CYTOPLASMIC LOCALIZATION OF A FUNCTIONALLY ACTIVE FANCONI-ANEMIA GROUP-A GREEN FLUORESCENT PROTEIN CHIMERA IN HUMAN 293-CELLS

Hypersensitivity to cross-linking agents and predisposition to maligna ncy 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 exp ected to localize in the nucleus as based on the presence of sequences homologous to a bipartite nuclear localization signal (NLS) and a leu cine repeat motif. In contrast to this expectation, we show here that a functionally active FAA-green fluorescent protein (GFP) hybrid resid es in the cytoplasmic compartment of human kidney 293 cells, In accord ance with this finding, disruption of the putative NLS by site-directe d mutagenesis failed to affect both subcellular localization and the c apacity to complement hypersensitivity to the cross-linking agent mito mycin 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 nuclea r translocation activity when fused to GFP, while the first 115 N-term inal amino acids appeared to be indispensable for the complementing ac tivity in FA-A cells. Similarly, mutagenesis studies of the putative l eucine repeat showed that, even though this region of the protein is i mportant for complementing activity, this activity does not depend on an intact leucine zipper motif. Finally, fusion of the NLS motif deriv ed from the SV40 large T antigen to FAA could not direct the hybrid pr otein into the nucleus of 293 cells, suggesting that FAA is somehow ma intained in the cytoplasm via currently unknown mechanisms, Thus, like the first identified FA protein, FAG, FAA seems to exert its function in the cytoplasmic compartment suggesting FA proteins to he active in a yet to be elucidated cytoplasmic pathway that governs hematopoiesis and protects against genomic instability. (C) 1997 by The American So ciety of Hematology.