Production of the recombinant human riboflavin transporters SLC52A1, 3 and functional assay in proteoliposomes

Riboflavin, the FMN and FAD precursor, is a crucial vitamin in cell metabolism. Its adsorption and tissue distribution are mediated by tree membrane transporters namely RFVT1-3. Mutations of their genes are associated with Riboflavin Transporter Deficiency. Moreover, derangements of the level of these transporters have been found in several human cancers. To obtain a suitable experimental tool for studying the function of the single proteins, for testing the effect of pathological mutations and for validating predicted ligands as candidate drugs, we have set up a proteoliposome system harbouring the functional RFVT1 or RFVT3. RFVT proteins have been produced in E. coli and purified to the homogeneity by affinity chromatography. The purified proteins show an apparent molecular mass of 45.6 or 48.4 kDa, which are very close to the theoretical mass of RFVT1 or RFVT3, respectively. The purified transporters have been reconstituted into proteoliposomes using a methodology previously pointed out for RFVT2. The transport of riboflavin shows cooperative kinetics with K_0.5 values of 0.86 or 1.13 μM and Hill coefficients of 1.19 or 1.3 for RFVT1 or RFVT3, respectively. The K_0.5 data of both the transporters are similar the Km reported in intact cell studies. The transporters are inhibited by the riboflavin analogues FMN and lumiflavin in agreement with the molecular docking simulations.