Sammanfattning: Aquaporins are integral membrane proteins that specifically transports water. In plants, aquaporins have been identified in the plasma membrane and in the vacuolar membrane. The presence of aquaporins provides the plant with an opportunity to regulate the flux of water between cells and within the cell by changing the abundance or activity of the aquaporins. One aquaporin, PM28A, and two aquaporin homologues, PM28B and So-dTIP were identified in spinach. PM28A and PM28B are located in the plasma membrane where they constitute approximately 15% of total protein, whereas So-dTIP is located in the vacuolar membrane. The water transport activity of PM28A was demonstrated by measuring the swelling rate of Xenopus laevis oocytes, transiently expressing PM28A. PM28A was shown to be phosphorylated in vivo and in vitro. The phosphorylated residue was identified and found to be serine-274 in the C-terminus, both in vivo and in vitro. Phosphorylation at serine-274 increased with increasing apoplastic water potential, as accomplished by incubating pieces of spinach leaves in buffers of different osmolarities. Experiments in oocytes showed that phosphorylation at this site results in increased water transport activity of PM28A. In these experiments, wild-type PM28A and mutated forms, where serines in potential phosphorylation sites had been replaced by alanines, were assayed in the presence or absence of a protein kinase inhibitor or an inhibitor of protein phosphatases. Our results suggest that PM28A is closed during water deficit, and opened when water is abundant and that this gating is regulated by phosphorylation of serine-274. When PM28A was expressed in oocytes, the phosphorylation of another residue, serine-115, was likewise shown to increase water transport activity. The protein kinase phosphorylating serine-274 of PM28A was shown to be plasma membrane associated and strictly Ca2+-dependent. Immunocytochemical techniques were used to determine the tissue- and cell type-specific expression pattern of So-dTIP. So-dTIP was found at high levels in most cell types in leaf, petiole, and root, but excluded from leaf epidermis and root meristem. The subcellular location of So-dTIP to the vacuolar membrane was demonstrated.
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