Expression and Function of Human and Plant Aquaporins

The aquaporins (AQPs) belong to a family of water permeable membrane channels found in virtually all living organisms. Thirteen isoforms of mammalian AQPs are found, whereas in Arabidopsis thaliana 35 genes encoding AQPs are found. The AQPs are distributed in different organs, cell types and in different subcellular membranes. The aquaporins form homotetramers where each monomer functions as an independent pore. In addition to water, some aquaporins are permeable to other small solutes, such as glycerol, urea, H2O2 and NH3. The objective of the work described in this thesis is to increase the knowledge of expression and function of human and plant aquaporins. Human AQP8 and AQP5 were heterologously expressed in Pichia pastoris. The proteins were purified and reconstituted into proteoliposomes to verify the functionality and were subsequently crystallized. The 2-D crystals of human AQP8 were analyzed by electron diffraction and diffracted to 3 Å, whereas the 3-D crystals of human AQP5 were analyzed by X-ray crystallography resulting in a high-resolution structure of 2.0 Å. A large amount of pure protein is needed in order to be able to crystallize a protein. Therefore, a method was developed to screen for multi-copy P. pastoris clones expressing selected aquaporins at a high level. The number of gene copies was verified by quantitative PCR and for each isoform it was correlated to the aquaporin protein expression level, although there is a large variation in expression between isoforms with the same copy number. Many aquaporins are known to be regulated at a post-translational level. Based on the high-resolution structure of the plant SoPIP2;1, we wanted to explore amino acid residues involved in stabilizing the open or closed conformation of the protein. The mutations we made were located in the D-loop, known to be involved in the gating mechanism of SoPIP2;1, or immediately after the D-loop. The mutant proteins were expressed in Xenopus laevis oocytes to determine their water transport rate. We identified several amino acid residues involved in stabilizing an open conformation or a closed conformation. In order for maintaining water homeostasis in chloroplasts and in order for the light-dependent photosynthetic reaction to occur, water needs to be transported into the chloroplast and into the thylakoid lumen. Arabidopsis chloroplast membranes were analyzed by immunoblots and mass spectrometric analysis to elucidate the aquaporin isoforms present in the chloroplast membranes. We identified the PIP2;1 isoform, one or several PIP1 isoforms and the TIP2;1 isoform in the thylakoid membranes, and the PIP2;1 isoform and the TIP2;1 isoform in the inner envelope membranes.