Role of cysteine residues in cell surface expression of the human riboflavin transporter-2 (hRFT2) in intestinal epithelial cells

Veedamali S. Subramanian, Laramie Rapp, Jonathan S. Marchant, Hamid M. Said

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)G100-G109
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume301
Issue number1
DOIs
StatePublished - Jul 2011

Keywords

  • Apical
  • Epithelia
  • Transport
  • Vitamin B2

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