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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.

Identification and Functional Characterization of the Caenorhabditis elegans Riboflavin Transporters rft-1 and rft-2