期刊
MOLECULAR MEMBRANE BIOLOGY
卷 31, 期 7-8, 页码 250-261出版社
TAYLOR & FRANCIS LTD
DOI: 10.3109/09687688.2014.990998
关键词
Cobalamin; inborn errors of metabolism; membrane proteins; protein-protein interactions; vitamin B-12 metabolism
资金
- Natural Sciences and Engineering Research Council [NSERC] [RGPIN 7289-06-07]
- Canadian Institutes of Health Research [CIHR] [MOP-15078, 200610PRG-165657-PRG-CFAA-11449]
- Canada Foundation for Innovation [CFI]
- AbbVie
- Canadian Institutes for Health Research
- Genome Canada
- GlaxoSmithKline
- Eli Lilly Canada
- Novartis Research Foundation
- Pfizer
- Takeda
- Canada Foundation for Innovation
- Ontario Ministry of Economic Development and Innovation
- Wellcome Trust [092809/Z/10/Z]
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.
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