Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan‐type N‐glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha‐1,2‐mannosyltransferase that synthesizes mannan‐type N‐glycans, by a tetracycline‐regulatable system. MNN2‐repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen‐activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2‐repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan‐type N‐glycans.     

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v‐SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)‐repressive conditions, although in wild‐type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post‐Golgi endosomes. The mislocalized Snc1 was co‐localized with an endocytic marker dye, FM4‐64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase‐activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP‐restricted form of Ypt32 GTPase. Furthermore, an endocytosis‐deficient mutant of Snc1 was localized to plasma membranes in PSS1‐repressed csg2Δ mutant cells as well as wild‐type cells. Thus, the PSS1‐repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post‐Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre‐vacuolar endosomes in the PSS1‐repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co‐ordinately involved in specific vesicular trafficking pathway.     

Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 fused to the green fluorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements confirmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K+ channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active Kin+ rectifier in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that Kin+ rectifier remains in clusters during trafficking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K+ channels preferentially in clusters during constitutive and pressure-driven exo- and endocytosis.     

Role of the protein kinase Kin1 and nuclear centering in actomyosin ring formation in fission yeast

Cytokinesis is the last step of the cell cycle, producing two daughter cells inheriting equal genetic information. This process involves the assembly of an actomyosin ring during mitosis. In the fission yeast Schizosaccharomyces pombe, cytokinesis occurs at the geometric cell centre, a position which is defined by the interphase nucleus and the anilin-related Mid1 protein. The pom1Δ, tea1Δ and tea4Δ mutants are defective in restricting Mid1 as a band around the nucleus and misplace the division site. We previously reported that inhibition of the protein kinase Kin1 promoted failure of cytokinesis in pom1Δ and tea1Δ cells but the mechanism involving Kin1 remained elusive. Here we investigated the contribution of Kin1 in cytokinesis. We show that Kin1-GFP has a dynamic cell-cycle regulated distribution. Like pom1Δ and tea1Δ, tea4Δ exhibits a strong genetic interaction with kin1Δ. Using a conditional repressible kin1 allele that only alters interphase nuclear centering, we observed that Kin1 down-regulation severely compromised actomyosin ring formation and septum synthesis in tea4Δ cells. In addition, nuclear displacement induced either by overexpression of a putative catalytically inactive Kin1 mutant, by chemically mediated microtubule depolymerization or by mutation in the par1Δ gene impaired cytokinesis in tea4Δ but not tea4+ cells. We propose that nuclear mispositioning exacerbates the tea4Δ, pom1Δ and tea1Δ cell division phenotype. Our work reveal that nuclear centering becomes essential when Pom1/Tea1/Tea4 function is compromised and that Kin1 expression level is a key regulatory element in this situation. Our results suggest the existence of distinct overlapping control mechanisms to ensure efficient cell division.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Sorting nexin 11 knockout mice exhibit enhanced thermosensing behaviour

Temperature sensing is an important adaptive mechanism for warm‐blooded animals such as humans. ThermoTRP ion channels are activated by distinct but overlapping physiological temperatures. Our previous research demonstrated that sorting nexin 11 (SNX11) regulates lysosomal degradation of plasma membrane TRPV3, one of ThermoTRP ion channel proteins. Here, we found that SNX11, a vesicular trafficking protein, modulates mouse behaviour in response to temperature changes. Snx11‐knockout mice exhibit a stronger preference for mild temperatures along with enhanced sensitivity to harmful heat. Mechanistically, keratinocytes from Snx11‐knockout mice exhibit a larger temperature‐gated TRPV3 membrane current and have enhanced thermoTRPV3 expression in the plasma membrane compared to wild‐type keratinocytes. Additionally, Snx11‐knockout mice show higher endogenous TRPV3 protein levels in skin tissues than wild‐type mice do. Therefore, our results indicate that SNX11 may regulate thermal perception via alteration of functional thermoTRPV3 on the plasma membrane of thermally sensitive cells, which is the first link between vesicular trafficking and thermal transduction.     

The Arabidopsis CERK1‐associated kinase PBL27 connects chitin perception to MAPK activation

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola. PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo. Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.     

The F‐box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen‐activated protein kinase (MAPK) pathways in Fusarium oxysporum

F‐box proteins determine substrate specificity of the ubiquitin–proteasome system. Previous work has demonstrated that the F‐box protein Fbp1, a component of the SCF^Fbp1 E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen‐activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

RHO1 and RHO2 share partially overlapping functions in the regulation of cell wall integrity and hyphal polarity in Neurospora crassa

Rho proteins are key regulators of cellular morphogenesis, but their function in filamentous fungi is poorly understood. By generating conditional rho‐1 mutants, we dissected the function of the essential GTPase RHO1 in cell polarization and maintenance of cell wall integrity in Neurospora crassa. We identified NCU00668/RGF1 as RHO1‐specific exchange factor, which controls actin organization and the cell wall integrity MAK1 MAP kinase pathway through the direct interaction of active RHO1 with the formin BNI1 and PKC1 respectively. The activity of RGF1 is controlled by an intramolecular interaction of its DEP and GEF domains that blocks the activation of the GTPase. Moreover, the N‐terminal region including the DEP domain of RGF1 interacts with the plasma membrane sensor NCU06910/WSC1, potentially to activate the cell wall integrity pathway. RHO1 also functions as regulatory subunit of the glucan synthase. N. crassa possesses a second GTPase, RHO2, that is highly homologous to RHO1. RHO2 is of minor importance for growth and does not interact with BNI1. Conditional rho‐1;rho‐2 double mutants display strong synthetic growth and cell polarity defects. We show that RHO2 does not regulate glucan synthase activity and the actin cytoskeleton, but physically interacts with PKC1 to regulate the cell wall integrity pathway.     

Expression of the yeast aquaporin Aqy2 affects cell surface properties under the control of osmoregulatory and morphogenic signalling pathways

Aquaporins mediate rapid and selective water transport across biological membranes. The yeast Saccharomyces cerevisiae possesses two aquaporins, Aqy1 and Aqy2. Here, we show that Aqy2 is involved in controlling cell surface properties and that its expression is controlled by osmoregulatory and morphogenic signalling pathways. Deletion of AQY2 results in diminished fluffy colony morphology while overexpression of AQY2 causes strong agar invasion and adherence to plastic surfaces. Hyper‐osmotic stress inhibits morphological developments including the above characteristics as well as AQY2 expression through the osmoregulatory Hog1 mitogen‐activated protein kinase. Moreover, two pathways known to control morphological developments are involved in regulation of AQY2 expression: the protein kinase A pathway derepresses AQY2 expression through the Sfl1 repressor, and the filamentous growth Kss1 mitogen‐activated protein kinase pathway represses AQY2 expression in a Kss1 activity‐independent manner. The AQY2 expression pattern resembles in many ways that of MUC1/FLO11, which encodes a cell surface glycoprotein required for morphological developments. Our observations suggest a potential link between aquaporins and cell surface properties, and relate to the proposed role of mammalian aquaporins in tumour cell migration and invasion.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

The DUF1996 and WSC domain‐containing protein Wsc1I acts as a novel sensor of multiple stress cues in Beauveria bassiana

Wsc1I homologues featuring both an N‐terminal DUF1996 (domain of unknown function 1996) and a C‐terminal WSC (cell wall stress‐responsive component) domain exist in filamentous fungi but have never been functionally characterized. Here, Wsc1I is shown to localize in the vacuoles and cell wall/membrane of the insect mycopathogen Beauveria bassiana and hence linked to cell membrane‐ and vacuole‐related cellular events. In B. bassiana, deletion of Wsc1I resulted in marked increases of hyphal and conidial sensitivities to hyperosmotic agents, oxidants, cell wall perturbing chemicals, and metal cations (Cu²⁺, Zn²⁺, Fe²⁺, and Mg²⁺) despite slight impact on normal growth and conidiation. Conidia produced by the deletion mutant showed not only reduced tolerance to both 45°C heat and UVB irradiation but also attenuated virulence to a susceptible insect through normal cuticle infection or cuticle‐bypassing infection. Importantly, phosphorylation of the mitogen‐activated protein kinase Hog1 was largely attenuated or nearly abolished in the Wsc1I‐free cells triggered with hyperosmotic, oxidative, or cell wall perturbing stress. All changes were well restored by targeted gene complementation. Our findings highlight a novel role of Wsc1I in sensing multiple stress cues upstream of the Hog1 signalling pathway and its pleiotropic effects in B. bassiana.     

A Candida albicans cell wall‐linked protein promotes invasive filamentation into semi‐solid medium

Growth of cells in contact with an abiotic or biological surface profoundly affects cellular physiology. In the opportunistic human pathogen, Candida albicans, growth on a semi‐solid matrix such as agar results in invasive filamentation, a process in which cells change their morphology to highly elongated filamentous hyphae that grow into the matrix. We hypothesized that a plasma membrane receptor‐type protein would sense the presence of matrix and activate a signal transduction cascade, thus promoting invasive filamentation. In this communication, we demonstrate that during growth in contact with a semi‐solid surface, activation of a MAP kinase, Cek1p, is promoted, in part, by a plasma membrane protein termed Dfi1p and results in invasive filamentation. A C. albicans mutant lacking Dfi1p showed reduced virulence in a murine model of disseminated candidiasis. Dfi1p is a relatively small, integral membrane protein that localizes to the plasma membrane. Some Dfi1p molecules become cross‐linked to the carbohydrate polymers of the cell wall. Thus, Dfi1p is capable of linking the cell wall to the plasma membrane and cytoplasm.     

Protein kinase C prevents oligodendrocyte differentiation: modulation of actin cytoskeleton and cognate polarized membrane traffic

In a previous study, we showed that activation of protein kinase C (PKC) prevents oligodendrocyte differentiation at the pro‐oligodendrocyte stage. The present study was undertaken to identify downstream targets of PKC action in oligodendrocyte progenitor cells. Activation of PKC induced the predominant phosphorylation of an 80‐kD protein, identified as myristoylated alanine‐rich C‐kinase substrate (MARCKS). Upon phosphorylation, MARCKS is translocated from the plasma membrane to the cytosol. Furthermore, PKC activation perturbed the organization of the actin cytoskeleton, causing a redistribution of actin filaments to the submembranous or cortical actin cytoskeleton. As a consequence, transport of a protein traffic marker, the vesicular stomatitis virus glycoprotein, from the trans‐Golgi network to the plasma membrane becomes perturbed. The effect of disruption of the actin filament network by cytochalasin D perfectly matched the effect of PKC. These data thus favor the existence of a causal relationship between actin rearrangement and docking and/or fusion of proteins to the plasma membrane. Interestingly, neither in control cells nor in PKC‐activated cells did another protein traffic marker, influenza hemagglutinin (HA), reach the cell surface. However, an eminent and specific accumulation of HA just underneath the plasma membrane became apparent upon PKC activation. Yet, this effect could not be simulated by cytochalasin D treatment. Therefore, these observations imply that although MARCKS represents a prominent PKC target site in regulating differentiation, another target involves the differential control of cognate polarized trafficking pathways, which are apparently operating in oligodendrocyte progenitor cells. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 385–398, 1999     

The Candida albicans ELMO homologue functions together with Rac1 and Dck1, upstream of the MAP Kinase Cek1, in invasive filamentous growth

Regulation of Rho G‐proteins is critical for cytoskeletal organization and cell morphology in all eukaryotes. In the human opportunistic pathogen Candida albicans, Rac1 and its activator Dck1, a member of the CED5, Dock180, myoblast city family of guanine nucleotide exchange factors, are required for the budding to filamentous transition during invasive growth. We show that Lmo1, a protein with similarity to human ELMO1, is necessary for invasive filamentous growth, similar to Rac1 and Dck1. Furthermore, Rac1, Dck1 and Lmo1 are required for cell wall integrity, as the deletion mutants are sensitive to cell wall perturbing agents, but not to oxidative or osmotic stresses. The region of Lmo1 encompassing the ELMO and PH‐like domains is sufficient for its function. Both Rac1 and Dck1 can bind Lmo1. Overexpression of a number of protein kinases in the rac1, dck1 and lmo1 deletion mutants indicates that Rac1, Dck1 and Lmo1 function upstream of the mitogen‐activated protein kinases Cek1 and Mkc1, linking invasive filamentous growth to cell wall integrity. We conclude that the requirement of ELMO/CED12 family members for Rac1 function is conserved from fungi to humans.