The plasma membrane aquaporin ZmPIP1;1 interacts with ZmPIP2s to increase the water membrane permeability of maize cells
(2022)
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- Authors
- Supervisors
- Chaumont, François
- Abstract
- Aquaporins (AQPs) are channels present in all living organisms. They share the hour-glass shape and are involved in many cellular processes. Plasma membrane intrinsic proteins (PIPs) are AQPs responsible for the facilitated diffusion of water and small solutes through plasma membrane (PM). Land plant PIP subfamily is composed of two distinguishable phylogenetic clusters: PIP1 and PIP2. They assemble as homo- or heterotetramers, the monomer being the functional unit. Previous works have demonstrated that PIPs present a dissimilar behavior regarding their subcellular localization and function. PIP2s are considered as efficient water channels and they localize in the PM. On the other hand, when expressed alone, most of the PIP1s are retained in the endoplasmic reticulum (ER) and are not efficient water channels. When co-expressed, PIP1s and PIP2s interact, a process leading to the relocalization of PIP1s from the ER to the PM and to an increase in the cell water permeability. In maize, this synergistic effect was observed for most of ZmPIP1s members, including ZmPIP1;2 when co-expressed with ZmPIP2;5. This unexpected synergy together with localization studies led researchers to further investigate the nature of ZmPIP1 and ZmPIP2 interaction. The heterotetramerization of PIP monomers is the mechanism responsible for the ZmPIP1 trafficking to the PM and for the modulation of the cell membrane permeability. ZmPIP1;1 is a ubiquitous and one of the highest expressed AQP in maize. It is the closest paralog of ZmPIP1;2. Despite their striking similarity, their function does not correlate. Previous studies showed that the co-expression of ZmPIP1;1 with ZmPIP2;5 did not lead to a synergistic effect in oocyte membrane water permeability, while both isoforms were physically interacting and found in the PM. This enigmatic isoform has been considered to be an inactive water channel until nowadays. In this thesis, we aimed at unraveling the role of ZmPIP1;1, using heterologous and homologous expression systems. To better understand ZmPIP1;1 role, we firstly compared the amino acid residues responsible for the PIP2 synergy driven by ZmPIP1;2. We identified two neighboring amino acid residues, located in the loop E, responsible for the activation of water transport in this isoform. In Xenopus oocytes, we swapped the two amino acids and we generated an activated mutant ZmPIP1;1QH/RD, which presented the same synergistic effect as ZmPIP1;2. We then inactivated this mutant by introducing an additional amino acid mutation ZmPIP1;1QH/RD-W94A resulting in the blockage of the water passage, thus confirming that ZmPIP1;1QH/RD could enable the water diffusion when expressed in Xenopus oocytes. To exploit a homologous model for studying ZmPIP1;1, we overexpressed (OE) it in Black Mexican Sweet (BMS) maize suspension cells. ZmPIP1;1 was detected in the PM in these cells, while in Xenopus oocytes it was located in the ER. Surprisingly, the membrane water permeability of ZmPIP1;1 OE protoplasts was similar to protoplasts overexpressing ZmPIP2;5. We were able to build two hypotheses, either ZmPIP1;1 was directly transporting water by itself, or it was leading to an enrichment of ZmPIP2s in the PM via heteromerization, indirectly affecting the PM water diffusion. The first hypothesis was tested by the generation of a BMS mutant line expressing ZmPIP1;1W94A, an inactive channel. The mutant cell line exhibited a PM water permeability similar to the negative control (WT cells), suggesting that ZmPIP1;1 has an intrinsic ability to facilitate water transport in its homologous environment. The second hypothesis was explored by PM purification of ZmPIP1;1 OE lines. We detected a significant increase in the ZmPIP2 content in the PM, therefore, possibly explaining the unexpected water permeability presented by these cell lines. Altogether, our data uncovered the role of two amino acids in loop E, being responsible for the water channel inactivation of ZmPIP1;1, compared to ZmPIP1;2. Also, BMS experiments suggested that ZmPIP1;1 may work as an active water channel in maize cells. Besides, ZmPIP1;1 was also able to regulate the water membrane permeability by increasing ZmPIP2 abundance in the PM via heteromerization. The mechanisms underlying this interaction, remains to be elucidated, for instance, with the structural determination of ZmPIP1 and ZmPIP2 heterotetramers.
- Affiliations
Citations
Teodoro Junqueira, B. R. (2022). The plasma membrane aquaporin ZmPIP1;1 interacts with ZmPIP2s to increase the water membrane permeability of maize cells. https://hdl.handle.net/2078.5/103362