title: Localization of an organic anion transporter-GFP fusion construct (rROAT1-GFP) in intact proximal tubules.
abstract: The organic anion transporter, rROAT1, is a dicarboxylate/organic anion exchanger, a function associated with the basolateral membrane in rat proximal tubule. To directly establish the subcellular localization of rROAT1 in renal epithelia, we made a rROAT1-green fluorescent protein (GFP) fusion construct (rROAT1-GFP). Plasma membrane-associated fluorescence was observed in rROAT1-GFP-expressing Xenopus oocytes examined by confocal microscopy. Uptake of 3H-labeled p-aminohippurate (PAH) increased 2. 5-fold in rROAT1-GFP-expressing Xenopus oocytes, and this increase was abolished by 1 mM probenecid. Thus the construct was capable of specific organic anion transport. Cultured renal epithelial cell lines (MDCK and LLC-PK1) transfected with the vector pEGFP-C3 showed a diffuse, evenly distributed cytoplasmic signal. However, when transfected with pEGFP-C3/rROAT1 (vector coding for rROAT1-GFP), both cell lines showed predominantly plasma membrane fluorescence. The expression and distribution of rROAT1-GFP in intact renal proximal tubules was also investigated. Isolated killifish (Fundulus heteroclitus) renal tubules transfected with pEGFP-C3/rROAT1 showed marked basal and lateral membrane-associated fluorescence, but no detectable signal in the nucleus or the apical pole of tubule cells. Tubules transfected with pEGFP-C3 showed diffuse cytoplasmic fluorescence. Function of the rROAT1-GFP construct was demonstrated in transfected killifish tubules by fluorescein transport assay. These results demonstrate the basolateral subcellular localization of rROAT1 in polarized renal epithelia and validate a new technique for localizing cloned transporters within intact renal tubules.
authors.affiliation: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
authors.forename: D H
authors.initials: DH
authors.lastname: Sweet
authors.forename: D S
authors.initials: DS
authors.lastname: Miller
authors.forename: J B
authors.initials: JB
authors.lastname: Pritchard
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D004285:Dogs; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007695:Killifishes; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D013552:Swine; D014018:Tissue Distribution; D014981:Xenopus
chemical_list: D027321:Anion Transport Proteins; D002352:Carrier Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins