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 M), 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.