Oligomerization state of water channels and glycerol facilitators. Involvement of loop E.
The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast or Xenopus oocytes behaves as an homotetramer when extracted by n-octyl beta-D-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes. The GlpF protein extracted either by SDS or by nondenaturing detergents, OG and Triton X-100, exhibits sedimentation coefficients only compatible with a monomeric form of the protein in micelles. We then substituted in loop E of AQPcic two amino acids predicted to play a role in the functional/structural properties of the MIPs. In two expression systems, yeast and oocytes, the mutant AQPcic-S205D is monomeric in OG and in SDS. The A209K mutation does not modify the tetrameric form of the heterologous protein in OG. This study shows that the serine residue at position 205 is essential for AQPcic tetramerization. Because the serine in this position is highly conserved among aquaporins and systematically replaced by an acid aspartic in GlpFs, we postulate that glycerol facilitators are monomers whereas aquaporins are organized in tetramers. Our data suggest that the role of loop E in MIP properties partly occurs through its ability to allow oligomerization of the proteins.
Lagrée
V
V
UPRES-A CNRS 6026, Biologie Cellulaire et Reproduction, "Canaux et Récepteurs Membranaires," Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 35042 Rennes cedex, Bretagne, France.
Froger
A
A
Deschamps
S
S
Pellerin
I
I
Delamarche
C
C
Bonnec
G
G
Gouranton
J
J
Thomas
D
D
Hubert
J F
JF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D019476:Insect Proteins; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014982:Xenopus laevis
C102524:AQPcic protein, Cicadella viridis; D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D019476:Insect Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C078043:GlpF protein, E coli