The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain

Abstract

Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.

Overexpression of X Intrinsic Protein 1;1 in Nicotiana tabacum and Arabidopsis reduces boron allocation to shoot sink tissues

“…After their discovery and the observation that this subfamily was lost in monocots and in some dicots such as the Brassicacea (Danielson & Johanson, 2008), XIP isoforms have been identified and their expression profile under different growth conditions was studied in various plant species (Giovannetti et al, 2012;Lopez et al, 2016;Noronha et al, 2016). Heterologous expression analysis revealed their ability to facilitate the diffusion of small solutes such as glycerol, hydrogen peroxide, urea or boric acid but no or only marginal water permeability (Ampah-Korsah et al, 2016;Bienert et al, 2011;Giovannetti et al, 2012;Lopez et al, 2016). So far, XIP-mediated boric acid permeability was only deduced based on XIP-expressing yeast growth inhibition experiments (Ampah-Korsah et al, 2016;Bienert et al, 2011;Noronha et al, 2016).…”

“…The first molecular characterization of plant XIPs demonstrated that NtXIP1;1 from tobacco is localized in the plasma membrane and that its promoter is widely active throughout the plant, both in roots and shoots (Bienert et al., ). In N. tabacum and Nicotiana benthamiana , the transcript of XIP1;1 is spliced in two distinct variants, XIP1;1 α and XIP1;1 β differing in only one amino acid in length (Ampah‐Korsah et al., , ; Bienert et al., ). Toxicity growth assays in the heterologous expression systems Saccharomyces cerevisiae and Pichia pastoris , respectively, suggest that NtXIP1;1α, NtXIP1;1β and NbXIP1;1α function as boric acid channels (Ampah‐Korsah et al., ; Bienert et al., ).…”

“…In N. tabacum and Nicotiana benthamiana , the transcript of XIP1;1 is spliced in two distinct variants, XIP1;1 α and XIP1;1 β differing in only one amino acid in length (Ampah‐Korsah et al., , ; Bienert et al., ). Toxicity growth assays in the heterologous expression systems Saccharomyces cerevisiae and Pichia pastoris , respectively, suggest that NtXIP1;1α, NtXIP1;1β and NbXIP1;1α function as boric acid channels (Ampah‐Korsah et al., ; Bienert et al., ). Additionally, NtXIP1;1 was shown to facilitate the membrane diffusion of glycerol, urea and probably hydrogen peroxide upon heterologous expression in S. cerevisiae cells and Xenopus laevis oocytes.…”

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

“…The detailed procedure of phosphorylation analysis and dephosphorylation is described in Kinoshita et al 20 with slight modication as mentioned in Ampah-Korsah et al 21 Cloning, expression and purication of human LIP5…”

Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

“…Since Nb XIP1;1αwt was found in the P. pastoris plasma membrane in a previous study [19], it is likely that the Nb XIP1:1α mutants are also localized to the plasma membrane of P. pastoris and therefore available for functional studies in spheroplasts.…”