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

: Abdul, J.Y.; Ritz, V.; Jakimenko, A.; Schmitz-Salue, C.; Siebert, H.; Awuah, D.; Kotthaus, A.; Kietzmann, T.; Ziemann, C.; Hirsch-Ernst, K.


American journal of physiology. Cell physiology 294 (2008), No.2, pp.C579-C590
ISSN: 0363-6143
ISSN: 1522-1563
Journal Article
Fraunhofer ITEM ()
ATP-binding cassette transporter; rat Abcb6; rat hepatocyte; Endosome; tolerance; LoVo; Lysosomes; copper

The clarification of subcellular localization represents an important basis towards 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 C-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 co-localized with LAMP1, a marker of lysosomes/late endosomes. Substantial co-localization of tagged rAbcb6 with lysosomal/late endosomal marker was confirmed with living, unfixed LoVo cells co-expressing rAbcb6 fused to enhanced green fluorescent protein (rAbcb6-EGFP). 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 towards transition metal toxicity. Applying MTT viability assays, we found that expression of either rAbcb6-V5 or untagged rAbcb6 conferred tolerance towards 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. Key words: LoVo, rat hepatocytes, lysosomes, endosomes, copper tolerance.