Role of transmembrane domain 3 in the trafficking of plant aquaporins to the plasma membrane

Introduction. Aquaporins are of major importance to plant physiology. In addition to being essential for plant water relations, aquaporins facilitate the diffusion of other small uncharged molecules and are involved in plant nutrition. This work focuses on the maize plasma membrane intrinsic protein (ZmPIP) family. Experiments using maize mesophyll protoplasts demonstrated that only ZmPIP2s are able to reach the plasma membrane on their own, ZmPIP1s being retained in the endoplasmic reticulum (ER). Previous results showed that several but not all ZmPIP2s reaching the plasma membrane contain a diacidic ER export motif. On the other hand, no ER retention signal has been identified in ZmPIP1s yet. The present work aims at uncovering new trafficking signals in ZmPIP proteins. Materials and Methods. Maize protoplasts were transfected with genetic constructs encoding fluorescently-tagged chimeric PIPs containing parts of both plasma membrane-localised ZmPIP2;5 and ER-retained ZmPIP1;2. The subcellular localisation of these chimeric proteins was analysed by confocal microscopy. Their water transport activity was determined by heterologous expression in Xenopus laevis oocytes. Results and Discussion. Six chimeric proteins were generated by individually replacing each transmembrane domain of mYFP:ZmPIP2;5 by the corresponding region of ZmPIP1;2. After maize protoplasts transfection, confocal microscopy analysis showed that the localisation of one them (mYFP:ZmPIP2;5-PIP1;2TM3) significantly differs from the wild type mYFP:ZmPIP2;5, as it is completely retained in the ER. In addition, mCFP:ZmPIP2;5-PIP1;2TM3 induced a partial retention of mYFP:ZmPIP2;5 in the ER upon co-expression in protoplasts. Biolistic bombardment of maize leaves allowed to validate the data in intact tissues. Preliminary results in Xenopus oocytes indicated that the chimeric protein ZmPIP2;5-PIP1;2TM3 does not induce any increase in the membrane water permeability. Conclusions. This work identified the third transmembrane domain of ZmPIPs as an important region regulating both the routing of these proteins to the plasma membrane and their water transport activity