Metal-binding ability of VIP1: a bZIP protein in Arabidopsis thaliana.
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.
Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo-shi, Tokyo, 188-0002, Japan.
Daisuke
D
Tsugama
Shenkui
S
Liu
Tetsuo
T
Takano
D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006639:Histidine; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D035165:Transcription Factors, General / Q000737:chemistry* / Q000378:metabolism*
D029681:Arabidopsis Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D011993:Recombinant Fusion Proteins; D035165:Transcription Factors, General; C523538:VIP2 protein, Arabidopsis; C033223:polyhistidine; D003035:Cobalt; D006639:Histidine; D004247:DNA