study on the factors determining nuclear translocation of metal-responsive transcription factor-1

• Main Authors: Hung-Chih Chen, 陳泓志
• Other Authors: Lih-Yuan Lin
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/34642008225658467246

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 95 === Metal responsive transcription factor 1 (MTF-1) is a transcription factor that contains six C2H2 zinc fingers, and is essential for the basal and zinc-inducing expression of metallothionein. It was shown previously that the nuclear entry of MTF-1 could be induced by zinc. However, the region serving as nuclear localization signal (NLS) and nuclear import mechanism remain unclear. Here we generated a series of eGFP-tagged deletion mutants of MTF-1 and investigated the subcellular localization of these fusion proteins by fluorescence microscopy. We found that zinc finger domain of MTF-1 might contain an NLS. Using GST pull-down assay, we were able to demonstrate that MTF-1 binds to a variety of importin α families. Further analysis of the individual zinc finger showed that only zinc finger 3 and 4 bound importin α1. We also found that R246 and K247 adjacent to the first constituent His of MTF-1 zinc finger 4 are crucial for the nuclear transport of MTF-1 since mutation of these sites impaired the function of nuclear translocation of MTF-1 and its binding ability to importin α1. Furthermore, fusion protein of 6 zinc fingers with the classical NLS ahead of the zinc finger showed a better nuclear import than 6 zinc fingers alone. Overall, our results indicate that nuclear entry of MTF-1 after zinc induction is mediated by the NLS within the zinc finger domain. Among which, zinc finger 4 plays the most important role. Besides, the classical NLS adjacent to zinc finger domain enhances the nuclear translocation. Presence of either importin family is required for the translocation of MTF-1.