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Planta

, 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

Abstract

It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), 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.

Keywords

Chloroplast Cucumis Ferrochelatase Green fluorescent protein Heme biosynthesis Mitochondrion 

Abbreviations

GFP

green fluorescent protein

LHCP

apoproteins for light harvesting chlorophyll a/b-binding protein

LSU

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

Mn-SOD

Mn-superoxide dismutase

SSU

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

Notes

Acknowledgements

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.

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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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