Role of PIP-Aquaporin Phosphorylation in Redox-Dependent Modulation of Osmotic Water Permeability in Plasmalemma from Roots of Pea Seedlings

Abstract

Investigations were directed to study the mechanism based on redox-dependent modulation of osmotic water permeability in plasmalemma from roots of pea (Pisum sativum L.) seedlings. This mechanism may be associated with oxidation of cysteine residues in PIP-aquaporins or involves redox-dependent phosphorylation of these proteins. Plasmalemma was isolated by partitioning of microsomal membranes in the aqueous polymer two-phase system and using the roots homogenization medium additionally contained SH-reagents, dithiothreitol or diamide, and also phenylarsin oxide, an inhibitor of tyrosine protein phosphatases. Water permeability of plasmalemma was estimated by the kinetics of light scattering changes of membrane vesicles due to their osmotic shrinkage and recorded by the stopped flow method. In order to elucidate whether PIP-aquaporins contain cysteines available for oxidation by SH-reagents, isolated plasmalemma was subjected to PEGylation (reaction, based on binding of 5 kD methoxypolyethylene glycol maleimide with available membrane proteins SH-groups) and further western blot analysis was performed to reveal shift of aquaporins molecular mass by 5 kD after their immunodetection with antibodies. Relative content of PIP-aquaporin phosphorylated forms in plasmalemma was determined using fluorescent labeling proteins in gels with ProQ Diamond after membrane proteins separation by two-dimensional electrophoresis (blue native (BN/PAGE) in first dimension and denaturing (SDS/PAGE) in second dimension). It was established that conservative cysteines in molecules of PIP-aquaporins are available for SH-reagents only under denaturing conditions. This fact excludes the possibility of these cysteines participation in modulation of plasmalemma osmotic water permeability. Comparative analysis of phosphorylated PIP-aquaporins pool with different redox status of membrane proteins and osmotic water permeability coefficients and also the inhibitor analysis data showed that redox-dependent modulation of these proteins activity is due to their phosphorylation with involvement of serine-threonine protein phosphatases of PP2C type and/or thyrosine protein phosphatases.