scholarly journals Receptor Internalization in Yeast Requires the Tor2-Rho1 Signaling Pathway

2003 ◽  
Vol 14 (11) ◽  
pp. 4676-4684 ◽  
Author(s):  
Amy K.A. deHart ◽  
Joshua D. Schnell ◽  
Damian A. Allen ◽  
Ju-Yun Tsai ◽  
Linda Hicke
Keyword(s):  
Cell Wall ◽  
Cell Cycle Progression ◽  
Structural Integrity ◽  
Nutrient Sensing ◽  
Receptor Internalization ◽  
Cell Integrity ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Similar Phenotype ◽  
Cell Growth And Differentiation

Efficient internalization of proteins from the cell surface is essential for regulating cell growth and differentiation. In a screen for yeast mutants defective in ligand-stimulated internalization of the α-factor receptor, we identified a mutant allele of TOR2, tor2G2128R. Tor proteins are known to function in translation initiation and nutrient sensing and are required for cell cycle progression through G1. Yeast Tor2 has an additional role in regulating the integrity of the cell wall by activating the Rho1 guanine nucleotide exchange factor Rom2. The endocytic defect in tor2G2128Rcells is due to disruption of this Tor2 unique function. Other proteins important for cell integrity, Rom2 and the cell integrity sensor Wsc1, are also required for efficient endocytosis. A rho1 mutant specifically defective in activation of the glucan synthase Fks1/2 does not internalize α-factor efficiently, and fks1Δ cells exhibit a similar phenotype. Removal of the cell wall does not inhibit internalization, suggesting that the function of Rho1 and Fks1 in endocytosis is not through cell wall synthesis or structural integrity. These findings reveal a novel function for the Tor2-Rho1 pathway in controlling endocytosis in yeast, a function that is mediated in part through the plasma membrane protein Fks1.

scholarly journals The citron homology domain as a scaffold for Rho1 signaling

2021 ◽  
Vol 118 (39) ◽  
pp. e2110298118
Author(s):  
Sergio G. Bartual ◽  
Wenfan Wei ◽  
Yao Zhou ◽  
Veronica M. Pravata ◽  
Wenxia Fang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Opportunistic Pathogen ◽  
Nucleotide Exchange ◽  
Chitin Synthesis ◽  
Fungal Cell ◽  
Guanine Nucleotide Exchange ◽  
Eukaryotic Genes ◽  
Nucleotide Exchange Factor ◽  
Domain Of Unknown Function ◽  
Important Regulator

Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate β-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed β-propeller fold containing three unusual loops. A model of the Rho1–Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis.

scholarly journals Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

2012 ◽  
Vol 12 (2) ◽  
pp. 288-298 ◽  
Author(s):  
Sweta Samantaray ◽  
Michael Neubauer ◽  
Christoph Helmschrott ◽  
Johannes Wagener
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Guanine Nucleotide Exchange Factor ◽  
Cell Wall Integrity ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Content Type ◽  
Guanine Nucleotide Exchange ◽  
Stress Sensors ◽  
Nucleotide Exchange Factor

ABSTRACTAspergillus fumigatusis 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 ofA. fumigatus. A conditionalrom2mutant 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 Δwsc1mutant and the Congo red, calcofluor white, and heat sensitivity of the ΔmidAmutant. 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.

scholarly journals Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

2001 ◽  
Vol 21 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Bevin Philip ◽  
David E. Levin
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Cell Surface ◽  
G Protein ◽  
Guanine Nucleotide Exchange Factor ◽  
Cell Wall Integrity ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in apmt2 mutant and that this modification is important for signaling by Mid2.

scholarly journals Susceptibility of Cryptococcus neoformans to Photodynamic Inactivation Is Associated with Cell Wall Integrity

2007 ◽  
Vol 51 (8) ◽  
pp. 2929-2936 ◽  
Author(s):  
Beth Burgwyn Fuchs ◽  
George P. Tegos ◽  
Michael R. Hamblin ◽  
Eleftherios Mylonakis
Keyword(s):  
Cell Wall ◽  
Photodynamic Therapy ◽  
Cryptococcus Neoformans ◽  
Cell Wall Integrity ◽  
Photodynamic Inactivation ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Kinase C ◽  
Nucleotide Exchange Factor ◽  
Cell Wall Integrity Pathway

ABSTRACT Photodynamic therapy is a rapidly developing antimicrobial technology which combines a nontoxic photoactivatable dye or photosensitizer with harmless visible light of the correct wavelength to excite the dye to its reactive triplet state to generate reactive oxygen species toxic to cells. In this report we present evidence that the fungal pathogen Cryptococcus neoformans is susceptible to photodynamic inactivation by use of a polycationic conjugate of polyethyleneimine and the photosensitizer chlorin(e6). A C. neoformans rom2 mutant, with a mutation involving a putative Rho1 guanyl nucleotide exchange factor that is part of the protein kinase C-cell wall integrity pathway, demonstrated a compromised cell wall and less (1,3)β-d glucan than the wild-type strain and increased accumulation of PEI-ce6 as assessed by fluorescence uptake and confocal microscopy. Interestingly, C. neoformans rom2 was hypersusceptible to photodynamic inactivation and coincubation of wild-type C. neoformans strain KN99α with caspofungin-enhanced photoinactivation. These studies demonstrated that C. neoformans is sensitive to photodynamic therapy and illustrated the significance of cell wall integrity in microbial susceptibility to antimicrobial photodynamic inactivation.

scholarly journals Involvement of Saccharomyces cerevisiae Avo3p/Tsc11p in Maintaining TOR Complex 2 Integrity and Coupling to Downstream Signaling

2008 ◽  
Vol 7 (8) ◽  
pp. 1328-1343 ◽  
Author(s):  
Hsiang-Ling Ho ◽  
Hsin-Yi Lee ◽  
Hsien-Ching Liao ◽  
Mei-Yu Chen
Keyword(s):  
Protein Interactions ◽  
Growth Control ◽  
The Other ◽  
Temperature Sensitive ◽  
Cell Integrity ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
Downstream Signaling ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Target-of-rapamycin proteins (TORs) are Ser/Thr kinases serving a central role in cell growth control. TORs function in two conserved multiprotein complexes, TOR complex 1 (TORC1) and TORC2; the mechanisms underlying their actions and regulation are not fully elucidated. Saccharomyces TORC2, containing Tor2p, Avo1p, Avo2p, Avo3p/Tsc11p, Bit61p, and Lst8p, regulates cell integrity and actin organization. Two classes of avo3 temperature-sensitive (avo3 ts) mutants that we previously identified display cell integrity and actin defects, yet one is suppressed by AVO1 while the other is suppressed by AVO2 or SLM1, defining two TORC2 downstream signaling mechanisms, one mediated by Avo1p and the other by Avo2p/Slm1p. Employing these mutants, we explored Avo3p functions in TORC2 structure and signaling. By observing binary protein interactions using coimmunoprecipitation, we discovered that the composition of TORC2 and its recruitment of the downstream effectors Slm1p and Slm2p were differentially affected in different avo3 ts mutants. These molecular defects can be corrected only by expressing AVO3, not by expressing suppressors, highlighting the role of Avo3p as a structural and signaling scaffold for TORC2. Phenotypic modifications of avo3 ts mutants by deletion of individual Rho1p-GTPase-activating proteins indicate that two TORC2 downstream signaling branches converge on Rho1p activation. Our results also suggest that Avo2p/Slm1p-mediated signaling, but not Avo1p-mediated signaling, links to Rho1p activation specifically through the Rho1p-guanine nucleotide exchange factor Tus1p.

scholarly journals Tuba, a Cdc42 GEF, is required for polarized spindle orientation during epithelial cyst formation

2010 ◽  
Vol 189 (4) ◽  
pp. 661-669 ◽  
Author(s):  
Yi Qin ◽  
Walter H. Meisen ◽  
Yi Hao ◽  
Ian G. Macara
Keyword(s):  
Cyst Formation ◽  
Cell Polarization ◽  
Spindle Orientation ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Lateral Plane ◽  
Similar Phenotype ◽  
Guanosine Triphosphatase

The Cdc42 guanosine triphosphatase is essential for cell polarization in several organisms and in vitro for the organization of polarized epithelial cysts. A long-standing question concerns the identity of the guanine nucleotide exchange factor (GEF) that controls this process. Using Madin–Darby canine kidney cells grown in Matrigel, we screened 70 GEFs by RNA interference. Of these, six positives were identified that caused a multilumen phenotype, including Tuba, a Cdc42-specific GEF localized below the apical cortex. Loss of Tuba abolishes Cdc42 enrichment at the apical cortex. Normal lumen formation is rescued by human Tuba or active Cdc42 but not by a GEF-negative Tuba mutant. Silencing Cdc42 causes a similar phenotype, including multilumen formation and reduced atypical protein kinase C (aPKC) activity. Lumen disorganization after depletion of Tuba or Cdc42 or inhibition of aPKC is caused by defective spindle orientation. Together, our findings implicate Tuba as a key activator of the Cdc42 GTPase during epithelial ductal morphogenesis, which in turn activates apical aPKC to ensure that spindles orient parallel to the lateral plane.

scholarly journals VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing

2020 ◽  
Vol 10 ◽  
Author(s):  
Arda Balci ◽  
Virtu Solano-Collado ◽  
Massimiliano Baldassarre ◽  
Stefania Spanò
Keyword(s):  
Typhoid Fever ◽  
Causative Agent ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Over Expression ◽  
Murine Macrophages ◽  
Ankyrin Repeat Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Similar Phenotype

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.

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