Lysosomal Vitamin B12 Trafficking

The C-propionate incorporation assay is a versatile technique which allows indirect determination of lysosomal cobalamin (Cbl) release by utilizing a metabolic pathway which requires the adenosyl-Cbl cofactor form. Vitamin B₁₂ is required as cofactor for essential human enzymes: cytosolic methionine synthase and mitochondrial methylmalonyl-CoA mutase. Using primary or immortalized cells, they allow determination of lysosomal vitamin B₁₂ function without requiring detailed knowledge of the individual steps involved, and can succeed at the very low level at which the endogenous proteins are expressed. From the point of view of lysosomal investigation, this allows the researcher to assay any condition that may result in alteration of lysosomal Cbl release. Free Cbl is then exported from the lysosome into the cytosol, where a series of metabolic steps results in the production of methyl-Cbl, the cofactor form for methionine synthase, or in transport into the mitochondria and the conversion to adenosyl-Cbl, the cofactor form for methylmalonyl-CoA mutase.