Vesicular localization of the rat ATP-binding cassette half-transporter rAbcb6

Abstract

The clarification of subcellular localization represents an important basis toward characterization of ATP-binding cassette (ABC) transporters and resolution of their roles in cellular physiology. Rat Abcb6 (rAbcb6) is a membrane-situated half-transporter belonging to the ABC protein superfamily. To investigate rAbcb6 subcellular distribution, the human colon adenocarcinoma line LoVo, which we found to be devoid of endogenous human ABCB6 mRNA, was employed for heterologous expression of rAbcb6 bearing a COOH-terminal epitope tag (rAbcb6-V5). Following subcellular fractionation, rAbcb6-V5 was observed as an N-glycosylated protein in fractions enriched with lysosomal/endosomal membrane proteins. Indirect immunofluorescence analyses of rAbcb6-V5 using antibodies against a rAbcb6-specific peptide or against the V5-tag revealed a punctate pattern that was colocalized with lysosome-associated membrane protein 1 (LAMP1), a marker of lysosomes/late endosomes. Substantial colocalization of tagged rAbcb6 with lysosomal/late endosomal marker was confirmed with living, unfixed LoVo cells coexpressing rAbcb6 fused to enhanced green fluorescent protein. Vesicular distribution in LoVo cells was consistent with localization of endogenous rAbcb6 expressed in rat primary hepatocyte cultures or in liver sections, as revealed by overlap of rat Lamp1 with rAbcb6 in double immunofluorescence analyses. Since several Abcb6-related half-transporters confer heavy metal tolerance, we investigated whether rAbcb6 expression in LoVo cells might affect sensitivity toward transition metal toxicity. Applying MTT viability assays, we found that expression of either rAbcb6-V5 or untagged rAbcb6 conferred tolerance toward copper, but not to cobalt or zinc. In summary, these results demonstrate that rAbcb6 is a glycosylated protein targeted to intracellular vesicular membranes and suggest involvement of rAbcb6 in transition metal homeostasis.

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

“…Since the mutations did not compromise overall expression levels, we analyzed the localization of ABCB6 variants in intracellular compartments using confocal microscopy. While initial reports indicated localization of ABCB6 in the outer mitochondrial membrane [7], [9], more recent studies detected ABCB6 in the plasma membrane [9], the Golgi apparatus [10], and in organelles of the vesicular system [11], [12], [13], [14], [15]. In line with the reports demonstrating the endolysosomal localization of ABCB6, our HA-tagged ABCB6 construct appeared in the lysosomal compartment of Hela cells, and not in the mitochondria (Figure 4).…”

“…Although initial findings suggested that loss of one Abcb6 allele in embryonic stem (ES) cells impairs porphyrin synthesis, mice derived from these stem cells were phenotypically normal [8]. Recent studies have challenged the paradigm linking the function of ABCB6 to mitochondria, and suggested that ABCB6 is localized in the endolysosomal continuum including the plasma membrane [9], the Golgi apparatus [10], and organelles of the vesicular system [11], [12], [13], [14]. ABCB6 is upregulated during erythroid differentiation, and although a proportion is lost during reticulocyte maturation through secreted exosomes, ABCB6 is expressed in mature red blood cells at relatively high levels [15].…”

Tissue-Specific Transcript Profiling for ABC Transporters in the Sequestering Larvae of the Phytophagous Leaf Beetle Chrysomela populi

“…Moreover, Cpabc7 is 3 times more highly expressed in the Malpighian tubules than in the gut. Cp ABC7 is most homologous to the human ABCB6 (Figure 3) which is reported to be localized in the Golgi apparatus [124], mitochondria, plasma membranes [125], vesicular structures [126], or endolysosomes and lysosomes of cells [79]. ABCB6 is discussed to play a role in the heme metabolism [78], [127]–[129], in the drug and arsenite resistance of cells [130]–[132].…”

Mitochondrial ABC transporters function: The role of ABCB10 (ABC-me) as a novel player in cellular handling of reactive oxygen species

“…In this regard, several studies have shown that ABCB6 is also located in the plasma membrane, ER and Golgi apparatus (as part of the secretory pathway) [41,43,65,82,85]. It was also shown that certain mutations in ABCB6 might cause an ocular disease named coloboma in humans [85].…”