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Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence

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Abstract

DNA topoisomerase II ofDictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even through its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins ofD. discoideum directly by analyzing deletion mutants.

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Abbreviations

ATP:

adenosine triphosphate

BSA:

bovine serum albumin

BPB:

bromophenol blue

DTT:

dithiothreitol

EDTA:

ethylenediaminetetraacetic acid

FITC:

fluorescein isothiocyanate

kDa:

kilodalton(s)

NP40:

Nonidet P-40

PAGE:

polyacrylamide gel electrophoresis

PCR:

polymerase chain reaction

PMSF:

phenylmethylsulfonyl fluoride

SDS:

sodium dodecylsulfate

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Author notes

  1. Kayoko Komori

    Present address: Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), Furuedai 6-2-3, Suita, 565, Osaka, Japan

Authors and Affiliations

  1. Institute of Biological Sciences, University of Tsukuba, Tsukuba, 305, Ibaraki, Japan

    Kayoko Komori, Fumiaki Maruo, Takahiro Morio, Hideko Urushihara & Yoshimasa Tanaka

  2. Center for TARA, University of Tsukuba, Tsukuba, 305, Ibaraki, Japan

    Yoshimasa Tanaka

Authors
  1. Kayoko Komori
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  2. Fumiaki Maruo
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  3. Takahiro Morio
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  4. Hideko Urushihara
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  5. Yoshimasa Tanaka
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Tsukuba Advanced Research Alliance (TARA researcher for the Sakabe project)

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Komori, K., Maruo, F., Morio, T. et al. Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence. J. Plant Res. 110, 65–75 (1997). https://doi.org/10.1007/BF02506844

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  • DOI: https://doi.org/10.1007/BF02506844

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