title: Identification and functional characterization of the Caenorhabditis elegans riboflavin transporters rft-1 and rft-2.
abstract: Two potential orthologs of the human riboflavin transporter 3 (hRFVT3) were identified in the C. elegans genome, Y47D7A.16 and Y47D7A.14, which share 33.7 and 30.5% identity, respectively, with hRFVT3. The genes are tandemly arranged, and we assign them the names rft-1 (for Y47D7A.16) and rft-2 (for Y47D7A.14). Functional characterization of the coding sequences in a heterologous expression system demonstrated that both were specific riboflavin transporters, although the rft-1 encoded protein had greater transport activity. A more detailed examination of rft-1 showed its transport of riboflavin to have an acidic pH dependence, saturability (apparent Km = 1.4 ± 0.5 µM), inhibition by riboflavin analogues, and Na(+) independence. The expression of rft-1 mRNA was relatively higher in young larvae than in adults, and mRNA expression dropped in response to RF supplementation. Knocking down the two transporters individually via RNA interference resulted in a severe loss of fertility that was compounded in a double knockdown. Transcriptional fusions constructed with two fluorophores (rft-1::GFP, and rft-2::mCherry) indicated that rft-1 is expressed in the intestine and a small subset of neuronal support cells along the entire length of the animal. Expression of rft-2 is localized mainly to the intestine and pharynx. We also observed a drop in the expression of the two reporters in animals that were maintained in high riboflavin levels. These results report for the first time the identification of two riboflavin transporters in C. elegans and demonstrate their expression and importance to metabolic function in worms. Absence of transporter function renders worms sterile, making them useful in understanding human disease associated with mutations in hRFVT3.
authors.affiliation: Department of Medicine, University of California Medical School, Irvine, California, USA.
authors.forename: Arundhati
authors.initials: A
authors.lastname: Biswas
authors.forename: Daniel
authors.initials: D
authors.lastname: Elmatari
authors.forename: Jason
authors.initials: J
authors.lastname: Rothman
authors.forename: Craig W
authors.initials: CW
authors.lastname: LaMunyon
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism; D007814:Larva / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D043562:Receptors, G-Protein-Coupled; D012256:Riboflavin / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D000097573:Red Fluorescent Protein
chemical_list: D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D043562:Receptors, G-Protein-Coupled; D012256:Riboflavin; D000097573:Red Fluorescent Protein; C583178:Rft-1 protein, C elegans; C583179:Rft-2 protein, C elegans; C540350:SLC52A2 protein, human