title: Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells.
abstract: The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, California 92697, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marchant
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Ian
authors.initials: I
authors.lastname: Parker
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D008870:Microtubules / Q000378:metabolism; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D049452:Green Fluorescent Proteins