Snyder, Floyd, Fritz, Julie-Anne, Li, Liang, Scheuchner, Deborah, Gravel, Roy, Lewis, Avalyn, Dharwadkar, Padmaja, McDonald, Megan
[
International Worm Meeting,
2011]
Vitamin B12 (cobalamin; Cbl) is a micronutrient essential to human health. Genetic defects in eight complementation groups involved in the pathway for intracellular utilization of Cbl in humans have led to delineation of much of the subcellular pathway leading to Cbl cofactor synthesis. Nevertheless, the pathway remains incomplete, with genes governing mitochondrial transport of Cbl and possibly a mitochondrial reductase yet to be identified. Recently, the ABC transporter, MRP-1, was identified as a Cbl intestinal efflux transporter. Mutant mice lacking MRP-1 (Mrp1(-/-)), however, display only diminished Cbl transport but no overt phenotype, suggesting multiple transport mechanisms involved in Cbl transport. We have chosen C. elegans as a model organism to define the role of ABC transporters in Cbl metabolism due to the organism's ease of use and wide availability of genetic tools. Importantly, the C. elegans genome contains 61 ABC transporters including an MRP-1 homolog and is ideal for modeling intestinal transport across the gut epithelium. We have screened 42 available ABC transporter mutant strains using a GC-MS based assay that detects the excretion of methylmalonic acid (MMA), which accumulates under Cbl deficiency. Null mutations in either MRP-1, MRP-2 or PGP-2 result in reduced excretion of MMA compared to wild-type worms. MRP-1 and MRP-2 are localized to the C. elegans intestine and may act redundantly as Cbl intestinal efflux transporters. PGP-2 is involved in intestinal granule biogenesis and may affect Cbl transport indirectly. In contrast, a null mutation in WHT-6 results in increased excretion of MMA compared to wild-type worms. WHT-6 is also localized to the intestine and may represent either a lysosomal or mitochondrial transporter. Further experiments, including the analysis of cytosolic homocysteine levels and the relevance to human Cbl transport, are underway. Overall, these studies suggest multiple ABC transporter involvement in Cbl transport and may lead to the identification of a mitochondrial Cbl transporter.