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Front Plant Sci
2018 Mar 26;9:382. doi: 10.3389/fpls.2018.00382.
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Heterotetramerization of Plant PIP1 and PIP2 Aquaporins Is an Evolutionary Ancient Feature to Guide PIP1 Plasma Membrane Localization and Function.
Bienert MD
,
Diehn TA
,
Richet N
,
Chaumont F
,
Bienert GP
.
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Aquaporins (AQPs) are tetrameric channel proteins regulating the transmembrane flux of small uncharged solutes and in particular water in living organisms. In plants, members of the plasma membrane intrinsic protein (PIP) AQP subfamily are important for the maintenance of the plant water status through the control of cell and tissue hydraulics. The PIP subfamily is subdivided into two groups: PIP1 and PIP2 that exhibit different water-channel activities when expressed in Xenopus oocytes or yeast cells. Most PIP1 and PIP2 isoforms physically interact and assemble in heterotetramers to modulate their subcellular localization and channel activity when they are co-expressed in oocytes, yeasts, and plants. Whether the interaction between different PIPs is stochastic or controlled by cell regulatory processes is still unknown. Here, we analyzed the water transport activity and the subcellular localization behavior of the complete PIP subfamily (SmPIP1;1, SmPIP2;1, and SmPIP2;2) of the lycophyte Selaginella moellendorffii upon (co-)expression in yeast and Xenopus oocytes. As observed for most of the PIP1 and PIP2 isoforms in other species, SmPIP1;1 was retained in the ER while SmPIP2;1 was found in the plasma membrane but, upon co-expression, both isoforms were found in the plasma membrane, leading to a synergistic effect on the water membrane permeability. SmPIP2;2 behaves as a PIP1, being retained in the endoplasmic reticulum when expressed alone in oocytes or in yeasts. Interestingly, in contrast to the oocyte system, in yeasts no synergistic effect on the membrane permeability was observed upon SmPIP1;1/SmPIP2;1 co-expression. We also demonstrated that SmPIP2;1 is permeable to water and the signaling molecule hydrogen peroxide. Moreover, growth- and complementation assays in the yeast system showed that heteromerization in all possible SmPIP combinations did not modify the substrate specificity of the channels. These results suggest that the characteristics known for angiosperm PIP1 and PIP2 isoforms in terms of their water transport activity, trafficking, and interaction emerged already as early as in non-seed vascular plants. The existence and conservation of these characteristics may argue for the fact that PIP2s are indeed involved in the delivery of PIP1s to the plasma membrane and that the formation of functional heterotetramers is of biological relevance.
FIGURE 1. Phylogeny of plasma membrane intrinsic proteins (PIPs) of Selaginella moellendorffii. Phylogenetic tree derived from all PIP amino acid sequences of S. moellendorffii (Sm), Zea mays (Zm), Arabidopsis thaliana (At), Coccomyxa sp. (Cc), and Physcomitrella patens (Pp) using Bayesian phylogenetic inference. Numbers next to branches indicate the percentage of node support for each branch. Only node support percentages <100 are shown. SmPIPs are highlighted in green. The clustering of PIPs into PIP1-, PIP2-, PIP3-, and algae PIP4-subgroups is indicated with a red, blue, purple, or orange line, respectively.
FIGURE 2. Water permeability coefficient (Pf) measurements and confocal microscopic images of Xenopus oocytes expressing SmPIPs. (A) Pf values for Xenopus oocytes injected with water or expressing SmPIP1;1, SmPIP2;1, or SmPIP2;2, or co-expressing SmPIP1;1 and SmPIP2;1, or SmPIP2;1 and SmPIP2;2. In total, 1 ng of SmPIP2;1, or 6 or 12 ng of SmPIP1;1 or SmPIP2;2 cRNA was injected. The results are expressed as the mean ± SD (water injected oocytes: n ≥ 10, SmPIP injected oocytes: n ≥ 9). Significance was calculated using t-test. Asterisks mark significant differences to the expression of SmPIP2;1 (∗∗∗p < 0.001, ∗∗p < 0.01). The experiment was repeated twice with consistent results. (B) Confocal microscopic images of fixed oocytes showing the localization of YFP-tagged SmPIP proteins. Oocytes were injected with 2 ng of YFP:SmPIP2;1 cRNA or 25 ng of YFP:SmPIP1;1, YFP:SmPIP2;2, single- or co-injected with 1 ng SmPIP2;1 cRNA, then observed 3 days after injection. Representative images are shown.
FIGURE 3. SmPIP-mediated osmotic water transport in yeast analyzed by stopped-flow spectrometry. SmPIP water transport capacity was determined using spheroplasts expressing GFP-tagged (A) or non-tagged (B) SmPIPs. Spheroplasts were prepared from the wild-type S. cerevisiae BY4741 yeast strain transformed with hAQP8 or empty vectors as controls or with indicated individual SmPIPs or SmPIP combinations. Spheroplasts were suspended in a 1.8 M sorbitol-based buffer at an OD600 of 1.5 and mixed in a fast kinetics instrument with an equal volume of a 1.2 M sorbitol-based buffer. Swelling kinetics were recorded by measuring the scattered light intensity over a time period of 6–8 s. Based on the swelling kinetics within the first second of swelling, rate constants were calculated for all trace recordings. To this aim traces were fitted to a one- or two-phase decay equation to obtain the best fit using GraphPad Prism6. Rate constant values for the fitted curves were determined and are displayed in (A) and (B). Gray bar charts represent mean values of rate constants ± SD (n = 22–28). Significance was calculated using t-test. Asterisks mark significant differences (∗∗∗p < 0.001) to the expression of GFP:SmPIP2;1 + ev in (A) or to SmPIP2;1 + ev in (B).
FIGURE 4. Localization of N-terminally GFP-tagged SmPIP proteins in yeast cells. Wild-type S. cerevisiae cells (BY4741) of the exponential growth phase (OD600 = 1–1.3) expressing GFP:SmPIP1;1 or GFP:SmPIP2;1 or GFP:SmPIP2;2 (A) or co-expressing GFP:SmPIP1;1 and SmPIP2;1, GFP:SmPIP1;1 and SmPIP2;2, or GFP:SmPIP2;2 and SmPIP2;1 (B) were examined by confocal microscopy. An overlay of the GFP channel and the Nomarski optical transmission is displayed. Scale bars = 5 μm.
FIGURE 5. Substrate specificity studies of SmPIPs in yeast by growth complementation- or toxicity growth assay. Hydrogen peroxide (A) and boric acid (D) toxicity growth assays and ammonium (B) and urea (C) complementation assays in the Δmep1-3
(A+B) and Δdur3
(C+D) mutant yeast strains expressing SmPIP isoforms. Cultures of mutant yeast cells co-transformed with the indicated combinations of empty vectors (pYeDP60u-ura or pYeDP60u-leu), pYeDP60u-leu, and pYeDP60u-ura carrying hAQP8, pYeDP60u-leu and pYeDP60u-ura carrying NtXIP1;1α, or pYeDP60u-ura and pYeDP60u-leu carrying the indicated SmPIP cDNA were diluted in sterile distilled water to an OD600 of 0.01 and spotted on medium containing the indicated concentrations of hydrogen peroxide (A), boric acid (D), ammonium (B), proline (B), arginine (C), or urea (C). The growth behavior and survival rates of the different transformants were recorded after 7–10 days at 30°C and were shown for the yeasts spotted at an OD600 of 0.01. All yeast growth assays were performed at least twice, with consistent results. Displayed images in (A–D) represent groups of sub-images assembled from different growth plates and conditions. Each “yeast growth spot” represents a sub-image within the image. This procedure does not alter any information and represents a usual presentation practice of yeast growth and complementation assays.
FIGURE 6. Subcellular localization of SmPIPs transiently expressed in Nicotiana benthamiana epidermis cells. (A) Abaxial tobacco epidermis cells infiltrated with the plasma membrane marker FM4-64 transiently expressing mYFP:SmPIP1;1, mYFP:SmPIP2;1, or mYFP:SmPIP2;2. Scale bars = 40 μm. (B) Abaxial tobacco epidermis cells co-expressing mYFP:SmPIP1;1 and mCFP:SmPIP2;1, mYFP:SmPIP1;1 and mCFP:SmPIP2;2, or mYFP:SmPIP2;2 and mCFP:SmPIP2;1. Scale bars = 50 μm. Representative images are displayed.
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