The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation

Cell separation in Schizosaccharomyces pombe is achieved by 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 ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.     

Characterizing the sphingolipid signaling pathway that remediates defects associated with loss of the yeast amphiphysin-like orthologs, Rvs161p and Rvs167p

Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267-277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.     

Control of β-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence

Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes β-glucan in the fungal cell wall. C. albicans β-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how β-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates β-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated β-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated β-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.     

Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall

A novel 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-beta-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1, 3-beta-glucan molecule forming a 1,3-beta linkage, resulting in the elongation of 1,3-beta-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-beta-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-beta-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.     

Rho4 GTPase is involved in secretion of glucanases during fission yeast cytokinesis

Rho GTPases are regulators of signaling pathways that control actin organization and cell polarity processes in all eukaryotic cells. In Schizosaccharomyces pombe, Rho4p is involved in the regulation of septum degradation during cytokinesis. Here we show that Rho4p participates in the secretion of the glucanases Eng1p and Agn1p, which are responsible for the septum degradation. First, eng1+ or agn1+ overexpression suppressed the rho4delta multiseptation phenotype, and simultaneous overproduction of Rho4p and Eng1p or of Rho4p and Agn1p caused a dramatic lysis. Second, Rho4p was not necessary for Eng1p-mediated glucanase activity as measured in cell extracts; however, rho4delta cells have a lower level of (1,3)-beta-D-glucanase activity in the culture medium. Additionally, Eng1- or Agn1-green fluorescent protein did not properly localize to the septum in rho4delta cells grown at 37 degrees C. There was a decreased amount of these enzymes in the cell wall and in the culture medium of rho4delta cells at 37 degrees C. These results provide evidence that Rho4p is involved in the regulation of Eng1p and Agn1p secretion during cytokinesis.     

Neurospora crassa FKS protein binds to the (1,3)beta-glucan synthase substrate,UDP-glucose

The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase. We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.     

Characterization and functional analysis of the β-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis

The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 ( Pb Gel3p) is presented here. Pb Gel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis . The recombinant Pb Gel3p was overexpressed in Escherichia coli , and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of Pb Gel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1 Δ mutant. The results indicated that Pb Gel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity.     

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. cerevisiae eng1 mutant cells, it was able to complement the separation defect of this mutant, indicating that these two proteins are true functional homologues.     

The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1(+) shut-off and bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-d-glucan and primary septum formation. bgs1(+) shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die.     

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

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-β-1,3-glucanase and the Agn1 endo-α-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 β-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 eng1 Δ 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.