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

Abstract

The basis for impaired reduced folate carrier (RFC) activity in methotrexate-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 ~ 85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cells, 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 (P1; changes glycine 44 → arginine) and 380 (P2; changes serine 127 → asparagine). A 4-base pair (CATG) insertion detected at position 191 (in 19-30% of cDNA clones) resulted in a frameshift and early translation termination. Wild-type RFC was also detected (0-9% of clones). Wild-type RFC and double-mutated RFC (RFC(P1+p2)) cDNAs were transfected into transport-impaired K562 and Chinese hamster ovary cells. Although RFC transcripts paralleled wild-type protein, for the RFC(P1+P2) transfectants, disproportionately low RFC(P1+P2) protein was detected. This reflected an increased turnover of RFC(P1+P2) over wild-type RFC. RFC(P1+P2) did not restore methotrexate transport; however, uptake was partially restored by constructs with single mutations at the P1 or P2 loci. Cumulatively, our results show that loss of transport function in CEM/Mtx-1 cells results from complete loss of RFC protein due to early translation termination and increased turnover of a mutant RFC protein.