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This Article Full Text Full Text (PDF) All Versions of this Article: 294/2/C579    most recent 00612.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Abdul Jalil, Y. Articles by Hirsch-Ernst, K. I. Search for Related Content PubMed PubMed Citation Articles by Abdul Jalil, Y. Articles by Hirsch-Ernst, K. I.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

Institutes of ¹Pharmacology and Toxicology and ²Neuropathology, University of Göttingen, Göttingen; ³Department of Biochemistry, Faculty of Chemistry, University of Kaiserslautern, Kaiserslautern, Germany; and ⁴Fraunhofer-Institute of Toxicology and Experimental Medicine, Hannover, Germany

Submitted 8 December 2006 ; accepted in final form 21 December 2007

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.

LoVo; rat hepatocytes; lysosomes; endosomes; glycosylation; copper tolerance

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