Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans

Two putative orthologs to the human reduced folate carrier (hRFC), folt-1 and folt-2, which share a 40 and 31% identity, respectively, with the hRFC sequence, have been identified in the Caenorhabditis elegans genome. Functional characterization of the open reading frame of the putative folt-1 and folt-2 showed folt-1 to be a specific folate transporter. Transport of folate by folt-1 expressed in a heterologous expression system showed an acidic pH dependence, saturability (apparent K(m) of 1.23 +/- 0.18 microM), a similar degree of inhibition by reduced and substituted folate derivatives, sensitivity to the anti-inflammatory drug sulfasalazine (apparent K(i) of 0.13 mM), and inhibition by anion transport inhibitors, e.g., DIDS. Knocking down (silencing) or knocking out the folt-1 gene led to a significant inhibition of folate uptake by intact living C. elegans. We also cloned the 5'-regulatory region of the folt-1 gene and confirmed promoter activity of the construct in vivo in living C. elegans. With the use of the transcriptional fusion construct (i.e., folt-1::GFP), the expression pattern of folt-1 in different tissues of living animal was found to be highest in the pharynx and intestine. Furthermore, folt-1::GFP expression was developmentally and adaptively regulated in vivo. These studies demonstrate for the first time the existence of a specialized folate uptake system in C. elegans that has similar characteristics to the folate uptake process of the human intestine. Thus C. elegans provides a genetically tractable model that can be used to study integrative aspects of the folate uptake process in the context of the whole animal level.