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Cell polarity is achieved by regulators such as small G proteins, exocyst members and phosphoinositides, with the latter playing a key role when bound to the exocyst proteins Sec3p and Exo70p, and Rho GTPases. This ensures asymmetric growth via the routing of proteins and lipids to the cell surface using actin cables. Previously, using a yeast mutant for a lysophosphatidylinositol acyl transferase encoded by the PSI1 gene, we demonstrated the role of stearic acid in the acyl chain of phosphoinositides in cytoskeletal organization and secretion. Here, we use a genetic approach to characterize the effect on late steps of the secretory pathway. The constitutive overexpression of PSI1 in mutants affecting kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane, which is essential for the function of the Cdc42p module. Decreasing the levels of stearic acid-containing phosphoinositides modifies the environment of the actors involved in the control of late steps in the secretory pathway. This leads to decreased interactions between Exo70p and Sec3p, with Cdc42p, Rho1p and Rho3p, because of disruption of the GTP/GDP ratio of at least Rho1p and Rho3p GTPases, thereby preventing activation of the exocyst

Influence des fongicides inhibiteurs de la biosynthese l'ergosterol chez Saccharomyces cerevisiae lors de la fermentation alcoolique. Etude et utilisation des genes impliques

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 80327 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Biomol NMR Assign

The protein Rgd1 is involved in the regulation of cytoskeleton formation and in signalling pathways that control cell polarity and growth in Saccharomyces cerevisiae. Rgd1p is composed of a F-BAR domain required for membrane binding and a RhoGAP domain responsible for activating Rho3p and Rho4p, two GTPases respectively involved in bud growth and cytokinesis. Rgd1p is recruited to the membrane through interactions with phosphoinositide lipids, which bind the two isolated domains and stimulate the RhoGAP activity on Rho4p. As previously shown by crystallography, the membrane-binding F-BAR domain contains a conserved inositol phosphate binding site, which explains the preferential binding of phosphoinositides. In contrast, RhoGAP domains are not expected to bind lipids. In order to unravel this puzzling feature, we solved the three-dimensional structure of the isolated protein and found a cryptic phosphoinositide binding site involving non conserved residues (Martinez et al. 2017). The assignment of the resonances and secondary structure of Rgd1-RhoGAP (aa 450-666) is presented here

Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain

Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners