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Engineering Plant Alternative Oxidase Function in Mammalian Cells: Substitution of the Motif-like Sequence ENV for QDT Diminishes Catalytic Activity of Arum concinnatum AOX1a Expressed in HeLa Cells

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Abstract

Alternative oxidase (AOX) is a nonproton motive quinol–oxygen oxidoreductase which is a component of the mitochondrial respiratory chain in higher plants. In this study, we have characterized the catalytic activity and regulatory behaviors of Arum concinnatum AOX isoforms, namely AcoAOX1a and AcoAOX1b, and their artificial mutants in HeLa cells. We demonstrated that substitution of the motif-like sequence ENV on the C-terminal half of AcoAOX1a for QDT diminishes its activity and proposed that the innate inactivity of AcoAOX1b in HeLa cells is, at least in part, attributable to its QDT motif. Furthermore, we show that introduction of F130L in the hydrophilic N-terminal extension of AcoAOX1a resulted in greater activity in the presence of pyruvate. This result indicates that functional significance of the N-terminal extension is not particular to the conventional regulatory cysteine. On the basis of these findings, we discuss new insights into the structural integrity of AOX in HeLa cells and the applicability of mammalian cells for functional analysis of this enzyme.

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Acknowledgments

This work was supported by JSPS KAKENHI (Grant-in-Aid for JSPS Fellows) grant number 22-224. YK is a JSPS Research Fellow. We thank the members of our laboratory for technical assistance and helpful discussions.

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Authors and Affiliations

  1. The United Graduate School of Agricultural Sciences, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan

    Yusuke Kakizaki

  2. Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate, 020-8550, Japan

    Kikukatsu Ito

Authors
  1. Yusuke Kakizaki
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  2. Kikukatsu Ito
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Corresponding authors

Correspondence to Yusuke Kakizaki or Kikukatsu Ito.

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ESM Fig. S1

Immunological detection of AOX proteins in the mitochondrial fraction of HeLa cells. In addition to AOX, beta-actin (ACTB) and a subunit of the respiratory complex I (NDUFB5) were detected as cytosolic and mitochondrial marker proteins, respectively. Cyt and Mit mean cytosolic and mitochondrial fractions, respectively. Approximate molecular weights of several standard proteins are indicated on the right of the images. Abbreviations: 1a, AcoAOX1a, 1b, AcoAOX1b. (PPT 2929 kb)

ESM Fig. S2

Quantitative comparison of AOX proteins in HeLa cells. Expression levels of AOX in 12 μg whole cell lysate proteins were determined by densitometry after immunological detection. The results are shown as mean ± SD (n = 3). Quantity can be compared within each pair only. The p values obtained by t-test are also shown for statistical significance of the results. Abbreviations: 1a, AcoAOX1a, 1b, AcoAOX1b. (PPT 100 kb)

ESM Fig. S3

Immunological detection of AOX proteins in HeLa cells expressing 1a, 1a-E83K, 1a-E110D or 1a-F130L. ACTB and NDUFB5 were also detected as cytosolic and mitochondrial marker proteins, respectively. The figure is presented as in Supplementary Fig. 1. Abbreviation: 1a, AcoAOX1a. (PPT 1016 kb)

ESM Table S1

PCR templates and primers used in this study to construct AcoAOX1a mutants. (DOCX 22 kb)

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Kakizaki, Y., Ito, K. Engineering Plant Alternative Oxidase Function in Mammalian Cells: Substitution of the Motif-like Sequence ENV for QDT Diminishes Catalytic Activity of Arum concinnatum AOX1a Expressed in HeLa Cells. Appl Biochem Biotechnol 170, 1229–1240 (2013). https://doi.org/10.1007/s12010-013-0235-x

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  • DOI: https://doi.org/10.1007/s12010-013-0235-x

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