Submitted on January 11, 2008
Revised on July 10, 2008
Accepted on July 10, 2008
Identification and Functional Characterization of a Novel Human and Rat Riboflavin Transporter, RFT1
Atsushi Yonezawa, Satohiro Masuda1, Toshiya Katsura, and Ken-ichi Inui2*
1 Kyoto University
2 Kyoto University Hospital
* To whom correspondence should be addressed. E-mail: inui{at}kuhp.kyoto-u.ac.jp.
Absorption of riboflavin is mediated by transporter(s). However, a mammalian riboflavin transporter has yet to be identified. In the present study, novel human and rat riboflavin transporters hRFT1 and rRFT1 were identified based on our rat kidney mRNA expression database [Horiba N, et al. Kidney Int 66: 29-45, 2004]. The hRFT1 and rRFT1 cDNAs have an open-reading frame encoding 448- and 450-amino acid proteins, respectively, that exhibit 81.1% identity and 96.4% similarity to each other. In addition, an inactive splice variant of hRFT1, hRFT1sv, was also cloned. The hRFT1sv cDNA, which encodes a 167-amino acid protein, retains an intron between exons 2 and 3 of hRFT1. Real-time PCR revealed that the sum of hRFT1 and hRFT1sv mRNAs was expressed strongly in the placenta and small intestine, and was detected in all tissues examined. In addition, hRFT1 and hRFT1sv were expressed in HEK293 and Caco-2 cells. HEK293 cells transfected with green fluorescent protein-tagged hRFT1 and rRFT1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [3H]riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [3H]riboflavin by HEK293 and Caco-2 cells. Riboflavin transport is Na+-, potential- and pH-independent. Kinetic analyses demonstrated that the Michaelis-Menten constants for the uptake by HEK293 and Caco-2 cells were 28.1 and 63.7 nM, respectively. We propose that hRFT1 and rRFT1 are novel mammalian riboflavin transporters, which belong to a new mammalian riboflavin transporter family.
