AbstractsBiology & Animal Science

Aquaporins (AQPs) are proteinaceous channels that facilitate the transport of water and small solutes across biological membranes. They assemble as homotetramers but might also form heterotetramers, in particular in plants. In Zea mays, the Plasma membrane Intrinsic Proteins cluster into two groups, PIP1 and PIP2, exhibiting different water channel activities when expressed in Xenopus oocytes. Interestingly, their physical interaction modulates their subcellular localization and channel activity. In the present work, chromatography purification showed that, when coexpressed in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We modeled such heterotetramers by homology to the spinach SoPIP2;1 three-dimensional structure to identify amino acid residues putatively interacting at the interfaces between monomers. We mutated some of the identified residues in ZmPIP1;2 and ZmPIP2;5 and investigated the effect of these mutations on the water channel activity, subcellular localization, protein abundance and physical interaction within an hetero-oligomer in Xenopus oocytes and maize leaf cells. In particular, we highlighted residue substitutions that inactivate ZmPIP1;2 and ZmPIP2;5, as well as independent point mutations that activate the non-functional ZmPIP1;2 and inhibit ZmPIP2;5 activity without preventing their physical interaction. We also demonstrated that ZmPIP1;2 is active in the heterotetramer. Finally, we expressed ZmPIP1;2-ZmPIP2;5 complexes in the yeast Pichia pastoris for further reconstitution and crystallization. Altogether, the data collected and analyzed in the scope of this thesis contribute to a better understanding of the interaction mechanisms between ZmPIPs and the role of heterotetramerization on their activity. (AGRO - Sciences agronomiques et ingénierie biologique)  – UCL, 2015