title: Erythroid expression and oligomeric state of the AQP3 protein.
abstract: Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems: Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.
authors.affiliation: Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Roudier
authors.forename: Pascal
authors.initials: P
authors.lastname: Bailly
authors.forename: Pierre
authors.initials: P
authors.lastname: Gane
authors.forename: Nicole
authors.initials: N
authors.lastname: Lucien
authors.forename: Renée
authors.initials: R
authors.lastname: Gobin
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cartron
authors.forename: Pierre
authors.initials: P
authors.lastname: Ripoche
mesh_terms: D000818:Animals; D051398:Aquaporin 1; D051400:Aquaporin 3; D020346:Aquaporins / Q000096:biosynthesis* / Q000737:chemistry*; D001789:Blood Group Antigens; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005614:Freeze Fracturing; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: C496020:AQP1 protein, human; C496027:AQP3 protein, human; C496021:Aqp1 protein, mouse; C496022:Aqp1 protein, rat; C496028:Aqp3 protein, mouse; C496029:Aqp3 protein, rat; D020346:Aquaporins; D001789:Blood Group Antigens; D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D013388:Succinimides; D051398:Aquaporin 1; D051400:Aquaporin 3; C019358:disuccinimidyl suberate