ENG1 (YNR067c), a gene encoding a new endo-1,3-␤-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-␤-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-␤ 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 motherdaughter septum.The yeast cell wall is a rigid structure that preserves the osmotic integrity of the cell and determines cellular morphology during the different stages of the life cycle. In Saccharomyces cerevisiae, the cell wall is essentially made up of highly mannosylated proteins and three different polysaccharide chains: (i) the predominant, linear 1,3-␤-glucan, (ii) a minor, highly branched 1,6-␤-glucan, and (iii) chitin. All of these components are covalently linked in vivo as part of a macromolecular structure composed of what has been called the "flexible building block," in which mannoproteins are linked to the 1,3-␤-glucan either directly (in the case of PIR proteins) or through a molecule of 1,6-␤-glucan (glycosylphosphatidylinositol proteins) (reviewed in references 8, 43, and 57).Glucans are the main components of the yeast cell wall and are responsible for the rigidity and mechanical strength of this structure. Although they do not undergo appreciable turnover during vegetative growth, it has been proposed that limited site-directed hydrolysis of the rigid skeletal wall ␤-glucans, mediated by endogenous ␤-glucanases, probably takes place during several morphogenetic processes, such as budding, wall growth, conjugation, and ascus formation (8, 42, 58).Two broad classes of 1,3-␤-glucanases occur in yeasts, the exoglucanases and the endoglucanases. As measured by their activity on the substrate laminarin (a linear 1,3-␤-glucan), exo-1,3-␤-glucanases account for the greater part of total glucanase activity in yeasts and hydrolyze the ␤-O-glycosidic linkages at the nonreducing end of the polymer chain, resulting in the release of glucose. These enzymes are not particularly specific because they usually also act on 1,6-␤ linkages...

Stimulation of GCN4 mRNA translation due to phosphorylation of the a-subunit of initiation factor 2 (eIF2) by its speci®c kinase, GCN2, requires binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like domain in GCN2. GCN2 function in vivo also requires GCN1 and GCN20, but it was unknown whether these latter proteins act directly to promote the stimulation of GCN2 by uncharged tRNA. We found that the GCN1±GCN20 complex physically interacts with GCN2, binding to the N-terminus of the protein. Overexpression of N-terminal GCN2 segments had a dominant-negative phenotype that correlated with their ability to interact with GCN1±GCN20 and impede association between GCN1 and native GCN2. Consistently, this Gcn ± phenotype was suppressed by overexpressing GCN2, GCN1±GCN20 or tRNA His . The requirement for GCN1 was also reduced by overexpressing tRNA His in a gcn1D strain. We conclude that binding of GCN1±GCN20 to GCN2 is required for its activation by uncharged tRNA. The homologous N-terminus of Drosophila GCN2 interacted with yeast GCN1±GCN20 and had a dominant Gcn ± phenotype, suggesting evolutionary conservation of this interaction.

Protein kinase GCN2 regulates translation initiation by phosphorylating eukaryotic initiation factor 2␣ (eIF2␣), impeding general protein synthesis but specifically inducing translation of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. GCN2 activity is stimulated in amino acid-deprived cells through binding of uncharged tRNA to a domain related to histidyl tRNA synthetase. We show that GCN2 is phosphorylated by another kinase on serine 577, located N-terminal to the kinase domain. Mutation of Ser-577 to alanine produced partial activation of GCN2 in nonstarved cells, increasing the level of phosphorylated eIF2␣, derepressing GCN4 expression, and elevating the cellular levels of tryptophan and histidine. The Ala-577 mutation also increased the tRNA binding affinity of purified GCN2, which can account for the elevated kinase activity of GCN2-S577A in nonstarved cells where uncharged tRNA levels are low. Whereas Ser-577 remains phosphorylated in amino acidstarved cells, its dephosphorylation could mediate GCN2 activation in other stress or starvation conditions by lowering the threshold of uncharged tRNA required to activate the protein.

Meiosis in Saccharomyces cerevisiae is followed by encapsulation of haploid nuclei within multilayered spore walls. Formation of this spore-specific wall requires the coordinated activity of enzymes involved in the biosynthesis of its components. Completion of late events in the sporulation program, leading to spore wall formation, requires the SWM1 gene. SWM1 is expressed at low levels during vegetative growth but its transcription is strongly induced under sporulating conditions, with kinetics similar to those of middle sporulation-specific genes. Homozygous swm1Delta diploids proceed normally through both meiotic divisions but fail to produce mature asci. Consistent with this finding, swm1Delta mutant asci display enhanced sensitivity to enzymatic digestion and heat shock. Deletion of SWM1 specifically affects the expression of mid-late and late sporulation-specific genes. All of the phenotypes observed are similar to those found for the deletion of SPS1 or SMK1, two putative components of a sporulation-specific MAP kinase cascade. However, epistasis analyses indicate that Swm1p does not form part of the Sps1p-Smk1p-MAP kinase pathway. We propose that Swm1p, a nuclear protein, would participate in a different signal transduction pathway that is also required for the coordination of the biochemical and morphological events occurring during the last phase of the sporulation program.