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Heterokaryon Technique for Analysis of Cell Type-specific Localization
Published on: March 11, 2011
Characterization of the nuclear import pathway for BLM protein
Zhiqiang Duan1, Jiafu Zhao1, Houqiang Xu1
1Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China; College of Animal Science, Guizhou University, Guiyang 550025, China.
Abstract:
Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin β1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin β1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin β1 through its NLS domain with the 14-16 HEAT repeats of importin β1. Furthermore, importin β1, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin β1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase.
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