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

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

Additional file 5: Table S4. of Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

Results of unpaired t-test for comparisons indicated in Fig. 5a. (PDF 44 kb

Additional file 3: Table S2. of Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

Estimated protein amounts, rate constants and specific activities for the first set of NbXIP1;1α mutants. (PDF 88 kb

Additional file 2: Figure S1. of Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

Expression of N-terminally His-tagged NbXIP1;1α mutants in P. pastoris. Western blots showing the expression levels of N-terminally His-tagged NbXIP1;1 mutants in P. pastoris X-33 clones. Blots were developed by enhanced chemiluminiscence in a Syngene PXi touch instrument. a. The first set of NbXIP1;1 α mutants. NbXIP1;1αst is an N-terminally truncated construct of NbXIP1;1α used as control for the western blot. b. The second set of NbXIP1;1α mutants. The control is 2 μg of purified NbXIP1;1αwt. (PDF 207 kb

Additional file 6: Table S5. of Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

Results of unpaired t-test for comparisons indicated in Fig. 5b. (PDF 45 kb

Additional file 7: Figure S2. of Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

Cartoon representations of the homology models of NbXIP1;1αwt and NbXIP1;1α mutants. The pores in the models are shown in mesh representation from blue (widest) to red (narrowest). The HOLE program [38] was used to estimate the radius of the pore in the models. Starting from the top left to the bottom right; NbXIP1;1αwt, NbXIP1;1αL79G/I102H/V242I (mutant 1), NbXIP1;1αL79G/I102H/T246I (mutant 2), NbXIP1;1αL79G (mutant 3), NbXIP1;1αI102H (mutant 4), NbXIP1;1αV242I (mutant 5), NbXIP1;1αL79G/I102H (mutant 6), NbXIP1;1αI102H/V242I (mutant 7) and NbXIP1;1αL79G/V242I (mutant 8), respectively. See also Fig. 7. (PDF 187 kb