Abstract
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.
Highlights
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 -D-glucopyranoside (OG) and as a monomer when extracted by SDS
Jung et al [14] reported that following expression in Xenopus oocytes, mutants in loop E sedimented with s values slightly higher than native AQP1 suggesting an importance of this loop in oligomerization and water channel function
Summary
The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Functional characterization data have distinguished two major subgroups of specific channels: aquaporins (AQPs), which transfer water, and glycerol facilitators (GlpFs), which transfer small neutral solutes. Jung et al [14] reported that following expression in Xenopus oocytes, mutants in loop E sedimented with s values slightly higher than native AQP1 suggesting an importance of this loop in oligomerization and water channel function. The structural feature of one aquaporin is known [12, 13], no data are yet available about the three-dimensional structure and oligomerization state of GlpFs. based on the similarities in their amino acid sequences and hydrophobicity profiles, the monomers of glycerol facilitators and of water channels likely exhibit similar three-dimensional structural organizations. One of these positions is located in loop C and the four others are found in or close to loop E
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