Multiple folate transport systems in L1210 cells.
Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 microM transporter is a non-glycosylated, integral membrane protein, while its nM counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.
Fan
J
J
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
Vitols
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KS
Huennekens
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D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D007939:Leukemia L1210 / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D051379:Mice; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; C075939:biotin-SS-folate; C075938:biotin-SS-methotrexate; D001710:Biotin; D005492:Folic Acid; D008727:Methotrexate