Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier

The basis for impaired reduced folate carrier (RFC) activity in methotrexat e-resistant CCRF-CEM (CEM/Mtx-1) cells was examined. Parental and CEM/Mtx-1 cells expressed identical levels of the 3.1-kilobase RFC transcript. A sim ilar to 85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cel ls, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and sequence analysis, G to A point mutations were identified in CEM/Mtx-1 transcripts at positions 130 (P-1; changes glycine 44 --> arginin e) and 380 (P-2; changes serine 127 --> asparagine), A 4-base pair (CATG) i nsertion detected at position 191 (in 19-30% of cDNA clones) resulted in a frameshift and early translation termination. Wild-type RFC was also detect ed (0-9% of clones). Wild-type RFC and double-mutated RFC (RFCP1+P2) cDNAs mere transfected into transport-impaired K562 and Chinese hamster ovary cel ls. Although RFC transcripts paralleled wild-type protein, for the RFCP1+P2 transfectants, disproportionately low RFCP1+P2 protein was detected, This reflected an increased turnover of RFCP1+P2 over wild type RFC, RFCP1+P2 di d not restore methotrexate transport; however, uptake was partially restore d by constructs with single mutations at the P-1 or P-2 loci. Cumulatively, our results show that loss of transport function in CEM/Mtx-1 cells result s from complete loss of RFC protein due to early translation termination an d increased turnover of a mutant RFC protein.