Transcription Factor Ace2p Research Articles

Conserved regulators of the cell separation process in Schizosaccharomyces

The fission yeasts (Schizosaccharomyces) representing a highly divergent phylogenetic branch of Fungi evolved from filamentous ancestors by gradual transition from mycelial growth to yeast morphology. For the transition, a mechanism had been developed that separates the sister cells after the completion of cytokinesis. Numerous components of the separation mechanism have been characterised in Schizosaccharomycespombe, including the zinc-finger transcription factor Ace2p and the fork-head transcription factor Sep1p. Here we show that both regulators have regions conserved within the genus. The most conserved parts contain the DNA-binding domains whose amino-acid sequences perfectly reflect the phylogenetic positions of the species. The less conserved parts of the proteins contain sequence blocks specific for the whole genus or only for the species propagating predominantly or exclusively as yeasts. Inactivation of either gene in the dimorphic species Schizosaccharomycesjaponicus abolished cell separation in the yeast phase conferring hypha-like morphology but did not change the growth pattern to unipolar and did not cause extensive polar vacuolation characteristic of the true mycelium. Neither mutation affected the mycelial phase, but both mutations hampered the hyphal fragmentation at the mycelium-to-yeast transition. Ace2pSj acts downstream of Sep1pSj and regulates the orthologues of the Ace2p-dependent S.pombe genes agn1+ (1,3-alpha-glucanase) and eng1+ (1,3-beta-glucanase) but does not regulate the orthologue of cfh4+ (chitin synthase regulatory factor). These results and the complementation of the cell separation defects of the ace2− and sep1− mutations of S.pombe by heterologously expressed ace2Sj and sep1Sj indicate that the cell separation mechanism is conserved in the Schizosaccharomyces genus.

Mutations in the Saccharomyces cerevisiae kinase Cbk1p lead to a fertility defect that can be suppressed by the absence of Brr1p or Mpt5p (Puf5p), proteins involved in RNA metabolism.

In Saccharomyces cerevisiae the protein kinase Cbk1p is a member of the regulation of Ace2p and cellular morphogenesis (RAM) network that is involved in cell separation after cytokinesis, cell integrity, and cell polarity. In cell separation, the RAM network promotes the daughter cell-specific localization of the transcription factor Ace2p, resulting in the asymmetric transcription of genes whose products are necessary to digest the septum joining the mother and the daughter cell. RAM and SSD1 play a role in the maintenance of cell integrity. In the presence of a wild-type SSD1 gene, deletion of any RAM component causes cell lysis. We show here that some mutations of CBK1 also lead to a reduced fertility and a reduced expression of some of the mating type-specific genes. As polarized growth is an integral part of the mating process, we have isolated suppressors of the fertility defect. Among these, mutations in BRR1 or MPT5 lead to a restoration of fertility and a more-or-less pronounced restoration of polarity; they also show genetic interactions with SSD1. Our experiments reveal a multilayered system controlling aspects of cell separation, cell integrity, mating, and polarized growth.

Mutations in a small region of the exportin Crm1p disrupt the daughter cell‐specific nuclear localization of the transcription factor Ace2p in Saccharomyces cerevisiae

The CBK1 gene of Saccharomyces cerevisiae encodes a protein kinase that is a member of the NDR (nuclear Dbf2-related) family of protein kinases, which are involved in morphogenesis and cell proliferation. Previous studies have shown that deletion of CBK1 leads to a loss of polarity and the formation of large aggregates of cells. This aggregation phenotype is due to the loss of the daughter cell-specific accumulation of the transcription factor Ace2p, which is responsible for the transcription of genes whose products are necessary for the final separation of the mother and the daughter at the end of cell division. We show that the daughter cell-specific localization of Ace2p does not occur via a specific localization of the ACE2 mRNA and that, in vivo, the transcription of CTS1, one of the principal targets of Ace2p, is daughter cell-specific. We have shown that extragenic suppressors of the Deltacbk1 aggregation phenotype are located in the nuclear exportin CRM1 and ACE2. These mutations disrupt the interaction of Ace2p and Crm1p, thus impairing Ace2p export and resulting in the accumulation of the protein in both mother and daughter cell nuclei. We propose that in the daughter cell nucleus Cbk1p phosphorylates the Ace2p nuclear export signal, and that this phosphorylation blocks the export of Ace2p via Crm1p, thus promoting the daughter cell-specific nuclear accumulation of Ace2p.

Transcription regulation of the α-glucanase gene agn1 by cell separation transcription factor Ace2p in fission yeast

During the final stage of the cell division cycle in the fission yeast Schizosaccharomyces pombe, transcription factor Ace2p activates expression of genes involved in the separation of newly formed daughter cells, such as agn1 +, which encodes the α-glucanase Agn1p. The agn1 promoter contains three copies of the nucleotide sequence motif CCAGCC, whose presence seems to correlate with Ace2p-mediated transcription activation. Here, we describe a simple plate-based assay utilizing as a reporter the secreted glucoamylase of Arxula adeninivorans to investigate the function of this motif. We show that not all three repeats, but only the two most proximal to the transcription start point, act as an upstream activating sequence (UAS). Finally, we demonstrate that this UAS is essential for agn1 promoter activity in vivo.

A Role for the Septation Initiation Network in Septum Assembly Revealed by Genetic Analysis of sid2-250 Suppressors

In the fission yeast Schizosaccharomyces pombe the septation initiation network (SIN) is required for stabilization of the actomyosin ring in late mitosis as well as for ring constriction and septum deposition. In a genetic screen for suppressors of the SIN mutant sid2-250, we isolated a mutation, ace2-35, in the transcription factor Ace2p. Both ace2Delta and ace2-35 show defects in cell separation, and both can rescue the growth defects of some SIN mutants at low restrictive temperatures, where the SIN single mutants lyse at the time of cytokinesis. By detailed analysis of the formation and constriction of the actomyosin ring and septum in the sid2-250 mutant at low restrictive temperatures, we show that the lysis phenotype of the sid2-250 mutant is likely due to a weak cell wall and septum combined with enzymatic activity of septum-degrading enzymes. Consistent with the recent findings that Ace2p controls transcription of genes involved in cell separation, we show that disruption of some of these genes can also rescue sid2-250 mutants. Consistent with SIN mutants having defects in septum formation, many SIN mutants can be rescued at the low restrictive temperature by the osmotic stabilizer sorbitol. The small GTPase Rho1 is known to promote cell wall formation, and we find that Rho1p expressed from a multi-copy plasmid can also rescue sid2-250 at the low restrictive temperature. Together these results suggest that the SIN has a role in promoting proper cell wall formation at the division septa.

Ace2p Controls the Expression of Genes Required for Cell Separation inSchizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Here we describe the identification of seven genes (adg1(+), adg2(+), adg3(+), cfh4(+), agn1(+), eng1(+), and mid2(+)) whose expression is induced by the transcription factor Ace2p. The expression of all of these genes varied during the cell cycle, maximum transcription being observed during septation. At least three of these proteins (Eng1p, Agn1p, and Cfh4p) localize to a ring-like structure that surrounds the septum region during cell separation. Deletion of the previously uncharacterized genes was not lethal to the cells, but produced defects or delays in cell separation to different extents. Electron microscopic observation of mutant cells indicated that the most severe defect is found in eng1Delta agn1Delta cells, lacking the Eng1p endo-beta-1,3-glucanase and the Agn1p endo-alpha-glucanase. The phenotype of this mutant closely resembled that of ace2Delta mutants, forming branched chains of cells. This suggests that these two proteins are the main activities required for cell separation to be completed.

Swm1p subunit of the APC/cyclosome is required for activation of the daughter-specific gene expression program mediated by Ace2p during growth at high temperature in Saccharomyces cerevisiae.

SWM1 was originally identified for its role in the late steps of the sporulation process, being required for spore wall assembly. This protein, recently identified as one of the core subunits of the anaphase-promoting complex (APC) is also required to complete cell separation in vegetative cells during growth at high temperature. Mutants lacking SWM1 show a thermosensitive growth defect that is suppressed by osmotic support in the culture medium. At the restrictive temperature, swm1 mutants are unable to complete separation, forming chains of cells that remain associated and, with prolonged incubation times, the stability of the cell wall is compromised, resulting in cell lysis. This separation defect is due to a reduction in expression of CTS1 (the gene encoding chitinase) and a group of genes involved in cell separation (such as ENG1,SCW11, DSE1 and DSE2). Interestingly, these genes are specifically regulated by the transcription factor Ace2p, suggesting that Swm1p is required for normal expression of Ace2p-dependent genes during growth at high temperatures. Although no defect in Ace2p localization can be observed at 28 degrees C, this transcription factor is unable to enter the nucleus of the daughter cell during growth at 38 degrees C. Under these growth conditions, swm1 cells undergo a delay in exit from mitosis, as determined by analysis of Clb2p degradation and Cdc28p-Clb2p kinase assays, and this could be the reason for the cytoplasmic localization of Ace2p.

Eng1p, an endo-1,3-beta-glucanase localized at the daughter side of the septum, is involved in cell separation in Saccharomyces cerevisiae.

ENG1 (YNR067c), a gene encoding a new endo-1,3-beta-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-beta-glucanases (Exglp, Exg2p, and Ssglp). 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-beta 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/G1 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.

Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

Cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae.

We have studied the CBK1 gene of Saccharomyces cerevisiae, which encodes a conserved protein kinase similar to the human myotonic dystrophy kinase. We have shown that the subcellular localization of the protein, Cbk1p, varies in a cell cycle-dependent manner. Three phenotypes are associated with the inactivation of the CBK1 gene: large aggregates of cells, round rather than ellipsoidal cells and a change from a bipolar to a random budding pattern. Two-hybrid and extragenic suppressor studies have linked Cbk1p with the transcription factor Ace2p, which is responsible for the transcription of chitinase. Cbk1p is necessary for the activation of Ace2p and we have shown that the aggregation phenotype is due to a lack of chitinase expression. The random budding pattern and the round cell phenotype of the CBK1 deletion strain show that in addition to its role in regulating chitinase expression via Ace2p, Cbk1p is essential for a wild-type morphological development of the cell.

Identification of Novel Saccharomyces cerevisiaeProteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

Identification of Novel Saccharomyces cerevisiaeProteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

Regulated nuclear localisation of the yeast transcription factor Ace2p controls expression of chitinase (CTS1) in Saccharomyces cerevisiae.

The yeast transcription factor Ace2p regulates expression of the chitinase gene CTS1 in a cell cycle-dependent manner. Nuclear localisation of Ace2p is restricted to late M and early G1 phases of the mitotic cell cycle. We show here that this nuclear localisation is directly associated with regulation of CTS1 expression. Using a version of Ace2p tagged with a c-myc epitope, we show that the protein is excluded from the nucleus of cells during most phases of the mitotic cell cycle. A mutant derivative in which one threonine and two serine residues, which are candidate phosphorylation sites, were replaced by alanine (to mimic constitutive dephosphorylation) is localised in the nucleus throughout the cell cycle. The mechanism of localisation of Ace2p therefore involves regulation of its phosphorylation state, and closely resembles that used by the homologous transcription factor Swi5p. The wild-type Ace2 protein associates with Cdc28p in vivo, suggesting this may be the kinase that mediates the phosphorylation event. The stability of the protein is greatly reduced in a mutant that is constitutively localised to the nucleus, but is restored in a deletion derivative which remains in the cytoplasm. Ace2p is therefore controlled throughout the cell cycle at three levels: transcription, nuclear localisation, and proteolysis.

Inappropriate expression of the yeast transcription factor Ace2p affects cell growth.

Inappropriate expression of the yeast transcription factor Ace2p affects cell growth.