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 va riety 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 differe nt reports to be exclusively cytoplasmic and predominantly nuclear. In the present study, we further confirm that FANCA localizes primarily to the nuc leus, Fusions of FANCA with the green fluorescent protein (GFP) showed a st rong nuclear signal and a weak cytoplasmic signal in several cell types, Co nfocal laser microscopy confirmed that FANCA is evenly distributed througho ut the nucleus, We also examined regions in FANCA that participate in its n uclear import, FANCA contains two bipartite nuclear localization signal (NL S) 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 del etion 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 w ith only a cytoplasmic signal, and with both a nuclear and cytoplasmic sign al. Deletion of the N-terminal 250 amino acids was required to block nuclea r 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 resu lts show that nuclear localization of FANCA involves several functional reg ions.