, Volume 217, Issue 4, pp 602–609 | Cite as

Subcellular localization of two types of ferrochelatase in cucumber

  • T. Masuda
  • T. Suzuki
  • H. Shimada
  • H. Ohta
  • K. Takamiya
Original Article


It is widely believed that ferrochelatase (protoheme ferrolyase, EC, which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.


Chloroplast Cucumis Ferrochelatase Green fluorescent protein Heme biosynthesis Mitochondrion 



green fluorescent protein


apoproteins for light harvesting chlorophyll a/b-binding protein


large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase


Mn-superoxide dismutase


small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase



This study was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We thank Dr. M. Shibasaka for providing the antibody against Mn-SOD. We are also grateful for Dr. Y. Niwa for providing GFP vectors. T. Masuda and T. Suzuki contributed equally to this work.


  1. Beale SI, Weinstein JD (1990) Tetrapyrrole metabolism in photosynthetic organisms. In: Dailey HA (ed) Biosynthesis of heme and chlorophylls, McGraw-Hill, New York, pp 287–391Google Scholar
  2. Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330PubMedGoogle Scholar
  3. Chow K-S, Singh DP, Roper JM, Smith AG (1997) A single precursor protein for ferrochelatase-I from Arabidopsis is imported in vitro into both chloroplasts and mitochondria. J Biol Chem 272:27565–27571CrossRefPubMedGoogle Scholar
  4. Chow K-S, Singh PD, Walker AR, Smith AG (1998) Two different genes encode ferrochelatase in Arabidopsis: mapping, expression and subcellular targeting of the precursor proteins. Plant J 15:531–541CrossRefPubMedGoogle Scholar
  5. Cleary SP, Tan F-C, Nakrieko K-A, Thompson SJ, Mullineaux PM, Creissen GP, von Stedingk E, Glaser E, Smith AG, Robinson C (2002) Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts. J Biol Chem 277:5562–5569CrossRefPubMedGoogle Scholar
  6. Cornah JE, Roper JM, Singh DP, Smith AG (2002) Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.). Biochem J 362:423–432CrossRefPubMedGoogle Scholar
  7. Douce R, Joyard J (1982) Purification of the chloroplast envelope. In: Edelman M, Hallick RB, Chua, N-H (eds) Methods in chloroplast molecular biology. Elsevier, Amsterdam, pp 239–256Google Scholar
  8. Fuesler TP, Castelfranco PA, Wong YS (1984) Formation of Mg-containing chlorophyll precursors from protoporphyrin IX, δ-aminolevulinic acid, and glutamate in isolated photosynthetically competent, developing chloroplasts. Plant Physiol 74:928–933Google Scholar
  9. Giglione C, Serero A, Pierre M, Boisson B, Meinnel T (2000) Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms. EMBO J 19:5916–5929Google Scholar
  10. Hansen J, Muldbjerg M, Cherest H, Surdin-Kerjan Y (1997) Siroheme biosynthesis in Saccharomyces cerevisiae required the products of both the MET1 and MET8 genes. FEBS Lett 401:20–24CrossRefPubMedGoogle Scholar
  11. Jones OTG (1967) Heme biosynthesis by isolated chloroplasts. Biochem Biophys Res Commun 28:671–674PubMedGoogle Scholar
  12. Jones OTG (1968) Ferrochelatase of spinach chloroplasts. Biochem J 107:113–119PubMedGoogle Scholar
  13. Kohler R, Hanson MR (2000) Plastid tubules of higher plants are tissue-specific and developmentally regulated. J Cell Sci 113:81–89PubMedGoogle Scholar
  14. Lange H, Kispel G, Lill R (1999) Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria. J Biol Chem 274:18989–18996CrossRefPubMedGoogle Scholar
  15. Lermontova I, Kruse E, Mock HP, Grimm B (1997) Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase. Proc Natl Acad Sci USA 94:8895–8900CrossRefPubMedGoogle Scholar
  16. Leustek T, Smith M, Murillo M, Singh DP, Smith AG, Woodcock S, Awan SJ, Warren MJ (1997) Siroheme biosynthesis in higher plants: Analysis of an S-adenosyl-l-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana. J Biol Chem 272:2744–2752CrossRefPubMedGoogle Scholar
  17. Lister R, Chew O, Rudhe C, Lee M-N, Whelan J (2001) Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria. FEBS Lett 506:291–295CrossRefPubMedGoogle Scholar
  18. Little HN, Jones OTG (1976) The subcellular localization and properties of the ferrochelatase of etiolated barley. Biochem J 156:309–314PubMedGoogle Scholar
  19. Menand B, Maréchal-Drouard L, Sakamoto W, Dietrich A, Wintz H (1998) A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl-tRNA synthetase in Arabidopsis thaliana. Proc Natl Acad Sci USA 95:11014–11019PubMedGoogle Scholar
  20. Miyamoto K, Tanaka R, Teramoto H, Masuda T, Tsuji H, Inokuchi H (1994) Nucleotide sequences of cDNA clones encoding ferrochelatase from barley and cucumber. Plant Physiol 105:769–770CrossRefPubMedGoogle Scholar
  21. Nishimura M, Douce R, Akazawa T (1982) Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts. Plant Physiol 69:916–920Google Scholar
  22. Papenbrock J, Mishra S, Mock H-P, Kruse E, Schmidt E-K, Petersman A, Braun H-P, Grimm B (2001) Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants. Plant J 28:41–50CrossRefPubMedGoogle Scholar
  23. Porra RJ, Jones OTG (1963) Studies on ferrochelatase. Biochem J 87:181–185Google Scholar
  24. Porra RJ, Lascelles J (1968) Studies on ferrochelatase: the enzymatic formation of haem in proplastids, chloroplasts and plant mitochondria. Biochem J 108:343–348PubMedGoogle Scholar
  25. Raux E, McVeigh T, Peters SE, Leustek T, Warren MJ (1999) The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis. Biochem J 338:701–708CrossRefPubMedGoogle Scholar
  26. Smith AG, Marsh O, Elder GH (1993) Investigation of the subcellular location of the tetrapyrrole-biosynthesis enzyme coproporphyrinogen oxidase in higher plants. Biochem J 292:503–508PubMedGoogle Scholar
  27. Smith AG, Santana MA, Wallace-Cook ADM, Roper JM, Labbe-Bois R (1994) Isolation of a cDNA encoding chloroplast ferrochelatase from Arabidopsis thaliana by functional complementation of a yeast mutant. J Biol Chem 269:13405–13413PubMedGoogle Scholar
  28. Spencer JB, Stolowich NJ, Roessner CA, Scott AI (1993) The Escherichia coli cysG gene encodes the multifunctional protein, sirohaem synthase. FEBS Lett 335:57–60CrossRefPubMedGoogle Scholar
  29. Suzuki T, Masuda T, Inokuchi H, Shimada H, Ohta H, Takamiya K (2000) Overexpression, enzymatic properties and tissue localization of a ferrochelatase of cucumber. Plant Cell Physiol 41:192–199CrossRefPubMedGoogle Scholar
  30. Suzuki T, Masuda T, Singh DP, Tan F-C, Tsuchiya T, Shimada H, Ohta H, Smith AG, Takamiya K (2002) Two types of ferrochelatase in photosynthetic and nonphotosynthetic tissues of cucumber. J Biol Chem 277:4731–4737CrossRefPubMedGoogle Scholar
  31. Taketani S, Tokunaga R (1984) Non-enzymatic heme formation in the presence of fatty acids and thiol reductants. Biochim Biophys Acta 798:226–230CrossRefPubMedGoogle Scholar
  32. Tanaka R, Yoshida K, Nakayashiki T, Masuda T, Tsuji H, Inokuchi H, Tanaka A (1996) Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings. Plant Physiol 110:1223–1230CrossRefPubMedGoogle Scholar
  33. Walker CJ, Willows RD (1997) Mechanism and regulation of Mg-chelatase. Biochem J 327:321–333PubMedGoogle Scholar
  34. Warren MJ, Bolt EL, Roessner CA, Scott AI, Spencer JB, WoodCock SC (1994) Gene dissection demonstrates that the Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase. Biochem J 265:725–729Google Scholar
  35. Watanabe N, Che F-S, Iwano M, Takayama S, Yoshida S, Isogai A (2001) Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons. J Biol Chem 276:20474–20481CrossRefPubMedGoogle Scholar
  36. Yu J, Hu S, Wang J et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79–92PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • T. Masuda
    • 1
  • T. Suzuki
    • 1
    • 2
  • H. Shimada
    • 1
  • H. Ohta
    • 1
  • K. Takamiya
    • 1
  1. 1.Graduate School of Bioscience and BiotechnologyTokyo Institute of TechnologyYokohamaJapan
  2. 2.National Institute of Health SciencesTokyoJapan

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