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.

Figure 1. Exonic and protein structure of riboflavin transporters.

(A) The tandem arrangement of rft-1 and rft-2 on chromosome V. Note that rft-2 is alternatively spliced. Hash marks along the chromosome indicate 1,000 bp intervals. (B) Amino acid alignment of rft-1, rft-2, and the putative human homolog, hRFVT3. Shaded regions indicate predicted transmembrane domains. (C) The locations of the transmembrane domains and intracellular and extracellular loops. The numbered bar indicates amino acid position.

Figure 2. Functional identification of the cloned rft-1 and rft-2 as a specific riboflavin transporter in ARPE-19 cells.

Confluent ARPE-19 cells expressing rft-1 and rft-2 were incubated with [³H] riboflavin (14 nM; 5 min at 37°C in Krebs-Ringer buffer, pH 5.5). Carrier mediated uptake was determined as described in the methods section. Induced uptake by the riboflavin transporter was determined by subtracting uptake in empty-vector transfected cells from the cells transfected with the rft-1 and rft-2 constructs and are expressed as means ± SE.

Figure 3. Characteristics of riboflavin uptake by ARPE-19 cells expressing rft-1.

Data are means ± SE of at least 3 separate uptake determinations, and uptake by the induced carrier was determined as determined in Fig. 2. (A) Effect of incubation buffer pH on the induced [³H]-riboflavin uptake. (B) Initial rate of [³H]-riboflavin uptake as a function of concentration by the induced riboflavin carrier. (C) Effect of unlabeled riboflavin and its structural analogues on [³H]-riboflavin uptake by the induced carrier compared to control. (D) Role of Na⁺ in [³H]-riboflavin uptake by the induced carrier; Na⁺ was replaced with either Li⁺ or choline.

Figure 4. Expression and phenotypes of the riboflavin transporters.

(A) Quantitative RT-PCR analysis of rft-1 expression. The data were normalized relative to the housekeeping gene β-actin (act-2). All worms were raised on HT115(DE3) bacteria harboring either the rft-1 RNAi feeding plasmid (rft-1 RNAi worms) or the empty vector, pPD129.36 (all other worms). Additionally, some worms were raised on plates supplemented with riboflavin. (B) The effects of RNAi on egg laying. Here, worms were reared on bacteria harboring the RNAi empty vector (Controls), the rft-1 RNAi feeding vector, the rft-2 RNAi feeding vector, or both RNAi feeding vectors. (C) Micrographs of a worm expressing the transcriptional reporters Prft-1::gfp and Prft-2::mCherry. Both reporters were expressed in the intestine, whereas only mCherry was found in the pharynx, and only GFP was found in neuronal support cells that spanned the length of the body.