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.

人LMBRD1和ABCD4中的突变阻止了溶酶体向细胞质输出维生素B ₁₂，损害了维生素B ₁₂依赖性酶蛋氨酸合酶和甲基丙二酰辅酶A突变酶。LMBRD1和ABCD4的基因产物与溶酶体膜上的维生素B ₁₂转运有关，并建议以复合体的形式起作用。解决溶酶体维生素B ₁₂的机制运输，我们报告了LMBD1和ABCD4的新型重组生产，用于详细的生物物理分析。使用蓝色本机PAGE，化学交联和尺寸排阻色谱联用多角度光散射（SEC-MALS），我们显示去污剂溶解的LMBD1和去污剂溶解的ABCD4均形成同二聚体。为了检查这些蛋白质的功能结合特性，无标记的表面等离振子共振（SPR）提供了直接的体外证据，即：（i）LMBD1和ABCD4以低纳摩尔亲和力相互作用；（ii）胞质维生素B ₁₂加工蛋白MMACHC还以低纳摩尔亲和力与LMBD1和ABCD4相互作用。因此，我们提出了一种模型，其中膜结合的LMBD1和ABCD4促进溶酶体维生素B ₁₂向细胞质MMACHC的矢量递送，从而防止辅因子稀释至细胞质环境并防止灭活副反应。