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The Protein Journal

, Volume 32, Issue 7, pp 526–532 | Cite as

Metal-Binding Ability of VIP1: A bZIP Protein in Arabidopsis thaliana

  • Daisuke Tsugama
  • Shenkui Liu
  • Tetsuo Takano
Article

Abstract

VirE2-interacting protein 1 (VIP1) is an Arabidopsis thaliana bZIP transcription factor which regulates pathogen responses and rehydration responses. VIP1 has transcriptional activation potential, DNA-binding ability, and a nuclear-cytoplasmic shuttling property. These functions are possibly regulated by cofactors and/or post-translational modifications. During an investigation of the functions of VIP1, we discovered that VIP1 can react with an Ni2+-activated derivative of horseradish peroxidase, HisProbe-HRP, suggesting that VIP1 can bind Ni2+. Using truncated versions and mutated versions of VIP1, the Ni2+-binding region was narrowed. Using VIP1 H145Q and H145R mutants, which have H → Q and H → R mutations at the amino acid position 145 of VIP1, a trihistidine site at the amino acid position 144–146 was confirmed to be responsible for the Ni2+-binding ability. Immobilized-metal affinity chromatography (IMAC) suggested that VIP1 can bind Zn2+ and Co2+ as well as Ni2+, which is consistent with the known metal-chelating property of polyhistidine. In IMAC, the levels of purified VIP1 were not significantly different between denaturing and non-denaturing conditions, suggesting that the trihistidine is located on the surface of the native form of VIP1. In gel shift assays, VIP1-dependent decreases of electrophoretic mobilities of DNA probes were further decreased by Co2+. Among wild-type VIP1 and the H145Q and H145R mutants, H145R was the least sensitive to the effect of Co2+ in the gel shift assays. These results suggest that the Co2+ and the metal-binding site of VIP1 affect the interaction between VIP1 and DNA.

Keywords

Arabidopsis thaliana Transcription factor Polyhistidine DNA–protein interaction 

Abbreviations

NLS

Nuclear localization signal

NES

Nuclear export signal

HRP

Horseradish peroxidase

TBS

Tris-buffered saline

IMAC

Immobilized-metal affinity chromatography

Notes

Acknowledgments

This work was supported by a Grant-in-aid for Scientific Research (22·2144) to D.T.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Asian Natural Environmental Science Center (ANESC)The University of TokyoNishitokyo-shiJapan
  2. 2.Alkali Soil Natural Environmental Science Center (ASNESC)Northeast Forestry UniversityHarbinPeople’s Republic of China

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