title: Restoration of transport activity by co-expression of human reduced folate carrier half-molecules in transport-impaired K562 cells: localization of a substrate binding domain to transmembrane domains 7-12.
abstract: Reduced folates such as 5-methyl tetrahydrofolate and classical antifolates such as methotrexate are actively transported into mammalian cells by the reduced folate carrier (RFC). RFC is characterized by 12 stretches of mostly hydrophobic, alpha-helix-promoting amino acids, internally oriented N and C termini, and a large central linker connecting transmembrane domains (TMDs) 1-6 and 7-12. Previous studies showed that deletion of the majority of the central loop domain between TMDs 6 and 7 abolished transport, but this segment could be replaced with mostly non-homologous sequence from the SLC19A2 thiamine transporter to restore transport function. In this report, we expressed RFC from separate TMD1-6 and TMD7-12 RFC half-molecule constructs, each with a unique epitope tag, in RFC-null K562 cells to restore transport activity. Restored transport exhibited characteristic transport kinetics for methotrexate, a capacity for trans-stimulation by pretreatment with leucovorin, and inhibition by N-hydroxysuccinimide methotrexate, a documented affinity inhibitor of RFC. The TMD1-6 half-molecule migrated on SDS gels as a 38-58 kDa glycosylated species and was converted to 27 kDa by N-glycosidase F or tunicamycin treatments. The 40 kDa TMD7-12 half-molecule was unaffected by these treatments. Using transfected cells expressing both TMDs 1-6 and TMDs 7-12 as separate polypeptides, the TMD7-12 half-molecule was covalently radiolabeled with N-hydroxysuccinimide [(3)H]methotrexate. No radioactivity was incorporated into the TMD1-6 half-molecule. Digestion with endoproteinase GluC decreased the size of the radiolabeled 40 kDa TMD7-12 polypeptide to approximately 20 kDa. Our results demonstrate that a functional RFC can be reconstituted with RFC half-molecules and localize a critical substrate binding domain to within TMDs 7-12.
authors.affiliation: Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Teah L
authors.initials: TL
authors.lastname: Witt
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Stapels
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry; D005492:Folic Acid / Q000737:chemistry; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D007700:Kinetics; D002955:Leucovorin / Q000494:pharmacology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000494:pharmacology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058978:Reduced Folate Carrier Protein; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D013831:Thiamine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000939:Epitopes; D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D010455:Peptides; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C039702:methotrexate N-hydroxysuccinimide ester; D014415:Tunicamycin; D012967:Sodium Dodecyl Sulfate; D005492:Folic Acid; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002955:Leucovorin; D013831:Thiamine; D008727:Methotrexate