scholarly journals Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

2008 ◽  
Vol 8 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Connie B. Nichols ◽  
Jessica Ferreyra ◽  
Elizabeth R. Ballou ◽  
J. Andrew Alspaugh
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Sexual Differentiation ◽  
Signaling Cascades ◽  
Ras Signaling ◽  
Signaling Specificity ◽  
Downstream Signaling ◽  
Functional Flexibility ◽  
Human Fungal Pathogen

ABSTRACT In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.

scholarly journals Three Separable Domains Regulate GTP-Dependent Association of H-ras with the Plasma Membrane

2004 ◽  
Vol 24 (15) ◽  
pp. 6799-6810 ◽  
Author(s):  
Barak Rotblat ◽  
Ian A. Prior ◽  
Cornelia Muncke ◽  
Robert G. Parton ◽  
Yoel Kloog ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Catalytic Domain ◽  
Attractive Force ◽  
Live Cells ◽  
Membrane Microdomains ◽  
Ras Signaling ◽  
Signaling Specificity ◽  
Linker Domain ◽  
Lipid Anchor

ABSTRACT The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

scholarly journals Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Shannon K. Esher ◽  
Kyla S. Ost ◽  
Lukasz Kozubowski ◽  
Dong-Hoon Yang ◽  
Min Su Kim ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Posttranslational Modification ◽  
Fungal Pathogen ◽  
Content Type ◽  
Processing Enzymes ◽  
Human Fungal Pathogen ◽  
Modification Process ◽  
Substrate Proteins ◽  
Processing Steps

ABSTRACT Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus. Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.

scholarly journals TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

2009 ◽  
Vol 20 (5) ◽  
pp. 1565-1575 ◽  
Author(s):  
Doris Berchtold ◽  
Tobias C. Walther
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Lipid Binding ◽  
Membrane Localization ◽  
Cellular Functions ◽  
Signaling Specificity ◽  
Downstream Signaling ◽  
Plasma Membrane Localization ◽  
Membrane Compartment ◽  
Plasma Membrane Domain

The conserved target of rapamycin (TOR) kinases regulate many aspects of cellular physiology. They exist in two distinct complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2), that posses both overlapping and distinct components. TORC1 and TORC2 respond differently to the drug rapamycin and have different cellular functions: whereas the rapamycin-sensitive TORC1 controls many aspects of cell growth and has been characterized in great detail, the TOR complex 2 is less understood and regulates actin polymerization, cell polarity, and ceramide metabolism. How signaling specificity and discrimination between different input signals for the two kinase complexes is achieved is not understood. Here, we show that TORC1 and TORC2 have different localizations in Saccharomyces cerevisiae. TORC1 is localized exclusively to the vacuolar membrane, whereas TORC2 is localized dynamically in a previously unrecognized plasma membrane domain, which we term membrane compartment containing TORC2 (MCT). We find that plasma membrane localization of TORC2 is essential for viability and mediated by lipid binding of the C-terminal domain of the Avo1 subunit. From these data, we suggest that the TOR complexes are spatially separated to determine downstream signaling specificity and their responsiveness to different inputs.

scholarly journals Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans

2015 ◽  
Vol 14 (7) ◽  
pp. 626-635 ◽  
Author(s):  
Connie B. Nichols ◽  
Kyla S. Ost ◽  
Dayton P. Grogan ◽  
Kaila Pianalto ◽  
Shirin Hasan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Temperature ◽  
Cryptococcus Neoformans ◽  
Genome Database ◽  
Temperature Growth ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Virulence Potential ◽  
Protein Palmitoylation ◽  
And Function

ABSTRACT The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans , Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S -acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence.

scholarly journals H-Ras Signaling and K-Ras Signaling Are Differentially Dependent on Endocytosis

2002 ◽  
Vol 22 (14) ◽  
pp. 5128-5140 ◽  
Author(s):  
Sandrine Roy ◽  
Bruce Wyse ◽  
John F. Hancock
Keyword(s):  
Plasma Membrane ◽  
Specific Activity ◽  
Mitogen Activated Protein Kinase ◽  
Membrane Microdomains ◽  
Ras Signaling ◽  
Wild Type ◽  
Endocytic Recycling ◽  
Membrane Recruitment ◽  
Downstream Signaling ◽  
Mitogen Activated Protein

ABSTRACT Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

scholarly journals Remodeling of Global Transcription Patterns of Cryptococcus neoformans Genes Mediated by the Stress-Activated HOG Signaling Pathways

2009 ◽  
Vol 8 (8) ◽  
pp. 1197-1217 ◽  
Author(s):  
Young-Joon Ko ◽  
Yeong Man Yu ◽  
Gyu-Bum Kim ◽  
Gir-Won Lee ◽  
Pil Jae Maeng ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Efflux Pump ◽  
Pathogenic Fungi ◽  
Global Gene Expression ◽  
Signaling Cascades ◽  
Ergosterol Biosynthesis ◽  
Hog Pathway ◽  
Potassium Efflux ◽  
Downstream Signaling ◽  
External Stresses

ABSTRACT The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.

scholarly journals UGE1 and UGE2 Regulate the UDP-Glucose/UDP-Galactose Equilibrium in Cryptococcus neoformans

2008 ◽  
Vol 7 (12) ◽  
pp. 2069-2077 ◽  
Author(s):  
Frédérique Moyrand ◽  
Ingrid Lafontaine ◽  
Thierry Fontaine ◽  
Guilhem Janbon
Keyword(s):  
Plasma Membrane ◽  
Carbon Source ◽  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Dependent Manner ◽  
Pathogenic Yeast ◽  
Intracellular Vesicles ◽  
Yeast Genomes ◽  
Fungal Kingdom ◽  
Affect Cell Growth

ABSTRACT The genome of the basidiomycete pathogenic yeast Cryptococcus neoformans carries two UDP-glucose epimerase genes (UGE1 and UGE2). UGE2 maps within a galactose cluster composed of a galactokinase homologue gene and a galactose-1-phosphate uridylyltransferase. This clustered organization of the GAL genes is similar to that in most of the hemiascomycete yeast genomes and in Schizosaccharomyces pombe but is otherwise not generally conserved in the fungal kingdom. UGE1 has been identified as necessary for galactoxylomannan biosynthesis and virulence. Here, we show that UGE2 is necessary for C. neoformans cells to utilize galactose as a carbon source at 30°C but is not required for virulence. In contrast, deletion of UGE1 does not affect cell growth on galactose at this temperature. At 37°C, a uge2Δ mutant grows on galactose in a UGE1-dependent manner. This compensation by UGE1 of UGE2 mutation for growth on galactose at 37°C was not associated with upregulation of UGE1 transcription or with an increase of the affinity of the enzyme for UDP-galactose at this temperature. We studied the subcellular localization of the two enzymes. Whereas at 30°C, Uge1p is at least partially associated with intracellular vesicles and Uge2p is on the plasma membrane, in cells growing on galactose at 37°C, Uge1p colocalizes with Uge2p to the plasma membrane, suggesting that its activity is regulated through subcellular localization.

scholarly journals Visualizing Wnt secretion from Endoplasmic Reticulum to Filopodia

2018 ◽  
Author(s):  
Naushad Moti ◽  
Jia Yu ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
David M Virshup
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Signaling Cascades ◽  
Downstream Signaling ◽  
Cell Compartments ◽  
A Cell ◽  
Cortical Regions ◽  
Rate Limiting ◽  
Wnt Signal

AbstractWnts are a family of secreted palmitoleated glycoproteins that play a key role in cell to cell communications during development and regulate stem cell compartments in adults. Wnt receptors, downstream signaling cascades and target pathways have been extensively studied while less is known about how Wnts are secreted and move from producing cells to receiving cells. We used the synchronization system called Retention Using Selective Hook (RUSH) to study Wnt trafficking from endoplasmic reticulum to Golgi and then to plasma membrane and filopodia in real time. Consistent with prior studies, inhibition of porcupine (PORCN) or knockout of Wntless (WLS) blocked Wnt exit from the ER. Indeed, WLS was rate-limiting for Wnt ER exit. Wnt-containing vesicles paused at sub-cortical regions of the plasma membrane before exiting the cell. Wnt-containing vesicles were transported to adjacent cells associated with filopodia. Increasing the number of filopodia by expression of LGR5 in the producing cell increased the ability of a cell to send a Wnt signal. The RUSH system is a powerful tool to provide new insights into the Wnt secretory pathway.

scholarly journals PTPN2 regulates the activation of KRAS and plays a critical role in proliferation and survival of KRAS-driven cancer cells

2020 ◽  
Vol 295 (52) ◽  
pp. 18343-18354
Author(s):  
Zhangsen Huang ◽  
Mingzhu Liu ◽  
Donghe Li ◽  
Yun Tan ◽  
Ruihong Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cancer Cells ◽  
Poor Prognosis ◽  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Membrane Association ◽  
Ras Signaling ◽  
Screening Assay ◽  
Downstream Signaling ◽  
Effective Signal

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

scholarly journals Internalization of the host alkaline pH signal in a fungal pathogen

2020 ◽  
Author(s):  
Hannah E. Brown ◽  
Kaila M. Pianalto ◽  
Caroline M. Fernandes ◽  
Katherine D. Mueller ◽  
Maurizio Del Poeta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Fungal Pathogen ◽  
Cellular Response ◽  
Microbial Pathogens ◽  
Dependent Manner ◽  
Human Host ◽  
Human Fungal Pathogen ◽  
Stress Signals

AbstractThe ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. This adaptability is especially important for microbial pathogens that must sense and respond to drastic changes when encountering the human host. Cryptococcus neoformans is an environmental fungus and opportunistic pathogen that naturally lives in slightly acidic reservoirs, but must adapt to the relative increase in alkalinity in the human host in order to effectively cause disease. The fungal-specific Rim alkaline response signaling pathway effectively converts this extracellular signal into an adaptive cellular response allowing the pathogen to survive in its new environment. The newly identified Rra1 protein, the most upstream component of the C. neoformans Rim pathway, is an essential component of this alkaline response. Previous work connected Rra1-mediated signaling to the dynamics of the plasma membrane. Here we identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner, and that this dynamic pattern of localization further depends on specific residues in its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. The data presented here continue to unravel the complex and intricate processes of pH-sensing in a relevant human fungal pathogen. These studies will further elucidate general mechanisms by which cells respond to and internalize extracellular stress signals.Author SummaryThe work described here explores the genetics and mechanics of a cellular signaling pathway in a relevant human fungal pathogen, Cryptococcus neoformans. The findings presented in this manuscript untangle the complex interactions involved in the activation of a fungal-specific alkaline response pathway, the Rim pathway. Specifically, we find that C. neoformans is able to sense an increase in pH within the human host, internalize a membrane-bound pH-sensor, and activate a downstream signaling pathway enabling this pathogen to adapt to a novel host environment and effectively cause disease. Revealing the mechanisms of Rim pathway activation within the larger context of the fungal cell allows us to understand how and when this microorganism interprets relevant host signals. Furthermore, understanding how this pathogenic organism converts extracellular stress signals into an adaptive cellular response will elucidate more general mechanisms of microbial environmental sensing and stress response.

Sign in / Sign up

Export Citation Format

Share Document