β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

Medina-Redondo, María de; Arnáiz‐Pita, Yolanda; Clavaud, Cécile; Fontaine, Thierry; Rey, Francisco del; Latgé, Jean Paul; Aldana, Carlos R. Vázquez de

doi:10.1371/journal.pone.0014046

Cited by 35 publications

(36 citation statements)

References 51 publications

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“…Similarly to what has been observed in yeast, 29 , 30 , 32 , 47 A. gossypii Aggas1B Δ mutants exhibited severe growth retardation and abnormal cells with granular cytoplasm and big vacuoles. Moreover, the Aggas1A Δ gas1B Δ double mutant presented sporulation deficiency and a high percentage of cellular lysis even when osmotic support was present.…”

Section: Discussionsupporting

confidence: 76%

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Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

et al. 2013

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To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.

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Section: Discussionsupporting

confidence: 76%

“…In A. fumigatus , abnormal conidiogenesis and slower growth were observed when GEL2 , a ortholog of the GAS gene family, was deleted 46 . These peculiar morphological alterations have been associated with altered structure and composition of the mutants cell wall 32 , 46 , 47 …”

Section: Discussionmentioning

confidence: 99%

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

et al. 2013

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“…Death of rho1-596 is quite similar to that caused by decreasing the amount of Rho1 in a "switch-off" strain (Arellano et al 1997) or by reducing Rho1 activity through overexpression of a rho1T20N dominant negative allele (Arellano et al 1997). These strains do not die by rapidly losing the intracellular content, as occurs to mutants with defects in cell wall components (Cortes et al 2005(Cortes et al , 2012 or cell wall organization (De Medina-Redondo et al 2010). While overexpression of rho1 + completely rescued rho1-596 growth at 36°, overexpression of other Rho GTPases coding genes like rho2 + or rho3 + could not rescue its thermosensitive growth and was deleterious, suggesting that both GTPases play different cellular roles than Rho1.…”

Section: Discussionmentioning

confidence: 98%

Negative Functional Interaction Between Cell Integrity MAPK Pathway and Rho1 GTPase in Fission Yeast

et al. 2013

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Rho1 GTPase is the main activator of cell wall glucan biosynthesis and regulates actin cytoskeleton in fungi, including Schizosaccharomyces pombe. We have obtained a fission yeast thermosensitive mutant strain carrying the rho1-596 allele, which displays reduced Rho1 GTPase activity. This strain has severe cell wall defects and a thermosensitive growth, which is partially suppressed by osmotic stabilization. In a global screening for rho1-596 multicopy suppresors the pmp1 + gene was identified. Pmp1 is a dual specificity phosphatase that negatively regulates the Pmk1 mitogen-activated protein kinase (MAPK) cell integrity pathway. Accordingly, elimination of Pmk1 MAPK partially rescued rho1-596 thermosensitivity, corroborating the unexpected antagonistic functional relationship of these genes. We found that rho1-596 cells displayed increased basal activation of the cell integrity MAPK pathway and therefore were hypersensitive to MgCl 2 and FK506. Moreover, the absence of calcineurin was lethal for rho1-596. We found a higher level of calcineurin activity in rho1-596 than in wild-type cells, and overexpression of constitutively active calcineurin partially rescued rho1-596 thermosensitivity. All together our results suggest that loss of Rho1 function causes an increase in the cell integrity MAPK activity, which is detrimental to the cells and turns calcineurin activity essential. RHO GTPases are conserved proteins that regulate the actin cytoskeleton organization in all eukaryots (Heasman and Ridley 2008). Additionally, in yeast and other fungi, these GTPases provide the coordinated regulation of cell wall biosynthesis and actin organization, which is required to maintain cell integrity and polarized growth (Levin 2005;Park and Bi 2007;Perez and Cansado 2010). The Schizosaccharomyces pombe genome contains six genes coding Rho GTPases. Among them, rho1 + is essential (Arellano et al. 1996). Rho1 function is mediated by its interaction with at least three different targets: the b(1,3)-glucan synthase (Arellano et al. 1996), which is responsible for the synthesis of the major cell wall component, and the kinases Pck1 and Pck2 Sayers et al. 2000). Through both kinases, Rho1 also regulates the cell wall synthesis. Rho2 also interacts with Pck2 and, therefore, both GTPases regulate the a-D-glucan synthesis through Pck2 (Katayama et al. 1999;Calonge et al. 2000). Lack of Rho1 is lethal, and this phenotype is not suppressed by osmotic stabilization (Arellano et al. 1997), suggesting that defective biosynthesis of the cell wall is not the unique cause of death. On the contrary, Rho2-less cells are viable, although they become slightly rounded and more sensitive to treatment with glucanases (Hirata et al. 1998). Rho2 and Pck2 participate in the activation of the cell integrity mitogen-activated protein kinase (MAPK) signaling pathway (Ma et al. 2006). This signaling cascade responds to different extracellular stress stimuli such as hyper-or hypotonic conditions, oxidative stress, cell wall damaging compounds, and g...

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“…Loss of psu1 + activity conferred resistance to (1,3)-β-glucanase suggesting that Psu1 may influence the amount or structure of (1,3)-β-glucan in the cell wall [55]. In addition, the (1,3)-β-glucanosyltransferase Gas2 has been shown to lengthen glucan chains during cell wall assembly and its overproduction is able to suppress the cell wall defect and lethality of gas1 Δ cells [56]. Then how does the overexpression of these cell wall-remodeling genes trigger flocculation in S. pombe cells?…”

Section: Discussionmentioning

confidence: 99%

Deciphering the Transcriptional-Regulatory Network of Flocculation in Schizosaccharomyces pombe

et al. 2012

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In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP–chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2+ and deletion of rfl1+ resulted in strong flocculation and transcriptional upregulation of gsf2 +/pfl1+ and several other putative flocculin genes (pfl2+–pfl9+). Overexpression of the pfl+ genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl+ overexpression. Among the pfl1+ genes, only loss of gsf2+ abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2+ or adn3+ overexpression was likely mediated by the transcriptional activation of cell wall–remodeling genes including gas2+, psu1+, and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2+ expression in an inhibitory feed-forward loop involving mbx2+. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall–remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.

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β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

Cited by 35 publications

References 51 publications

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

Negative Functional Interaction Between Cell Integrity MAPK Pathway and Rho1 GTPase in Fission Yeast

Deciphering the Transcriptional-Regulatory Network of Flocculation in Schizosaccharomyces pombe

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