Abstract
Metal-responsive transcription factor 1 (MTF-1) is a transcription factor that contains six C2H2¬¬ zinc fingers and is essential for basal and heavy metal-induced MT gene expression by binding to MRE (TGCRCNC) in the MT promoter. It was previously shown that MTF-1 rapidly translocates to the nucleus in response to zinc exposure. However, regions within the MTF-1 protein that serve as nuclear localization signal (NLS) remain unclear. In our study, enhanced green fluorescent protein (eGFP) was fused with MTF-1, and their subcellular localization was examined by fluorescence microscopy and Western blot. Fusion of eGFP with the first two zinc fingers of MTF-1 and with the last four zinc fingers of MTF-1 respectively led the protein to accumulate in the nucleus. Thus, we suggest that there may be two NLS with independent functions in MTF-1. Furthermore, we demonstrated neither the tertiary structure of the zinc fingers nor the DNA binding activity of the protein are necessary for nuclear localization. We also noted that the N-terminal domain of MTF-1 (1-136 amino acid) appears to inhibit nuclear localization. We create the mutant of MTF-1 containing histidine to alanine mutations that disrupt the structure of each zinc individually to identify the different significance of each zinc figure. At the same time, we observe tertiary structure of zinc finger also involved in regulating nuclear import. These data suggest that two nuclear localization signals in the zinc finger domain direct MTF-1 to nucleus and that N-terminal domain and six zinc fingers regulate nuclear import of MTF-1 cooperatively.