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Molecular Characterisation of the INTRAcellular Plant Aquaporin Trafficking and Hetero-oligomerisation

Final Report Summary - INTRA PATH (Molecular Characterisation of the INTRAcellular Plant Aquaporin Trafficking and Hetero-oligomerisation)

In plants, the movement of water and small neutral solutes (ranging from gases to metalloids) across the plasma membrane (PM) is dependent on the number and activity of water channels, named aquaporins (AQP). Higher plants encode for five subfamilies of AQPs, the Plasma membrane Intrinsic Proteins (PIPs), Tonoplast Intrinsic Proteins (TIPs), Nodulin26-like Intrinsic Proteins (NIPs), Small basic Intrinsic Proteins (SIPs), and the recently identified X-Intrinsic Proteins (XIPs). In plant PM, PIPs seem to function as highly specialized water channels and fulfil essential key roles in water transport. Recent studies have shown that PIPs, belonging to two subgroups, PIP1 and PIP2, physically interact to regulate their trafficking and the cell membrane water permeability: When transiently expressed singly in plant cells, PIP1s and PIP2s differ in their subcellular localization. PIP1s are retained in the secretory pathway and, more specifically, in the endoplasmic reticulum (ER), whereas PIP2s are targeted to the PM. Upon co-expression, ZmPIP1s are localized in the PM as a result of their physical interaction with ZmPIP2s. These data indicate that ZmPIP2s, but not ZmPIP1s, possess signals that allow them to be transported to the PM and that hetero-oligomerization of the two proteins is required for the trafficking of PIP1s to the PM and therewith for the control of plant membrane water permeability.
Therefore, one main objective of the project was to shed light into the mechanisms regulating the intracellular trafficking of PIP1s and PIP2s and the implication of their mutual interaction. PIPs from a rather drought resistant crop plant were chosen as objects for investigation to potentially transfer and apply the obtained knowledge to increase the resistance of drought sensitive plants. In accordance with data from other proteins we could show that a known ER export signal, the so-called di-acidic motif in the N-termini of ZmPIP2;6 and ZmPIP2;7 influences ER export. To determine why ZmPIP2;1 is PM-localised when expressed in maize cells, although it does not contain any known ER export motif, and to identify putative dominant ER retention motifs in ZmPIP1s, we generated a complete set of fluorescently-tagged chimeric proteins in which the N- and C-termini and the five loops were singly or in combination exchanged between ZmPIP1;2 and ZmPIP2;1. Trafficking and localisation were followed using confocal microscopy. In addition, we tested the functionality of these chimeric proteins in the oocyte system to ensure that the chimeric proteins still form functional water channels. Expression of some of the constructs resulted in a different ratio of internal to PM-localized PIP-labelled membranes. However, until now, no dominant ER export or retention signal could be attributed to a single terminus, transmembrane spanning helix or a cytoplasmic or apoplastic loop or to a structural cooperation between them. These results demonstrate that the ER export or retention of ZmPIP2;1 and ZmPIP1;2, respectively, is not, unlike for other membrane proteins, dependent on sequence-wise narrowed protein trafficking motifs but rather on the spatially collaborative arrangement of various protein parts.
The vast set of generated chimeric PIP2s was still able to interact with non-mutated PIP1 proteins, as demonstrated through their correct co-trafficking to the PM. These results show, that no single ZmPIP1;2 or ZmPIP2;1-specific terminus or loop is essential for their mutual interaction, but that rather several distributed amino acid residues cause the interaction between PIP1s and PIP2s. Moreover, it suggests that the capacity to interact as hetero-oligomers is probably isoform unspecific, as the sequence similarity within isoforms of the PIP1 and the PIP2 group is mostly higher than between the two different groups.