Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer.

Aquaporins are channels facilitating the diffusion of water and/or small uncharged solutes across biological membranes. They assemble as homotetramers but some of them also form heterotetramers, especially in plants. In Zea mays, aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily are clustered into two groups, PIP1 and PIP2, which exhibit different water-channel activities when expressed in Xenopus oocytes. When PIP1 and PIP2 isoforms are co-expressed, they physically interact to modulate their subcellular localization and channel activity. Here, we demonstrated by affinity chromatography purification that, when co-expressed in Xenopus oocytes, the maize PIP1;2 and PIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We built the 3D structure of such heterotetramers by comparative modeling on the basis of the spinach SoPIP2;1 X-ray structure and identified amino acid residues in the transmembrane domains which putatively interact at the interfaces between monomers. Their roles in the water-channel activity, subcellular localization, protein abundance, and physical interaction were investigated by mutagenesis. We highlighted single-residue substitutions that either inactivated PIP2;5 or activated PIP1;2 without affecting their interaction. Interestingly, the Phe220Ala mutation in the transmembrane domain 5 of PIP1;2 activated its water-channel activity and, at the same time, inactivated PIP2;5 within a heterotetramer. Altogether, these data contribute to a better understanding of the interaction mechanisms between PIP isoforms and the role of heterotetramerization on their water-channel activity.

The grape aquaporin VvSIP1 transports water across the ER membrane.

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity.

A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers.

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.