title: 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.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany.
authors.forename: Youssef Abdul
authors.initials: YA
authors.lastname: Jalil
authors.forename: Vera
authors.initials: V
authors.lastname: Ritz
authors.forename: Ana
authors.initials: A
authors.lastname: Jakimenko
authors.forename: Christoph
authors.initials: C
authors.lastname: Schmitz-Salue
authors.forename: Heike
authors.initials: H
authors.lastname: Siebert
authors.forename: David
authors.initials: D
authors.lastname: Awuah
authors.forename: André
authors.initials: A
authors.lastname: Kotthaus
authors.forename: Thomas
authors.initials: T
authors.lastname: Kietzmann
authors.forename: Christina
authors.initials: C
authors.lastname: Ziemann
authors.forename: Karen I
authors.initials: KI
authors.lastname: Hirsch-Ernst
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation / Q000502:physiology*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D004351:Drug Resistance / Q000235:genetics; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008670:Metals / Q000378:metabolism* / Q000633:toxicity; D019216:Metals, Heavy / Q000378:metabolism / Q000633:toxicity; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D018528:ATP-Binding Cassette Transporters; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D051907:Lysosomal Membrane Proteins; D008670:Metals; D019216:Metals, Heavy; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C491540:Abcb6 protein, rat; C489117:Lamp1 protein, rat; D052118:Lysosomal-Associated Membrane Protein 1