Characterization of regions functional in the nuclear localization of the Fanconi anemia group A protein

Fanconi anemia (FA) is an autosomal recessive disease characterized by a variety of congenital abnormalities, Cells from FA patients show chromosomal instability and are hypersensitive to DNA cross-linking agents, though the basic cellular defect in FA is not known. The FANCA gene encodes a protein with an M-r of 162 kDa and with unknown function. The cellular localization of the FANCA protein has been controversial, and has been shown in different reports to be exclusively cytoplasmic and predominantly nuclear. In the present study, we further confirm that FANCA localizes primarily to the nucleus, Fusions of FANCA with the green fluorescent protein (GFP) showed a strong nuclear signal and a weak cytoplasmic signal in several cell types, Confocal laser microscopy confirmed that FANCA is evenly distributed throughout the nucleus, We also examined regions in FANCA that participate in its nuclear import, FANCA contains two bipartite nuclear localization signal (NLS) motifs at the extreme N-terminus. Deletion of amino acids N-terminal to the NLS motifs had no effect on the nuclear localization of FANCA or on its ability to correct mitomycin C sensitivity in an FA-A cell line, while deletion of both motifs impeded but did not prevent nuclear import. Deletions of 75, 90 and 150 residues from the N-terminus yielded a mixture of cells with only a cytoplasmic signal, and with both a nuclear and cytoplasmic signal. Deletion of the N-terminal 250 amino acids was required to block nuclear localization completely. Fusion of GFP to the N-terminal 250 amino acids showed a localization pattern similar to FANCA. Mutant forms of FANCA with deletions of the C-terminal 70 or 260 residues localized to the cytoplasm, although the C-terminal 260 amino acids alone lacked NLS activity, The results show that nuclear localization of FANCA involves several functional regions.