Purification and interaction analyses of two human lysosomal vitamin B₁₂ transporters: LMBD1 and ABCD4

Abstract

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B₁₂ to the cytoplasm, impairing the vitamin B₁₂-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B₁₂ transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B₁₂ transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B₁₂-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B₁₂ to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.

• Cobalamin
• inborn errors of metabolism
• membrane proteins
• protein–protein interactions
• vitamin B₁₂ metabolism

Supplementary material available online

Supplementary Figures 1 and 2

Supplementary Tables 1 and 2.