Transmembrane helix 6 of ABCD4 is indispensable for cobalamin transport

Abstract ABCD4, which belongs to the ABC protein subfamily D, plays a role in the transport of cobalamin from lysosomes to the cytosol by cooperating with ATP‐binding and ATP‐hydrolysis. Pathogenic variants in the ABCD4 gene lead to an inherited metabolic disorder characterized by cobalamin deficiency. However, the structural requirements for cobalamin transport in ABCD4 remain unclear. In this study, six proteoliposomes were prepared, each containing a different chimeric ABCD4 protein, wherein each of the six transmembrane (TM) helices was replaced with the corresponding ABCD1. We analyzed the cobalamin transport activities of the ABCD mutants. In the proteoliposome with chimeric ABCD4 replacing TM helix 6, the cobalamin transport activity disappeared without a reduction in ATPase activity, indicating that TM helix 6 contributes to substrate recognition. Furthermore, the substitution of aspartic acid at position 329 or threonine at position 332 in TM helix 6 with the basic amino acid lysine led to a decrease in cobalamin‐transport activity without causing a reduction in ATPase activity. The amino acids in TM helix 6 may be critically involved in substrate recognition; the charged state in the C‐terminal half of TM helix 6 of ABCD4 is responsible for cobalamin transport activity.