Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

Abstract

ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6.

Structures and functions of mitochondrial ABC transporters

“…ABCB6 was also found in the plasma membrane in Nglycosylated form [27,28] and in the Golgi apparatus [30]. Recently, evidence has been presented for an endolysosomal localization of ABCB6 [31][32][33], whereas ABCB7, ABCB8 and ABCB10 were found exclusively in mitochondria [31].…”

Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter

“…[6], others have not been able to demonstrate either [20]. In our case, presence of basal activity and absence of stimulated activity may be explained e.g.…”

“…While we did not purify the protein further to demonstrate that both of these activities are directly mediated by ABCB6 molecules, we believe that the overall consistence of subcellular distribution of effects adds further credence to this postulate: not only do ABCB6-expressing cells differ in the expected manner from control ones, but within the overexpressing cells these activities are concentrated in intracellular compartments, where we have shown that human ABCB6 expression is stronger than in the plasma membrane. Thus, the increased chlorin content in the ABCB6-expressing strain is in line with expected direction of ABCB6-mediated transport: ABCB6, as most ABC transporters, functions as an exporter [20], so while plasma membrane ABCB6 potentially removes chlorin from the cytoplasm, the much more…”

“…While it is known that human ABCB6 is glycosylated in vivo and this modification has a potential role in protein trafficking and stability [33], studies on the potential role of glycosylation in functional activity have been hindered by the lack of suitable models: e.g. Sf9 cells produce only core-glycosylated ABCB6 [20].…”

“…This model is reliable and physiologically relevant, but very non-cost-effective if it were to be used for direct biochemical assays. A more relevant heterologous expression system that was used in some biochemical studies [20] is the baculovirus-insect cell system. For this reason, we selected this system for a quantitative comparison:…”