title: Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins.
abstract: We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.
authors.affiliation: UMR 6026, CNRS, Equipe Canaux et Récepteurs Membranaires, Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 35042 Rennes cedex, Bretagne, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Duchesne
authors.forename: Isabelle
authors.initials: I
authors.lastname: Pellerin
authors.forename: Christian
authors.initials: C
authors.lastname: Delamarche
authors.forename: Stephane
authors.initials: S
authors.lastname: Deschamps
authors.forename: Valerie
authors.initials: V
authors.lastname: Lagree
authors.forename: Alexandrine
authors.initials: A
authors.lastname: Froger
authors.forename: Georgette
authors.initials: G
authors.lastname: Bonnec
authors.forename: Daniel
authors.initials: D
authors.lastname: Thomas
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Hubert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014981:Xenopus
chemical_list: D020346:Aquaporins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C078043:GlpF protein, E coli