Cytoplasmic localization of a functionally active Fanconi anemia group A-green fluorescent protein chimera in human...

Hypersensitivity to cross-linking agents and predisposition to malignancy are characteristic of the genetically heterogeneous inherited bone marrow failure syndrome, Fanconi anemia (FA). The protein encoded by the recently cloned FA complementation group A gene, FAA, has been expected to localize in the nucleus as based on the presence of sequences homologous to a bipartite nuclear localization signal (NLS) and a leucine repeat motif. In contrast to this expectation, we show here that a functionally active FAA-green fluorescent protein (GFP) hybrid resides in the cytoplasmic compartment of human kidney 293 cells. In accordance with this finding, disruption of the putative NLS by site-directed mutagenesis failed to affect both subcellular localization and the capacity to complement hypersensitivity to the cross-linking agent mitomycin C in FA-A lymphoblasts. Furthermore, the N-terminal part of FAA with the putative NLS at amino acid position 18 to 35 showed no nuclear translocation activity when fused to GFP, while the first 115 N-terminal amino acids appeared to be indispensable for the complementing activity in FA-A cells. Similarly, mutagenesis studies of the putative leucine repeat showed that, even though this region of the protein is important for complementing activity, this activity does not depend on an intact leucine zipper motif. Finally, fusion of the NLS motif derived from the SV40 large T antigen to FAA could not direct the hybrid protein into the nucleus of 293 cells, suggesting that FAA is somehow maintained in the cytoplasm via currently unknown mechanisms. Thus, like the first identified FA protein, FAC, FAA seems to exert its function in the cytoplasmic compartment suggesting FA proteins to be active in a yet to be elucidated cytoplasmic pathway that governs hematopoiesis and protects against genomic instability.