Interazione tra le proteine 14-3-3 e l'H+-ATPasi di membrana plasmatica: ruolo delle poliammine e via di trasduzione indotta dagli zuccheri

14-3-3 proteins are a family of dimeric proteins found in in every eukaryotic organism. Over 100 proteins bind to 14-3-3, including various protein kinases and phosphatases: proteins involved in cell cycle control and in transcriptional control of gene expression, metabolism, and apoptosis. 14-3-3 proteins are known in plants as regulators of the proton pump H+-ATPase. Plasma membrane H+-ATPases constitute a family of proton pumps driven by hydrolysis of ATP and are found exclusively in the plasma membrane of plants and fungi. The enzyme's C-terminus regulatory domain is an autoinhibitory domain that interacts directly with regulatory protein 14-3-3. The study carried out in this thesis has been divided in two parts: in the first one we investigated the possible role of the polyamines in modulating 14-3-3 association with the H+-ATPase. Polyamines are abundant polycationic compounds involved in many plant physiological processes such as cell division, dormancy breaking, plant morphogenesis and response to environmental stresses. In vivo experiments demonstrate that, among the different polyamines, spermine brings about two fold stimulation of the H+-ATPase activity and this effect is due to an increasing of 14-3-3 levels associated with the enzyme. In vivo administration of polyamines synthesis inhibitors causes a significant decrease of the H+-ATPase phosphohydrolitic activity, demonstrating a physiological role of the polyamines in regulating the enzyme activity. Spermine stimulates the activity of the H+-ATPase AHA1 expressed in yeast ER, in the presence of exogenous 14-3-3 proteins. Moreover, spermine strongly stabilizes the 14-3-3 association with regulatory C-terminal domain of the proton pump. Comparison of spermine with Mg2+, necessary for binding of 14-3-3s to different target proteins, shows that polyamine effect is stronger and additive to that of the divalent cation. In the second part of this thesis we analyzed a sugar-mediated regulation of plasma membrane H+-ATPase. Since the involvement of 14-3-3 proteins in sugar sensing regulated processes has recently emerged, in this thesis we address the question as to whether sugars sensing plays a role in the regulation of the H+-ATPase. The data reported show that sugar depletion inhibits the association of 14-3-3 proteins to the H+-ATPase by hampering phosphorylation of the 14-3-3 binding site of the enzyme. By using non-metabolizable disaccharides it is proven that H+-ATPase regulation by 14-3-3 proteins can involve a specific sugar perception and transduction mechanism