Tonoplast Water Channel Protein TIP’

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 yTIP, one of the MIP homologs found in the vacuolar membrane of plant cells, i s an aquaporin or water channel protein (C. Maurel, J. Reizer, 1.1. Schroeder, M.J. Chrispeels [1993] EMBO J 12: 22412247). RD28, another MIP homolog in Arabidopsis thaliana, was first identified as being encoded by a turgor-responsive transcript. To find out i f RD28 i s a water channel protein, rd28 cRNA was injected into Xenopus laevis oocytes. Expression of RD28 caused a 10to 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 i s an aquaporin just like i t s homolog ?-TIP. Although RD28 has several cysteine residues, i ts activity is not inhibited by mercury, and in this respect it differs from y-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 i s 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 i t s 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 (?-TIP).