Rho signaling participates in membrane fluidity homeostasis.
ABSTRACT: Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast's sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.
Project description:Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1-mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2-protein phosphatase 2A(Cdc55) (PP2A(Cdc55)) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2-PP2A(Cdc55) promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1-Mpk1 activity inhibits cortical PP2A(Cdc55), ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2A(Cdc55) specifies Rho1 signaling output and that reciprocal antagonism between Rho1-PP2A(Cdc55) and Rho1-Pkc1 explains how only one signaling pathway is robustly activated at a time.
Project description:Aspergillus fumigatus is a mold and the causal agent of invasive aspergillosis, a systemic disease with high lethality. Recently, we identified and functionally characterized three stress sensors implicated in the cell wall integrity (CWI) signaling of this pathogen, namely, Wsc1, Wsc3, and MidA. Here, we functionally characterize Rom2, a guanine nucleotide exchange factor with essential function for the cell wall integrity of A. fumigatus. A conditional rom2 mutant has severe growth defects under repressive conditions and incorporates all phenotypes of the three cell wall integrity sensor mutants, e.g., the echinocandin sensitivity of the ?wsc1 mutant and the Congo red, calcofluor white, and heat sensitivity of the ?midA mutant. Rom2 interacts with Rho1 and shows a similar intracellular distribution focused at the hyphal tips. Our results place Rom2 between the cell surface stress sensors Wsc1, Wsc3, MidA, and Rho1 and their downstream effector mitogen-activated protein (MAP) kinase module Bck1-Mkk2-MpkA.
Project description:Candida albicans cells homozygous at the mating-type locus stochastically undergo the white-to-opaque switching to become mating-competent. This switching is regulated by a core circuit of transcription factors organized through interlocking feedback loops around the master regulator Wor1. Although a range of distinct environmental cues is known to induce the switching, the pathways linking the external stimuli to the central control mechanism remains largely unknown. By screening a C. albicans haploid gene-deletion library, we found that SAC7 encoding a GTPase-activating protein of Rho1 is required for the white-to-opaque switching. We demonstrate that Sac7 physically associates with Rho1-GTP and the constitutively active Rho1G18V mutant impairs the white-to-opaque switching while the inactive Rho1D124A mutant promotes it. Overexpressing WOR1 in both sac7?/? and rho1 G18V cells suppresses the switching defect, indicating that the Sac7/Rho1 module acts upstream of Wor1. Furthermore, we provide evidence that Sac7/Rho1 functions in a pathway independent of the Ras/cAMP pathway which has previously been positioned upstream of Wor1. Taken together, we have discovered new regulators and a signaling pathway that regulate the white-to-opaque switching in the most prevalent human fungal pathogen C. albicans.
Project description:In the yeast Saccharomyces cerevisiae the guanosine triphosphatase (GTPase) Rho1 controls actin polarization and cell wall expansion. When cells are exposed to various environmental stresses that perturb the cell wall, Rho1 activates Pkc1, a mammalian Protein Kinase C homologue, and Mpk1, a mitogen activated protein kinase (MAPK), resulting in actin depolarization and cell wall remodeling. In this study, we demonstrate a novel feedback loop in this Rho1-mediated Pkc1-MAPK pathway that involves regulation of Rom2, the guanine nucleotide exchange factor of Rho1, by Mpk1, the end kinase of the pathway. This previously unrecognized Mpk1-dependent feedback is a critical step in regulating Rho1 function. Activation of this feedback mechanism is responsible for redistribution of Rom2 and cell wall synthesis activity from the bud to cell periphery under stress conditions. It is also required for terminating Rho1 activity toward the Pkc1-MAPK pathway and for repolarizing actin cytoskeleton and restoring growth after the stressed cells become adapted.
Project description:The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.
Project description:Stearoyl-CoA desaturase enzyme 1 (SCD1) is a lipogenic enzyme that is upregulated in obesity, insulin resistance, and cancer. Since glucose is a substrate for both de novo fatty acid synthesis and deoxyribose synthesis, we hypothesized that SCD1 affects these multiple synthetic pathways through changes in glucose utilization. This study determined glucose utilization for fatty acid synthesis and cell proliferation in 3T3-L1 preadipocytes during SCD1 inhibition. The effects of SCD1 on cellular metabolism as mediated by its monounstaurated fatty acid products (palmitoleate and oleate) were also observed. 3T3-L1 preadipocytes underwent differentiation induction in conjunction with one of the following treatments for 4 days: (A) no treatment, (B) SCD1 inhibitor CGX0290, (C) CGX0290 + palmitoleate, or (D) CGX0290 + oleate. All cells received medium with 50 % [U(13)C]-glucose. Cells were harvested on day 7 for studies of fatty acid metabolism, tricarboxylic acid (TCA) cycle activities, and deoxyribose synthesis. CGX0290 decreased fatty acid desaturation, glucose utilization for fatty acid synthesis (acetyl-CoA enrichment), and de novo synthesis. CGX0290 treatment also led to decreased cell density through increased cell death. Further analysis showed that deoxyribose new synthesis and oxidative pentose phosphate pathway activity were unchanged, while non-oxidative transketolase pathway activity was stimulated. Palmitoleate and oleate supplementation each partially ameliorated the effects of CGX0290. In 3T3-L1 cells, SCD1 promotes glucose utilization for fatty acid synthesis. In cell proliferation, SCD1 may promote cell survival, but does not impact the oxidative pathway of deoxyribose production. These effects may be mediated through the production of palmitoleate and oleate.
Project description:The RHO1 gene encodes a homolog of the mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. Multicopy suppressors of a temperature-sensitive, dominant negative mutant allele of RHO1, RHO1(G22S, D125N), were isolated and named ROM (RHO1 multicopy suppressor). Rom1p and Rom2p were found to contain a DH (Dbl homologous) domain and a PH (pleckstrin homologous) domain, both of which are conserved among the GDP/GTP exchange proteins (GEPs) for the Rho family small GTP binding proteins. Disruption of ROM2 resulted in a temperature-sensitive growth phenotype, whereas disruption of both ROM1 and ROM2 resulted in lethality. The phenotypes of deltarom1deltarom2 cells were similar to those of deltarho1 cells, including growth arrest with a small bud and cell lysis. Moreover, the temperature-sensitive growth phenotype of deltarom2 was suppressed by overexpression of RHO1 or RHO2, but not of CDC42. The glutathione-S-transferase (GST) fusion protein containing the DH domain of Rom2p showed the lipid-modified Rholp-specific GDP/GTP exchange activity which was sensitive to Rho GDP dissociation inhibitor. These results indicate that Rom1p and Rom2p are GEPs that activate Rho1p in S.cerevisiae.
Project description:Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.
Project description:The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.
Project description:Mtl1 is a member of the cell wall integrity (CWI) pathway of Saccharomyces cerevisiae, which functions as a cell wall sensor for oxidative stress. Genome-wide transcriptional analysis revealed a cluster of genes that were down-regulated in the absence of Mtl1. Many of these genes were potentially regulated by the general stress response factor Msn2/Msn4. In response to rapamycin, caffeine, glucose starvation and oxidative stress provoked by H(2)O(2), mtl1 presents a significant loss of viability as well as a deficiency in the transcriptional response mediated by Msn2/Msn4. The Mtl1 function was required (i) to induce ribosomal gene repression, (ii) to induce the general stress response driven by the transcription factor Msn2/Msn4, and (iii) to activate the CWI pathway in response to both glucose starvation and oxidative stress. We also detected higher cAMP levels in the mtl1 mutant than in wild type cells indicative of up-regulated RAS2-PKA activity. Disruption of TOR1, disruption of RAS2, or hyperactivation of Rho1 restored both the viability and the transcriptional function (both ribosomal and Msn2/Msn4-dependent gene expression) in the mtl1 mutant to almost wild type levels when cells were starved of glucose or stressed with H(2)O(2). Taking our results together, we propose an essential role for Mtl1 in signaling oxidative stress and quiescence to the CWI pathway and to the general stress response through Rho1 and the inhibition of either the TOR1 or RAS2 functions. These mechanisms would be required to allow cells to adapt to both oxidative and nutritional stresses.