Daniels M J, Mirkov T E, Chrispeels M J
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Plant Physiol. 1994 Dec;106(4):1325-33. doi: 10.1104/pp.106.4.1325.
Plant cells contain proteins that are members of the major intrinsic protein (MIP) family, an ancient family of membrane channel proteins characterized by six membrane-spanning domains and two asparagine-proline-alanine (NPA) amino acid motifs in the two halves of the protein. We recently demonstrated that gamma-TIP, one of the MIP homologs found in the vacuolar membrane of plant cells, is an aquaporin or water channel protein (C. Maurel, J. Reizer, J.I. Schroeder, M.J. Chrispeels [1993] EMBO J 12: 2241-2247). RD28, another MIP homolog in Arabidopsis thaliana, was first identified as being encoded by a turgor-responsive transcript. To find out if RD28 is a water channel protein, rd28 cRNA was injected into Xenopus laevis oocytes. Expression of RD28 caused a 10- to 15-fold increase in the osmotic water permeability of the oocytes, indicating that the protein creates water channels in the plasma membrane of the oocytes and is an aquaporin just like its homology gamma-TIP. Although RD28 has several cysteine residues, its activity is not inhibited by mercury, and in this respect it differs from gamma-TIP and all but one of the mammalian water channels that have been described. Introduction of a cysteine residue next to the second conserved NPA motif creates a mercury-sensitive water channel, suggesting that this conserved loop is critical to the activity of the protein. Antibodies directed at the C terminus of RD28 were used in combination with a two-phase partitioning method to demonstrate that RD28 is located in the plasma membrane. The protein is present in leaves and roots of well-watered plants, suggesting that its presence in plants does not require a specific desiccation regime. These results demonstrate that plant cells contain constitutively expressed aquaporins in their plasma membranes (RD28), as well as in their tonoplasts (gamma-TIP).
植物细胞含有属于主要内在蛋白（MIP）家族的蛋白质，这是一个古老的膜通道蛋白家族，其特征是具有六个跨膜结构域以及在蛋白质的两个半部分中各有一个天冬酰胺 - 脯氨酸 - 丙氨酸（NPA）氨基酸基序。我们最近证明，γ-TIP是在植物细胞液泡膜中发现的MIP同源物之一，是一种水通道蛋白（C. 莫雷尔、J. 赖泽、J.I. 施罗德、M.J. 克里斯皮尔斯 [1993] 《欧洲分子生物学组织杂志》12: 2241 - 2247）。RD28是拟南芥中的另一个MIP同源物，最初被鉴定为由一个膨压响应转录本编码。为了确定RD28是否为水通道蛋白，将rd28 cRNA注射到非洲爪蟾卵母细胞中。RD28的表达使卵母细胞的渗透水通透性增加了10至15倍，这表明该蛋白在卵母细胞质膜中形成水通道，并且与它的同源物γ-TIP一样是一种水通道蛋白。尽管RD28有几个半胱氨酸残基，但其活性不受汞抑制，在这方面它不同于γ-TIP以及除一种已描述的哺乳动物水通道蛋白之外的所有其他哺乳动物水通道蛋白。在第二个保守的NPA基序旁边引入一个半胱氨酸残基会产生一个对汞敏感的水通道，这表明这个保守环对该蛋白的活性至关重要。针对RD28 C末端的抗体与两相分配法结合使用，以证明RD28位于质膜中。该蛋白存在于水分充足的植物的叶片和根中，这表明它在植物中的存在不需要特定的干燥条件。这些结果表明，植物细胞在其质膜（RD28）以及液泡膜（γ-TIP）中含有组成型表达的水通道蛋白。
