A novel 1,3-␤-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-␤-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1,3-␤-glucan molecule forming a 1,3-␤ linkage, resulting in the elongation of 1,3-␤-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-␤-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-␤-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.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1⌬ mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1⌬ mutant cells. In these cells, the level of alkali-insoluble 1,6-␤-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1⌬ cells was found to be essential since the chs3⌬ ggp1⌬ mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3⌬ ggp1⌬ cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1⌬ cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1⌬ kre6⌬ and the ggp1⌬ pkc1⌬ combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-␤-D-glucans with 1,6-␤-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.

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The first fungal glycosylphosphatidylinositol anchored beta(1-3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI-anchored protein and shares the same beta(1-3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Deltagas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Deltagel2 mutant and the double mutant Deltagel1Deltagel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Deltagel2 and the Deltagel1Deltagel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that beta(1-3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.

This paper reports a phenotypic characterization of ggpl mutants. The cloned GGPI (GAS)) gene, which encodes a mawjor GPI-anchored glycoprotein (gpllS)

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1⌬. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1⌬ mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.Yeast cells are surrounded by a matrix composed of ␤(1,3)/ (1,6)-glucans and mannoproteins as major components and chitin as a minor one (21). Chitin constitutes only 1 to 2% of the cell wall dry weight, but it plays a key role in yeast morphogenesis and is essential for the viability of yeast and fungal cells. During vegetative growth chitin is deposited at the site of bud emergence, forms a ring that surrounds the neck between the mother and daughter cells, and constitutes the primary septum. On the surface of mother cells a chitin ring is still recognizable after cell division, the so-called bud scar, and in the corresponding site on the daughter surface a birth scar is present. A tiny amount of chitin is also layered over the whole of the lateral cell wall, and this occurs in the mother cell.Three chitin synthase (CS) activities, CSI, CSII, and CSIII, are responsible for the deposition of cell wall chitin. The three isoenzymes differ in certain properties, such as the optimum pH, metal specificity, and susceptibility to inhibitors (6). CSI and CSII activities are determined only by the product of CHS1 and CHS2 genes, respectively, which encode the polypeptides containing the catalytic domain of each chitin synthases. Chs1p is responsible for the synthesis of chitin after cell separation. It plays a repair function, since it counterbalances the acid-induced increase in the chitinase activity that hydrolyzes the chitin present in the primary septum at the end of cytokinesis (3-5, 17, 18). CSI represents about 90% of the in vitr...

Gas1p is a glycosylphosphatidylinositol‐anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1‐2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be β‐(1,3)‐glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys‐rich domain; and a highly O‐glycosylated serine‐rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of ≈ 10 mg·L−1 of medium, and this value was unaffected by the further removal of the serine‐rich region or by fusion to a 6 × His tag. Purified soluble Gas1 protein showed β‐(1,3)‐glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane‐bound Gas1p were analyzed in S. cerevisiae. The Gas1‐C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.