title: 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 B(12) to the cytoplasm, impairing the vitamin B(12)-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B(12) transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B(12) 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 B(12)-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 B(12) to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.
authors.affiliation: Department of Microbiology and Immunology, McGill University , Montreal , Canada .
authors.forename: Justin C
authors.initials: JC
authors.lastname: Deme
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hancock
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Xia
authors.forename: Chitra A
authors.initials: CA
authors.lastname: Shintre
authors.forename: Maria
authors.initials: M
authors.lastname: Plesa
authors.forename: Jaeseung C
authors.initials: JC
authors.lastname: Kim
authors.forename: Elisabeth P
authors.initials: EP
authors.lastname: Carpenter
authors.forename: David S
authors.initials: DS
authors.lastname: Rosenblatt
authors.forename: James W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008958:Models, Molecular; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010088:Oxidoreductases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D020349:Surface Plasmon Resonance; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: C489273:ABCD4 protein, human; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; C502888:LMBRD1 protein, human; D029543:Nucleocytoplasmic Transport Proteins; D011994:Recombinant Proteins; C508359:MMACHC protein, human; D010088:Oxidoreductases; D014805:Vitamin B 12
keywords: Cobalamin; inborn errors of metabolism; membrane proteins; protein–protein interactions; vitamin B12 metabolism