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. 2000 Mar 1;346(Pt 2):509–518.

Mutations in the reduced-folate carrier affect protein localization and stability.

H Sadlish 1, R C Murray 1, F M Williams 1, W F Flintoff 1
PMCID: PMC1220880  PMID: 10677373

Abstract

The reduced-folate-carrier (rfc) gene has been shown to be functionally important for reduced-folate transport in mammalian cells. In the present paper we describe the identification of alterations in both alleles of the rfc gene in a mutant Chinese-hamster ovary cell line deficient in methotrexate transport. One allele of the rfc gene contains a point mutation resulting in a Gly(345)-->Arg substitution in the predicted amino acid sequence. In this case, a protein of similar size to the wild-type protein is produced, although it remains as an immature, core-glycosylated, form. The second allele contains a point mutation in the last base of intron 5 that results in the utilization of a cryptic splice site leading to a seven-base deletion in the mRNA. The use of an alternate splice site changes the reading frame to yield a truncated protein with 68 different C-terminal amino acids as compared with the wild-type. Both of these altered gene products were monitored by fusion with green fluorescent protein and found to be non-functional with an increased rate of turnover. The protein with the point mutation is trapped in the endoplasmic reticulum with subsequent degradation, whereas the product of the splice mutation is not membrane-associated and is partially degraded. Thus mutations in both alleles of the rfc gene in this resistant cell line account for the loss of reduced-folate transport. The observations made regarding the degradation of these mutant gene products also provide support for putative checkpoints in the endoplasmic reticulum.

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Selected References

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  1. Brigle K. E., Spinella M. J., Sierra E. E., Goldman I. D. Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line. J Biol Chem. 1995 Sep 29;270(39):22974–22979. doi: 10.1074/jbc.270.39.22974. [DOI] [PubMed] [Google Scholar]
  2. Brigle K. E., Spinella M. J., Sierra E. E., Goldman I. D. Organization of the murine reduced folate carrier gene and identification of variant splice forms. Biochim Biophys Acta. 1997 Aug 7;1353(2):191–198. doi: 10.1016/s0167-4781(97)00082-1. [DOI] [PubMed] [Google Scholar]
  3. Chang X. B., Cui L., Hou Y. X., Jensen T. J., Aleksandrov A. A., Mengos A., Riordan J. R. Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis. Mol Cell. 1999 Jul;4(1):137–142. doi: 10.1016/s1097-2765(00)80196-3. [DOI] [PubMed] [Google Scholar]
  4. Denning G. M., Anderson M. P., Amara J. F., Marshall J., Smith A. E., Welsh M. J. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature. 1992 Aug 27;358(6389):761–764. doi: 10.1038/358761a0. [DOI] [PubMed] [Google Scholar]
  5. Dick L. R., Cruikshank A. A., Destree A. T., Grenier L., McCormack T. A., Melandri F. D., Nunes S. L., Palombella V. J., Parent L. A., Plamondon L. Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells. J Biol Chem. 1997 Jan 3;272(1):182–188. doi: 10.1074/jbc.272.1.182. [DOI] [PubMed] [Google Scholar]
  6. Dixon K. H., Lanpher B. C., Chiu J., Kelley K., Cowan K. H. A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells. J Biol Chem. 1994 Jan 7;269(1):17–20. [PubMed] [Google Scholar]
  7. Ferguson P. L., Flintoff W. F. Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion. J Biol Chem. 1999 Jun 4;274(23):16269–16278. doi: 10.1074/jbc.274.23.16269. [DOI] [PubMed] [Google Scholar]
  8. Flintoff W. F., Davidson S. V., Siminovitch L. Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells. Somatic Cell Genet. 1976 May;2(3):245–261. doi: 10.1007/BF01538963. [DOI] [PubMed] [Google Scholar]
  9. Flintoff W. F., Nagainis C. R. Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding. Arch Biochem Biophys. 1983 Jun;223(2):433–440. doi: 10.1016/0003-9861(83)90607-0. [DOI] [PubMed] [Google Scholar]
  10. Flintoff W. F., Spindler S. M., Siminovitch L. Genetic characterization of methotrexate-resistant chinese hamster ovary cells. In Vitro. 1976 Nov;12(11):749–757. doi: 10.1007/BF02835450. [DOI] [PubMed] [Google Scholar]
  11. Galivan J. 5-Methyltetrahydrofolate transport by hepatoma cells and methotrexate-resistant sublines in culture. Cancer Res. 1981 May;41(5):1757–1762. [PubMed] [Google Scholar]
  12. Goldman I. D. The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann N Y Acad Sci. 1971 Nov 30;186:400–422. doi: 10.1111/j.1749-6632.1971.tb46996.x. [DOI] [PubMed] [Google Scholar]
  13. Goldman I. D. Transport energetics of the folic acid analogue, methotrexate, in L1210 leukemia cells. Enhanced accumulation by metabolic inhibitors. J Biol Chem. 1969 Jul 25;244(14):3779–3785. [PubMed] [Google Scholar]
  14. Gong M., Yess J., Connolly T., Ivy S. P., Ohnuma T., Cowan K. H., Moscow J. A. Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line. Blood. 1997 Apr 1;89(7):2494–2499. [PubMed] [Google Scholar]
  15. Gorlick R., Goker E., Trippett T., Waltham M., Banerjee D., Bertino J. R. Intrinsic and acquired resistance to methotrexate in acute leukemia. N Engl J Med. 1996 Oct 3;335(14):1041–1048. doi: 10.1056/NEJM199610033351408. [DOI] [PubMed] [Google Scholar]
  16. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  17. Jackson R. C., Hart L. I., Harrap K. R. Intrinsic resistance to methotrexate of cultured mammalian cells in relation to the inhibition kinetics of their dihydrololate reductases. Cancer Res. 1976 Jun;36(6):1991–1997. [PubMed] [Google Scholar]
  18. Jansen G., Mauritz R., Drori S., Sprecher H., Kathmann I., Bunni M., Priest D. G., Noordhuis P., Schornagel J. H., Pinedo H. M. A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance. J Biol Chem. 1998 Nov 13;273(46):30189–30198. doi: 10.1074/jbc.273.46.30189. [DOI] [PubMed] [Google Scholar]
  19. Jentsch S., Schlenker S. Selective protein degradation: a journey's end within the proteasome. Cell. 1995 Sep 22;82(6):881–884. doi: 10.1016/0092-8674(95)90021-7. [DOI] [PubMed] [Google Scholar]
  20. Loo T. W., Clarke D. M. Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein. J Biol Chem. 1998 Dec 4;273(49):32373–32376. doi: 10.1074/jbc.273.49.32373. [DOI] [PubMed] [Google Scholar]
  21. Matherly L. H., Czajkowski C. A., Angeles S. M. Identification of a highly glycosylated methotrexate membrane carrier in K562 human erythroleukemia cells up-regulated for tetrahydrofolate cofactor and methotrexate transport. Cancer Res. 1991 Jul 1;51(13):3420–3426. [PubMed] [Google Scholar]
  22. Moscow J. A., Gong M., He R., Sgagias M. K., Dixon K. H., Anzick S. L., Meltzer P. S., Cowan K. H. Isolation of a gene encoding a human reduced folate carrier (RFC1) and analysis of its expression in transport-deficient, methotrexate-resistant human breast cancer cells. Cancer Res. 1995 Sep 1;55(17):3790–3794. [PubMed] [Google Scholar]
  23. Murray R. C., Williams F. M., Flintoff W. F. Structural organization of the reduced folate carrier gene in Chinese hamster ovary cells. J Biol Chem. 1996 Aug 9;271(32):19174–19179. doi: 10.1074/jbc.271.32.19174. [DOI] [PubMed] [Google Scholar]
  24. Nguyen T. T., Dyer D. L., Dunning D. D., Rubin S. A., Grant K. E., Said H. M. Human intestinal folate transport: cloning, expression, and distribution of complementary RNA. Gastroenterology. 1997 Mar;112(3):783–791. doi: 10.1053/gast.1997.v112.pm9041240. [DOI] [PubMed] [Google Scholar]
  25. Niethammer D., Jackson R. C. Changes of molecular properties associated with the development of resistance against methotrexate in human lymphoblastoid cells. Eur J Cancer. 1975 Dec;11(12):845–854. doi: 10.1016/0014-2964(75)90083-3. [DOI] [PubMed] [Google Scholar]
  26. Prasad P. D., Ramamoorthy S., Leibach F. H., Ganapathy V. Molecular cloning of the human placental folate transporter. Biochem Biophys Res Commun. 1995 Jan 17;206(2):681–687. doi: 10.1006/bbrc.1995.1096. [DOI] [PubMed] [Google Scholar]
  27. Roy K., Tolner B., Chiao J. H., Sirotnak F. M. A single amino acid difference within the folate transporter encoded by the murine RFC-1 gene selectively alters its interaction with folate analogues. Implications for intrinsic antifolate resistance and directional orientation of the transporter within the plasma membrane of tumor cells. J Biol Chem. 1998 Jan 30;273(5):2526–2531. doi: 10.1074/jbc.273.5.2526. [DOI] [PubMed] [Google Scholar]
  28. Said H. M., Nguyen T. T., Dyer D. L., Cowan K. H., Rubin S. A. Intestinal folate transport: identification of a cDNA involved in folate transport and the functional expression and distribution of its mRNA. Biochim Biophys Acta. 1996 Jun 11;1281(2):164–172. doi: 10.1016/0005-2736(96)00005-3. [DOI] [PubMed] [Google Scholar]
  29. Sirotnak F. M. Obligate genetic expression in tumor cells of a fetal membrane property mediating "folate" transport: biological significance and implications for improved therapy of human cancer. Cancer Res. 1985 Sep;45(9):3992–4000. [PubMed] [Google Scholar]
  30. Tolner B., Roy K., Sirotnak F. M. Organization, structure and alternate splicing of the murine RFC-1 gene encoding a folate transporter. Gene. 1997 Apr 11;189(1):1–7. doi: 10.1016/s0378-1119(96)00676-2. [DOI] [PubMed] [Google Scholar]
  31. Tolner B., Roy K., Sirotnak F. M. Structural analysis of the human RFC-1 gene encoding a folate transporter reveals multiple promoters and alternatively spliced transcripts with 5' end heterogeneity. Gene. 1998 May 12;211(2):331–341. doi: 10.1016/s0378-1119(98)00123-1. [DOI] [PubMed] [Google Scholar]
  32. Tse A., Brigle K., Taylor S. M., Moran R. G. Mutations in the reduced folate carrier gene which confer dominant resistance to 5,10-dideazatetrahydrofolate. J Biol Chem. 1998 Oct 2;273(40):25953–25960. doi: 10.1074/jbc.273.40.25953. [DOI] [PubMed] [Google Scholar]
  33. Underhill T. M., Flintoff W. F. Mutant Chinese hamster ovary cells with defective methotrexate uptake are distinguishable by reversion analysis. Somat Cell Mol Genet. 1989 Jan;15(1):49–59. doi: 10.1007/BF01534669. [DOI] [PubMed] [Google Scholar]
  34. Ward C. L., Omura S., Kopito R. R. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 1995 Oct 6;83(1):121–127. doi: 10.1016/0092-8674(95)90240-6. [DOI] [PubMed] [Google Scholar]
  35. Williams F. M., Flintoff W. F. Isolation of a human cDNA that complements a mutant hamster cell defective in methotrexate uptake. J Biol Chem. 1995 Feb 17;270(7):2987–2992. doi: 10.1074/jbc.270.7.2987. [DOI] [PubMed] [Google Scholar]
  36. Williams F. M., Flintoff W. F. Structural organization of the human reduced folate carrier gene: evidence for 5' heterogeneity in lymphoblast mRNA. Somat Cell Mol Genet. 1998 May;24(3):143–156. doi: 10.1023/b:scam.0000007117.50428.63. [DOI] [PubMed] [Google Scholar]
  37. Williams F. M., Murray R. C., Underhill T. M., Flintoff W. F. Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake. J Biol Chem. 1994 Feb 25;269(8):5810–5816. [PubMed] [Google Scholar]
  38. Wong S. C., Proefke S. A., Bhushan A., Matherly L. H. Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells. J Biol Chem. 1995 Jul 21;270(29):17468–17475. doi: 10.1074/jbc.270.29.17468. [DOI] [PubMed] [Google Scholar]
  39. Wong S. C., Zhang L., Witt T. L., Proefke S. A., Bhushan A., Matherly L. H. Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier. J Biol Chem. 1999 Apr 9;274(15):10388–10394. doi: 10.1074/jbc.274.15.10388. [DOI] [PubMed] [Google Scholar]
  40. Zhang L., Wong S. C., Matherly L. H. Transcript heterogeneity of the human reduced folate carrier results from the use of multiple promoters and variable splicing of alternative upstream exons. Biochem J. 1998 Jun 15;332(Pt 3):773–780. doi: 10.1042/bj3320773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhao R., Assaraf Y. G., Goldman I. D. A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function. J Biol Chem. 1998 Jul 24;273(30):19065–19071. doi: 10.1074/jbc.273.30.19065. [DOI] [PubMed] [Google Scholar]
  42. Zhao R., Assaraf Y. G., Goldman I. D. A reduced folate carrier mutation produces substrate-dependent alterations in carrier mobility in murine leukemia cells and methotrexate resistance with conservation of growth in 5-formyltetrahydrofolate. J Biol Chem. 1998 Apr 3;273(14):7873–7879. doi: 10.1074/jbc.273.14.7873. [DOI] [PubMed] [Google Scholar]
  43. Zhao R., Sharina I. G., Goldman I. D. Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis. Mol Pharmacol. 1999 Jul;56(1):68–76. [PubMed] [Google Scholar]

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