Search

In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP‐DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1‐acyl lysoPA and subsequent acylation of 1‐acyl lysoPA at the sn‐2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1‐acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn‐1 and/or sn‐2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn‐1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids. [ABSTRACT FROM AUTHOR]

Phosphoinositides (PIPs) are present in very small amounts but are essential for cell signaling, morphogenesis, and polarity. By mass spectrometry, we demonstrated that some PIPs with stearic acyl chains were strongly disturbed in a psi1Δ Saccharomyces cerevisiae yeast strain deficient in the specific incorporation of a stearoyl chain at the sn-1 position of phosphatidylinositol. The absence of PIPs containing stearic acid induced disturbances in intracellular trafficking, although the total amount of PIPs was not diminished. Changes in PIPs also induced alterations in the budding pattern and defects in actin cytoskeleton organization (cables and patches). Moreover, when the PSI1 gene was impaired, a high proportion of cells with bipolar cortical actin patches that occurred concomitantly with the bipolar localization of Cdc42p was specifically found among diploid cells. This bipolar cortical actin phenotype, never previously described, was also detected in a bud9Δ/bud9Δ strain. Very interestingly, overexpression of PSI1 reversed this phenotype. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spiroplasma citri is a plant-pathogenic mollicute phylogenetically related to Gram-positive bacteria. Spiroplasma cells are restricted to the phloem sieve tubes and are transmitted from plant to plant by the leafhopper vector Circulifer haematoceps. In the plant sieve tubes, S. citri grows on glucose and fructose, whereas in the leafhopper haemolymph the spiroplasma must grow on trehalose, the major sugar in insects. Previous studies in this laboratory have shown that fructose utilization was a key factor of spiroplasmal pathogenicity. To further study the implication of sugar metabolism in the interactions of S. cirri with its plant host and its leafhopper vector, genes encoding permease enzymes II (Ell[sup Glc] and EII[sup Tre]) of the S. citri phosphoenolpyruvate:glucose and phosphoenolpyruvate:trehalose phosphotransferase systems (PTS) were characterized. Mapping studies revealed that the Ell[sup Glc] complex was split into two distinct polypeptides, IIA[sup Glc] and IICB[sup Glc], encoded by two separate genes, crr and ptsG, respectively. As expected, S. citri polypeptides IIA[sup Glc] and IICB[sup Glc] were more phylogenetically related to their counterparts from Gram-positive than to those from Gram-negative bacteria. The trehalose operon consisted of three genes treR, treP and treA, encoding a transcriptional regulator, the PTS permease (EII[sup Tre]) and the amylase, respectively. However, in contrast to the fructose-PTS permease, which is encoded as a single polypeptide (IIABC[sup Fru]) containing the three domains A, B and C, the trehalose-PTS permease (IIBC[sup Tre]) lacks its own IIA domain. No trehalose-specific IIA could be identified in the spiroplasmal genome, suggesting that the IIBC[sup Tre] permease probably functions with the IIA[sup Glc] domain. In agreement with this statement, yeast two-hybrid system experiments revealed that the IIA[sup Glc] domain interacted not only with IIB[sup Glc] but also with the IIB[sup Tre] domain. The results are discussed with respect to the ability of the spiroplasma to adapt from the phloem sap of the host plant to the haemolymph and salivary gland cells of the insect vector. [ABSTRACT FROM AUTHOR]

Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal anti- fetidin serum, we have cloned a cDNA that encoded the 40-Wa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen. [ABSTRACT FROM AUTHOR]

Abstract: Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population. [Copyright &y& Elsevier]

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Abstract: The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides. [Copyright &y& Elsevier]

Abstract: Rho proteins are essential regulators of polarized growth in eukaryotic cells. These proteins are down-regulated in vivo by specific Rho GTPase Activating Proteins (RhoGAP). We investigated the role of Rgd1 RhoGAP, encoded by the Candida albicans RGD1 gene. We demonstrated that CaCdc42, CaRho3 and CaRho4 proteins had an intrinsic GTPase activity and that CaRgd1 stimulates in vitro GTP hydrolysis of these GTPases. Deletion of RGD1 in C. albicans results in sensitivity to low pH as already described for rgd1Δ in Saccharomyces cerevisiae. The role of Rgd1 in survival at low pH is conserved in the two yeast species as the CaRGD1 gene complements the Scrgd1Δ sensitivity. By tagging the RhoGAP with GFP, we found that CaRgd1 is localized at the tip and cortex of growing cells and during cytokinesis at the septation sites in yeast and filamentous forms. We investigated the effect of CaRgd1 on the control of the polarized growth. Removing CaRGD1 alleles increased filamentous growth and cells lacking CaRgd1 presented longer germ tubes. Conversely, RGD1 overexpression restricted hyphae growth. Our results demonstrate that Rgd1 is critical for filamentous formation in C. albicans especially for filamentous elongation. [Copyright &y& Elsevier]

Cell polarity is a key element of development in most eukaryotes. The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p contains an F-BAR domain at its N-terminal end in addition to its RhoGAP domain at its C-terminal end. We demonstrate here that phospholipids discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity on Rho4p. The central region of the protein contiguous to the F-BAR domain is required for this stimulation. The F-BAR region binds to phosphoinositides in vitro and also plays a key role in the localization of Rgd1p to the bud tip and neck during the cell cycle. Studies of heat-sensitive mutants lacking phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate suggested that Rgd1p initially binds to Golgi membranes via phosphatidylinositol 4-phosphate and is then transported to the plasma membrane, where it binds phosphatidylinositol 4,5-biphosphate. We demonstrate here the dual effects of phosphoinositides on a RhoGTPase-activating protein. Phosphoinositides both regulate the recruitment and trafficking of Rgd1p to membranes via the F-BAR domain and specifically stimulate GTPase-activating protein activity, consistent with functional interplay between lipids, RhoGAP, and its related GTPases in yeast growth. [ABSTRACT FROM AUTHOR]

The article proposes that Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae, participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the protein kinase C-mitogen-activated protein kinase pathway. It details the yeast strains and genetic procedures. The article also discusses the production of Rho3 and Rho4 GTPases.

Abstract: The RhoGAP Rgd1p is involved in different signal transduction pathways in Saccharomyces cerevisiae through its regulatory activity upon the Rho3 and Rho4 GTPases. The rgd1Δ mutant, which presents a mortality at the entry into the stationary phase in minimal medium, is sensitive to medium acidification caused by biomass augmentation. We showed that low-pH shock leads to abnormal intracellular acidification of the rgd1Δ mutant. Transcriptional regulation of RGD1 was studied in several stress conditions and we observed an activation of RGD1 transcription at low pH and after heat and oxidative shocks. The transcription level at low pH and after heat shock was demonstrated to depend on the STRE box located in the RGD1 promoter. The general stress-activated transcription factors Msn2p and Msn4p as well as the HOG pathway were shown to mainly act on the basal RGD1 transcriptional level in normal and stress conditions. [Copyright &y& Elsevier]

The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH–coiled coil–RhoGAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1 Δ and vrp1 Δ mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1–VRP1 co-lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo . Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied. These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity. [ABSTRACT FROM AUTHOR]