title: Role of cysteine residues in cell surface expression of the human riboflavin transporter-2 (hRFT2) in intestinal epithelial cells.
abstract: The water-soluble vitamin B2 (riboflavin, RF) is an essential micronutrient for normal cell function and survival. Recent studies have identified a role for the human riboflavin transporter-2 (hRFT2) in normal intestinal RF absorption. However, little is known about the cell biology of this transporter and specifically about the molecular determinant(s) that dictate its cell surface expression in human intestinal epithelial cells. Here we show that the full-length hRFT2 protein fused to green fluorescent protein (GFP) (GFP-hRFT2) is expressed exclusively at the apical membrane domain of Caco-2 cells. COOH-terminal sequence was essential in dictating cell surface expression with a specific role for conserved cysteine residues (C463 and C467). Mutation of C463 and C467 ablated RF uptake, explained by retention of the constructs within the endoplasmic reticulum. Modeling analysis suggested a potential disulfide bridge between C463 and C386. Consistent with this prediction, mutating the C386 site in the context of the full-length transporter resulted in intracellular retention, whereas mutation of another conserved cysteine (C326A) was without effect on hRFT2 targeting. Intracellular trafficking of hRFT2 was also examined and appeared to involve distinct vesicular structures, the motility of vesicles critically dependent on an intact microtubule network. These results demonstrate a potential role for specific cysteine residues in the cell surface expression of the hRFT2 in human intestinal epithelial cells.
authors.affiliation: Department of Medicine, University of California Medical School, Irvine, USA.
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Laramie
authors.initials: L
authors.lastname: Rapp
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marchant
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D026901:Membrane Transport Proteins; C548553:SLC52A3 protein, human; D003545:Cysteine