Carlos R. Vázquez de Aldana

Consejo Superior de Investigaciones Científicas

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Francisco del Rey

Universidad Complutense de Madrid

Jaime Correa‐Bordes

Universidad de Extremadura

Yolanda Arnáiz‐Pita

Universidad de Salamanca

Encarnación Dueñas‐Santero

Consejo Superior de Investigaciones Científicas

Alberto González‐Novo

Pompeu Fabra University

Sara Orellana‐Muñoz

University of Oslo

M. Belén Suárez

Instituto de Biología Funcional y Genómica

Alan G. Hinnebusch

Eunice Kennedy Shriver National Institute of Child Health and Human Development

Pilar Gutiérrez-Escribano

MRC London Institute of Medical Sciences

Encarnación Dueñas

Consejo Superior de Investigaciones Científicas

Cooperative Institutions

Universidad de Salamanca

Consejo Superior de Investigaciones Científicas

Wellcome Sanger Institute

Institut Pasteur

Instituto de Biología Funcional y Genómica

Centre National de la Recherche Scientifique

Universidad de Extremadura

National Institutes of Health

Universidad Complutense de Madrid

J. Craig Venter Institute

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The endo-β-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation inSchizosaccharomyces pombe

Journal of Cell Science (2003)

Ana Belén Martín‐Cuadrado Encarnación Dueñas Matthias Sipiczki Carlos R. Vázquez de Aldana Francisco del Rey

Schizosaccharomyces pombe cells divide by medial fission throughout contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomoysin ring and septum assembly,little information is available concerning the mechanism of cell separation. Here we describe the characterization of eng1+, a new gene that encodes a protein with detectable endo-β-1,3-glucanase activity and whose deletion is not lethal to the cells but does interfere in their separation. Electron microscopic observation of mutant cells indicated that this defect is mainly due to the failure of the cells to degrade the primary septum, a structure rich in β-1,3-glucans, that separates the two sisters cells. Expression of eng1+ varies during the cell cycle,maximum expression being observed before septation, and the protein localizes to a ring-like structure that surrounds the septum region during cell separation. This suggests that it could also be involved in the cleavage of the cylinder of the cell wall that covers the division septum. The expression of eng1+ during vegetative growth is regulated by a C2H2 zinc-finger protein (encoded by the SPAC6G10.12c ORF), which shows significant sequence similarity to the Saccharomyces cerevisiae ScAce2p,especially in the zinc-finger region. Mutants lacking this transcriptional regulator (which we have named ace2+) show a severe cell separation defect, hyphal growth being observed. Thus, ace2p may regulate the expression of the eng1+ gene together with that of other genes whose products are also involved in cell separation.

Schizosaccharomyces

Cleavage (geology)

Interphase

10.1242/jcs.00377

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Citations (171)

Generation of null alleles for the functional analysis of six genes from the right arm ofSaccharomyces cerevisiae chromosome II

Yeast (1999)

Encarnación Dueñas Carlos R. Vázquez de Aldana T. Cos Carmen Castro M. Henar Valdieso

Using PCR-ligated long flanking homology cassettes, null alleles of six open reading frames (ORFs) from chromosome II have been created in Saccharomyces cerevisiae. Deletants were constructed in three genetic backgrounds: FY1679, W303 and CEN.PK2. Tetrad analysis of heterozygous deletants revealed that none of the ORFs is essential for vegetative growth. Basic phenotypic analysis of haploid deletants showed that deletion of the YBR283c ORF causes a slight growth defect at 30 degrees C and 37 degrees C on glycerol-complete, glucose-complete, and glucose-minimal media only in the FY1679 and W303 backgrounds. Transformation of these deletants with the corresponding cognate gene in a centromeric plasmid complements the defects. Deletion of the YBR287w ORF leads to poor growth on glucose-minimal medium at 15 degrees C in the FY 1679 background. None of the six ORFs seems to be involved in mating or sporulation.

ORFS

Null allele

Homology

Genetic Analysis

10.1002/(sici)1097-0061(199905)15:7<615::aid-yea385>3.0.co;2-x

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Eng1p, an Endo-1,3-β-Glucanase Localized at the Daughter Side of the Septum, Is Involved in Cell Separation in Saccharomyces cerevisiae

Eukaryotic Cell (2002)

Victoriano Baladrón Sandra Ufano Encarnación Dueñas Ana Belén Martín‐Cuadrado Francisco del Rey

ABSTRACT ENG1 ( YNR067c ), a gene encoding a new endo-1,3-β-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-β-glucanases (Exg1p, Exg2p, and Ssg1p). Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2 gene, involved in actin assembly “in vitro,” and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-β linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds. ENG1 transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G 1 transition and is dependent on the transcription factor Ace2p. Interestingly, eng1 deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum.

10.1128/ec.1.5.774-786.2002

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Citations (153)

Dinámica de septinas y regulación de la separación celular durante el crecimiento hifal de Candida albicans ¿una diana de antifúngicos?

Carlos R. Vázquez de Aldana Diana Calderón-Noreña Alberto González‐Novo Pilar Gutiérrez-Escribano M. Belén Suárez

Resumen del trabajo presentado al IV Congreso GEBMP: Simposio Hongos Patogenos celebrado en Badajoz el 6 de julio del 2012.

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The Schizosaccharomyces pombe endo‐1,3‐β‐glucanase Eng1 contains a novel carbohydrate binding module required for septum localization

Molecular Microbiology (2008)

Ana Belén Martín‐Cuadrado Javier Encinar del Dedo María de Medina-Redondo Thierry Fontaine Francisco del Rey

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro, the purified CBM has affinity for beta-1,3-glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1Delta mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, as the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans-factors (septins and the exocyst complex) but also a cis-element localized to the C-terminus of the protein.

Septin

Glucanase

Exocyst

Schizosaccharomyces

10.1111/j.1365-2958.2008.06275.x

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Citations (36)

GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3.

Genes & Development (1995)

Minerva T. Garcia-Barrio Tatjana Naranda Carlos R. Vázquez de Aldana Rafael Cuesta Alan G. Hinnebusch

GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(iMet) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIF-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.

Initiation factor

Eukaryotic translation

Eukaryotic initiation factor

Translational regulation

EIF4A1

Eukaryotic Ribosome

eIF2

10.1101/gad.9.14.1781

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Citations (73)

SSG1, a gene encoding a sporulation-specific 1,3-beta-glucanase in Saccharomyces cerevisiae

Journal of Bacteriology (1993)

Pedro A. San-Segundo Jaime Correa‐Bordes Carlos R. Vázquez de Aldana Francisco del Rey

In Saccharomyces cerevisiae, the meiotic process is accompanied by a large increase in 1,3-beta-glucan-degradative activity. The molecular cloning of the gene (SSG1) encoding a sporulation-specific exo-1,3-beta-glucanase was achieved by screening a genomic library with a DNA probe obtained by polymerase chain reaction amplification using synthetic oligonucleotides designed according to the nucleotide sequence predicted from the amino-terminal region of the purified protein. DNA sequencing indicates that the SSG1 gene specifies a 445-amino-acid polypeptide (calculated molecular mass, 51.8 kDa) showing extensive similarity to the extracellular exo-1,3-beta-glucanases encoded by the EXG1 gene (C. R. Vazquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebreda, E. Mendez, and F. del Rey, Gene 97:173-182, 1991). The N-terminal domain of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins. Northern (RNA) analysis reveals a unique SSG1-specific transcript, 1.7 kb long, which can be detected only in sporulating diploids (MATa/MAT alpha) but does not appear in vegetatively growing cells or in nonsporulating diploids (MAT alpha/MAT alpha) when incubated under nitrogen starvation conditions. The meiotic time course of SSG1 induction indicates that the gene is transcribed only in the late stages of the process, beginning at the time of meiosis I and reaching a maximum during spore formation. Homozygous ssg1/ssg1 mutant diploids are able to complete sporulation, although with a significant delay in the appearance of mature asci.

Glucanase

genomic DNA

10.1128/jb.175.12.3823-3837.1993

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Citations (43)

CDK-dependent phosphorylation of Mob2 is essential for hyphal development inCandida albicans

Molecular Biology of the Cell (2011)

Pilar Gutiérrez-Escribano Alberto González‐Novo M. Belén Suárez Chang-Run Li Yue Wang

Nuclear Dbf2-related (NDR) protein kinases are essential components of regulatory pathways involved in cell morphogenesis, cell cycle control, and viability in eukaryotic cells. For their activity and function, these kinases require interaction with Mob proteins. However, little is known about how the Mob proteins are regulated. In Candida albicans, the cyclin-dependent kinase (CDK) Cdc28 and the NDR kinase Cbk1 are required for hyphal growth. Here we demonstrate that Mob2, the Cbk1 activator, undergoes a Cdc28-dependent differential phosphorylation on hyphal induction. Mutations in the four CDK consensus sites in Mob2 to Ala significantly impaired hyphal development. The mutant cells produced short hyphae with enlarged tips that displayed an illicit activation of cell separation. We also show that Cdc28 phosphorylation of Mob2 is essential for the maintenance of polarisome components at hyphal tips but not at bud tips during yeast growth. Thus we have found a novel signaling pathway by which Cdc28 controls Cbk1 through the regulatory phosphorylation of Mob2, which is crucial for normal hyphal development.

10.1091/mbc.e11-03-0205

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Citations (46)

The Int1 anillin and the Sep7 septin collaborate to maintain cellular ploidy in Candida albicans

Sara Orellana‐Muñoz Encarnación Dueñas‐Santero Yolanda Arnáiz‐Pita Francisco del Rey Iglesias Jaime Correa‐Bordes

Trabajo presentado en la 11o Reunion de la Red Espanola de Levaduras, celebrada en El Escorial (Espana), del 13 al 15 de diciembre de 2017

Septin

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Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation.

Proceedings of the National Academy of Sciences (1993)

Carlos R. Vázquez de Aldana Thomas Dever Alan G. Hinnebusch

Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA. We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein. All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation. We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation. These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B.

Initiation factor

eIF2

Eukaryotic translation

Eukaryotic initiation factor

EIF4E

Alpha (finance)

10.1073/pnas.90.15.7215

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Citations (56)