Identification of ABC transporters acting in vitamin B12 metabolism in Caenorhabditis elegans

Vitamin B-12 (cobalamin, Cb1) is a micronutrient essential to human health. Cb1 is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cb1 for methionine synthase, a cytosolic enzyme; and adenosyl-Cb1 for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cb1 to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cb1 utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cb1 transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [C-14]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B-12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cb1 into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cb1, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.