Francisco del Rey

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

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.

Characterization of the CaENG1 Gene Encoding an Endo-1,3-β-Glucanase Involved in Cell Separation in Candida albicans

The Candida albicans CaENG1 gene encoding an endo-1,3-β-glucanase was cloned by screening a genomic library with a DNA probe obtained by polymerase chain reaction using synthetic oligonucleotides designed according to conserved regions found between two Saccharomyces cerevisiae endo-1,3-β-glucanases (Eng1p and Eng2p). The gene contains a 3435-bp open reading frame (ORF), capable of encoding a protein of 1145 amino acids (124,157 Da), that contains no introns. Comparison of the ScEng1p sequence with partial C. albicans genomic sequences revealed the presence of a second protein with sequence similarity (the product of the Ca20C1.22c ORF, which was named CaENG2). Disruption of the CaENG1 gene in C. albicans had no dramatic effects on the growth rate of the strains, but it resulted in the formation of chains of cells, suggesting that the protein is involved in cell separation. Expression of CaENG1 in S. cerevisiae cells afforded a 12-fold increase in the 1,3-β-glucanase activity detected in culture supernatants, showing that the protein has similar enzymatic activity to that of the S. cerevisiae Eng1p. In addition, when the C. albicans protein was expressed under its native promoter in S. cerevisiaeeng1 mutant cells, it was able to complement the separation defect of this mutant, indicating that these two proteins are true functional homologues.

Purification and partial characterization of a new, sporulation specific, exo-β-glucanase from Saccharomyces cerevisiae

Abstract A new exo-β-glucanase, sporulation-specific, was purified from sporulating S. cerevisiae ( AP 1 , a α ). Characterization of this new activity shows that the enzyme is a glycoprotein with substrate specificities, kinetic parameters and aminoacid composition clearly different from those of its vegetative counterpart.

Cloning and characterization of the EXG1 gene from the yeast Yarrowia lipolytica

The YlEXG1 gene of Yarrowia lipolytica, encoding an exo‐1,3‐β‐glucanase, was isolated by screening a genomic library with a DNA probe obtained by PCR amplification, using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. YlEXG1 consists of a 1263 bp open reading frame encoding a protein of 421 amino acids with a calculated molecular weight of 48 209 Da. Northern blot analysis revealed a unique YlEXG1‐specific transcript, 1·4 kb long. A putative pre(signal)‐peptide of 15 amino acids is proposed at the N‐terminal domain of the primary translation product. The deduced amino acid sequence shares a high degree of homology with exo‐1,3‐β‐glucanases from other yeast species, including S. cerevisiae, Kluyveromyces lactis, Pichia angusta and Debaryomyces occidentalis. YlExg1p contains the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases. Chromoblot analysis indicated that YlEXG1 is located on chromosome VI. Disruption of YlEXG1 did not result in a phenotype under laboratory conditions and did not prevent the yeast–hypha transition. The sequence data reported in this paper have been assigned EMBL Accession No. Z46872. Copyright

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-ΔNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.

A Single Nucleotide Polymorphism Uncovers a Novel Function for the Transcription Factor Ace2 during Candida albicans Hyphal Development

Candida albicans is a major invasive fungal pathogen in humans. An important virulence factor is its ability to switch between the yeast and hyphal forms, and these filamentous forms are important in tissue penetration and invasion. A common feature for filamentous growth is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is regulated developmentally. In C. albicans, the formation of filaments during hyphal growth requires changes in septin ring dynamics. In this work, we studied the functional relationship between septins and the transcription factor Ace2, which controls the expression of enzymes that catalyze septum degradation. We found that alternative translation initiation produces two Ace2 isoforms. While full-length Ace2, Ace2L, influences septin dynamics in a transcription-independent manner in hyphal cells but not in yeast cells, the use of methionine-55 as the initiation codon gives rise to Ace2S, which functions as the nuclear transcription factor required for the expression of cell separation genes. Genetic evidence indicates that Ace2L influences the incorporation of the Sep7 septin to hyphal septin rings in order to avoid inappropriate activation of cell separation during filamentous growth. Interestingly, a natural single nucleotide polymorphism (SNP) present in the C. albicans WO-1 background and other C. albicans commensal and clinical isolates generates a stop codon in the ninth codon of Ace2L that mimics the phenotype of cells lacking Ace2L. Finally, we report that Ace2L and Ace2S interact with the NDR kinase Cbk1 and that impairing activity of this kinase results in a defect in septin dynamics similar to that of hyphal cells lacking Ace2L. Together, our findings identify Ace2L and the NDR kinase Cbk1 as new elements of the signaling system that modify septin ring dynamics in hyphae to allow cell-chain formation, a feature that appears to have evolved in specific C. albicans lineages.

The sequence of a 20·3 kb DNA fragment from the left arm of Saccharomyces cerevisiae chromosome IV contains the KIN28, MSS2, PHO2, POL3 and DUN1 genes, and six new open reading frames

We report the sequence of a 20 300 bp DNA fragment from the left arm of Saccharomyces cerevisiae chromosome IV. This segment contains 13 complete open reading frames (ORFs) and part of another ORF, altogether covering 84·2% of the entire sequence, five of which correspond to the previously characterized KIN28, MSS2, PHO2, POL3/CDC2 and DUN1 genes. One putative protein, D2358p, shares considerable homology with an O‐sialoglycoprotein endopeptidase from Pasteurella haemolytica serotype A1. The putative product of D2325 contains the characteristic consensus motif of triacylglycerol lipases. D2320p and D2352p have a putative ‘leucine‐zipper’ structure and a RNA‐binding region Rnp‐1 signature, respectively. The sequence data have been submitted to EMBL data library under Accession Number X95644.

Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe.

The division cycle of unicellular yeasts is completed with the activation of a cell separation program that results in the dissolution of the septum assembled during cytokinesis between the 2 daughter cells, allowing them to become independent entities. Expression of the eng1+ and agn1+ genes, encoding the hydrolytic enzymes responsible for septum degradation, is activated at the end of each cell cycle by the transcription factor Ace2. Periodic ace2+ expression is regulated by the transcriptional complex PBF (PCB Binding Factor), composed of the forkhead-like proteins Sep1 and Fkh2 and the MADS box-like protein Mbx1. In this report, we show that Ace2-dependent genes contain several combinations of motifs for Ace2 and PBF binding in their promoters. Thus, Ace2, Fkh2 and Sep1 were found to bind in vivo to the eng1+ promoter. Ace2 binding was coincident with maximum level of eng1+ expression, whereas Fkh2 binding was maximal when mRNA levels were low, supporting the notion that they play opposing roles. In addition, we found that the expression of eng1+ and agn1+ was differentially affected by mutations in PBF components. Interestingly, agn1+ was a major target of Mbx1, since its ectopic expression resulted in the suppression of Mbx1 deletion phenotypes. Our results reveal a complex regulation system through which the transcription factors Ace2, Fkh2, Sep1 and Mbx1 in combination control the expression of the genes involved in separation at the end of the cell division cycle.

Eng2 Is a Component of a Dynamic Protein Complex Required for Endocytic Uptake in Fission Yeast

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2‐GFP dynamics did not match the pattern of other endocytic proteins. Eng2‐GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3‐YFP, a WASP‐interacting protein, interacted with Eng2 by co‐immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.

The sequence of a 21.3 kb DNA fragment from the left arm of yeast chromosome XIV reveals LEU4, MET4, POL1, RAS2, and six new open reading frames.

The nucleotide sequence of a fragment of 21 308 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been determined. Analysis of the sequence revealed 13 open reading frames (ORFs) longer than 300 bp, four of which correspond to the previously identified genes LEU4, MET4, POL1 and RAS2. One putative protein, N2160, shares considerable homology (32% identity) with a hypothetical protein encoded by a gene located on chromosome XV as well as with human OCRL protein (36% identity), involved in Lowes syndrome. N2185 contains ten predicted transmembrane segments and is similar to another putative protein (YKL146) from yeast. The sequence of the reported DNA fragment has been submitted to the EMBL data library under Accession Number Z50161.