution. J Mol Biol 286:105–120

    Article  CAS  PubMed  Google Scholar 

  • Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J (2003) TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34:374–378

    CAS  PubMed  Google Scholar 

  • Small I, Peeters N, Legeai F, Lurin C (2004) Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4:1581–1590

    Article  CAS  PubMed  Google Scholar 

  • Tanudji M, Dessi P, Murcha M, Whelan J (2001) Protein import into plant mitochondria: precursor proteins differ in ATP and membrane potential requirements. Plant Mol Biol 45:317–325

    Article  CAS  PubMed  Google Scholar 

  • Taylor NL, Heazlewood JL, Day DA, Millar AH (2004) Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis. Plant Physiol 134:838–848

    Article  CAS  PubMed  Google Scholar 

  • Thirkettle-Watts D, McCabe TC, Clifton R, Moore C, Finnegan PM, Day DA, Whelan J (2003) Analysis of the alternative oxidase promoters from soybean. Plant Physiol 133:1158–1169

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    CAS  PubMed  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 25:4876–4882

    Article  CAS  PubMed  Google Scholar 

  • Tian GW, Mohanty A, Chary SN, Li S, Paap B, Drakakaki G, Kopec CD, Li J, Ehrhardt D, Jackson D, Rhee SY, Raikhel NV, Citovsky V (2004) High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol 135:25–38

    Article  CAS  PubMed  Google Scholar 

  • Toufighi K, Brady SM, Austin R, Ly E, Provart NJ (2005) The botany array resource: e-Northerns, expression angling, and promoter analyses. Plant J 43:153–163

    Article  CAS  PubMed  Google Scholar 

  • Tsukamoto T, Hata S, Yokota S, Miura S, Fujiki Y, Hijikata M, Miyazawa S, Hashimoto T, Osumi T (1994) Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor. J Biol Chem 269:6001–6010

    CAS  PubMed  Google Scholar 

  • van Roermund CW, Waterham HR, Ijlst L, Wanders RJ (2003) Fatty acid metabolism in Saccharomyces cerevisiae. Cell Mol Life Sci 60:1838–1851

    Article  PubMed  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 27:769–778

    Article  CAS  PubMed  Google Scholar 

  • Winning BM, Sarah CJ, Purdue PE, Day CD, Leaver CJ (1992) The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria. Plant J 2:763–773

    Article  CAS  PubMed  Google Scholar 

  • Yamada K, Lim J, Dale JM, Chen H, Shinn P, Palm CJ, Southwick AM, Wu HC, Kim C, Nguyen M, Pham P, Cheuk R, Karlin-Newmann G, Liu SX, Lam B, Sakano H, Wu T, Yu G, Miranda M, Quach HL, Tripp M, Chang CH, Lee JM, Toriumi M, Chan MM, Tang CC, Onodera CS, Deng JM, Akiyama K, Ansari Y, Arakawa T, Banh J, Banno F, Bowser L, Brooks S, Carninci P, Chao Q, Choy N, Enju A, Goldsmith AD, Gurjal M, Hansen NF, Hayashizaki Y, Johnson-Hopson C, Hsuan VW, Iida K, Karnes M, Khan S, Koesema E, Ishida J, Jiang PX, Jones T, Kawai J, Kamiya A, Meyers C, Nakajima M, Narusaka M, Seki M, Sakurai T, Satou M, Tamse R, Vaysberg M, Wallender EK, Wong C, Yamamura Y, Yuan S, Shinozaki K, Davis RW, Theologis A, Ecker JR (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302:842–846

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was funded through grants from the Australian Research Council (ARC) Centre of Excellence Programme to JW, SS and AHM. AHM is funded as an ARC Queen Elizabeth II Fellow and SS as an ARC Federation Fellow.

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Correspondence to James Whelan.

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Carrie, C., Murcha, M.W., Millar, A.H. et al. Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria. Plant Mol Biol 63, 97–108 (2007). https://doi.org/10.1007/s11103-006-9075-1

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