Climbing the yeast cell wall.

Here is a life in three countries, with different cultures, different political structures and even different skies. The constant through all these changes is the addiction of the subject of this story to science and laboratory work. Perhaps the tale that unfolds here will show to some beginners in research that persistence, seasoned with a little luck, can bring results and satisfaction in the long run.

How carbohydrates sculpt cells: chemical control of morphogenesis in the yeast cell wall.

In budding yeast, the neck that connects the mother and daughter cell is the site of essential functions such as organelle trafficking, septum formation and cytokinesis. Therefore, the morphology of this region, which depends on the surrounding cell wall, must be maintained throughout the cell cycle. Growth at the neck is prevented, redundantly, by a septin ring inside the cell membrane and a chitin ring in the cell wall.

Crosslinks in the cell wall of budding yeast control morphogenesis at the mother-bud neck.

Previous work has shown that, in cla4Δ cells of budding yeast, where septin ring organization is compromised, the chitin ring at the mother-daughter neck becomes essential for prevention of neck widening and for cytokinesis. Here, we show that it is not the chitin ring per se, but its linkage to β(1-3)glucan that is required for control of neck growth. When in a cla4Δ background, crh1Δ crh2Δ mutants, in which the chitin ring is not connected to β(1-3)glucan, grew very slowly and showed wide and growing necks, elongated buds and swollen cells with large vacuoles.

Presence of a large β(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control.

Previous results suggested that the chitin ring present at the yeast mother-bud neck, which is linked specifically to the nonreducing ends of β(1-3)glucan, may help to suppress cell wall growth at the neck by competing with β(1-6)glucan and thereby with mannoproteins for their attachment to the same sites. Here we explored whether the linkage of chitin to β(1-3)glucan may also prevent the remodeling of this polysaccharide that would be necessary for cell wall growth. By a novel mild procedure, β(1-3)glucan was isolated from cell walls, solubilized by carboxymethylation, and fractionated by size exclusion chromatography, giving rise to a very high-molecular-weight peak and to highly polydisperse material.

Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall.

Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with beta(1-3)- or beta(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to beta(1-6)glucan. With this technique, crh1Delta crh2Delta mutants still appeared to contain a substantial percentage of chitin linked to beta(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall.

Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro.

The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to beta(1-3)glucose branches of beta(1-6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors.

Hyperpolarized growth of Saccharomyces cerevisiae cak1P212S and cla4 mutants weakens cell walls and renders cells dependent on chitin synthase 3.

In a screen for cell wall defects in Saccharomyces cerevisiae, we isolated a strain carrying a mutation in the Cdc28-activating kinase CAK1. The cak1P212S mutant cells exhibit multiple, elongated and branched buds, beta(1,3)glucan-poor regions of the cell periphery and lysed upon osmotic shock after treatment with the chitin synthase III inhibitor Nikkomycin Z. Ultrastructural examination of cak1P212S mutants revealed a thin, uneven cell wall and marked abnormalities in septum formation.

Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall.

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan.

Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast.

In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to beta(1-3)glucan or beta(1-6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site.

Chitin synthase III activity, but not the chitin ring, is required for remedial septa formation in budding yeast.

Chitin is a minor but essential component of the Saccharomyces cerevisiae cell wall. In wild-type, chitin synthase II is required for the formation of primary septa and chitin synthase III (CSIII) is not essential. However, in chs2 mutants CSIII becomes essential for the formation of aberrant septa.

Septins, under Cla4p regulation, and the chitin ring are required for neck integrity in budding yeast.

CLA4, encoding a protein kinase of the PAK type, and CDC11, encoding a septin, were isolated in a screen for synthetic lethality with CHS3, which encodes the chitin synthase III catalytic moiety. Although Ste20p shares some essential function with Cla4p, it did not show synthetic lethality with Chs3p. cla4 and cdc11 mutants exhibited similar morphological and septin localization defects, including aberrant and ectopic septa.

The septation apparatus, an autonomous system in budding yeast.

Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast. By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously. Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal.

Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall.

In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p.

In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each other.

Saccharomyces cerevisiae chs2 mutants are unable to synthesize primary septum chitin, and myo1 mutants cannot construct a functional contractile ring. The morphology of the two mutants, as observed by electron microscopy, is very similar. In both cases, neither an invagination of the plasma membrane, which normally results from contraction of the actomyosin ring, nor generation of a chitin disc, the primary septum, is observed.