THE FANCONI-ANEMIA GROUP-C GENE-PRODUCT IS LOCATED IN BOTH THE NUCLEUS AND CYTOPLASM OF HUMAN-CELLS

The Fanconi anemia (FA) complementation group C (FAC) protein gene enc odes a cytoplasmic protein with a predicted M-r of 63,000. The protein 's function is unknown, but it has been hypothesized that it either me diates resistance to DNA cross-linking agents or facilitates repair af ter exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E). Attr ibuting a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has establ ished the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Pur ified recombinant Escherichia coli-derived FAC antigens were used to c reate antisera able to specifically identify an M-r = 58,000 protein i n lysates from human Epstein-Barr virus (EBV) transformed cell lines b y immunoblot analysis. Subcellular fractionation of the cell lysates f ollowed by immunoblot analysis revealed that the majority of the FAC p rotein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. Th ese results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 2 93 cells engineered to express FAC clearly demonstrated that FAC prote in is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene trans fer resulted in a substantial increase in nuclear FAC protein. Therefo re, while cytoplasmic localization of this protein appears to be funct ionally important, it may also exert some essential nuclear function. (C) 1998 by The American Society of Hematology.