Lagrée V, Froger A, Deschamps S, Pellerin I, Delamarche C, Bonnec G, Gouranton J, Thomas D, Hubert J F
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.
J Biol Chem. 1998 Dec 18;273(51):33949-53. doi: 10.1074/jbc.273.51.33949.
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.
主要内在蛋白（MIP）家族包括水通道水孔蛋白（AQP）和小分子溶质转运蛋白如甘油转运蛋白（GlpF）。蔗糖梯度速度沉降实验表明，在酵母或非洲爪蟾卵母细胞中表达的异源AQPcic，用正辛基-β-D-吡喃葡萄糖苷（OG）提取时表现为同四聚体，用SDS提取时则表现为单体。我们对从大肠杆菌膜或注射mRNA的非洲爪蟾卵母细胞中溶解的GlpF进行了分析。用SDS或非变性去污剂OG和Triton X-100提取的GlpF蛋白，其沉降系数仅与蛋白质在胶束中的单体形式相符。然后，我们在AQPcic的E环中替换了两个预测在MIP功能/结构特性中起作用的氨基酸。在酵母和卵母细胞这两种表达系统中，突变体AQPcic-S205D在OG和SDS中均为单体。A209K突变不会改变OG中异源蛋白的四聚体形式。这项研究表明，205位的丝氨酸残基对于AQPcic的四聚化至关重要。由于该位置的丝氨酸在水孔蛋白中高度保守，而在GlpF中系统地被酸性的天冬氨酸取代，我们推测甘油转运蛋白是单体，而水孔蛋白则以四聚体形式存在。我们的数据表明，E环在MIP特性中的作用部分是通过其允许蛋白质寡聚化的能力实现的。
