Localization of a human reduced folate carrier protein in the mitochondrial as well as the cell membrane of leukemia cells

IgG polyclonal antiserum was generated in New Zealand White rabbits immuniz ed with a 16-mer peptide consisting of a specific amino acid sequence at re sidues corresponding to the sixth to seventh predicted transmembrane domain of the human reduced folate carrier (RFC), Using Western immunoblotting to examine the cytosolic and membrane fractions of the human CCRF-CEM T-cell lymphoblastic leukemia cell line, polyclonal antihuman RFC antiserum recogn ized two bands in the cytosolic fraction (approximately 60 kDa and approxim ately 70 kDa) on 10% polyacrylamide gels. In the membrane fraction, an appr oximately 60-kDa protein was identified. Comparative studies of a panel of human tumor cell lines including the HT1080 fibrosarcoma, 8805 malignant fi brous histiocytoma, and the MCF breast cancer cell lines revealed similar f indings. Likewise, a recombinant approximately 60-kDa membrane protein was identified after expression of baculovirus-infected Sf9 insect cells contai ning cDNA of the human RFC, In the CEM-7A cell line, a variant of the CCRF- CEM cell fine that overexpresses the RFC, 21-fold overexpression of the app roximately 60-kDa membrane protein (RFC) was shown by Western analysis. To characterize further the cellular distribution of the human RFC, immunohist ochemical analyses were performed in CCRF-CEM T-cell lymphoblastic leukemia cells. Predominantly membrane localization of the antibody reacting sites was detected; however, a cytoplasmic component was noted as well. By confoc al microscopy and by immunogold electron microscopy, the cytoplasmic expres sion was found to be largely of mitochondrial origin. These findings were c orroborated by Western immunoblotting of mitochondrial membrane isolates fr om the CCRF-CEM cell line, which demonstrate an approximately 60-kDa protei n, The localization of the human RFC to the mitochondrial membrane is a nov el finding, and it suggests a role for the mitochondrial membrane in the tr ansport of folates.