Edwin Vink

Low external pH induces HOG1‐dependent changes in the organization of the Saccharomyces cerevisiae cell wall

Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to β1,3‐glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins, namely an alkali‐labile linkage to β1,3‐glucan. This novel type of modification for Cwp1p did not require the presence of a GPI‐derived structure connecting the protein with β1,6‐glucan. In addition, we found high levels of Cwp1p, which was double‐anchored through both the novel alkali‐sensitive bond to β1,3‐glucan and the alkali‐resistant GPI‐derived linkage to β1,6‐glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the β1,3‐glucan framework by an alkali‐sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali‐sensitive type of linkage of cell wall proteins to β1,3‐glucan was induced by low pH. The low pH‐induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p‐mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high‐osmolarity glycerol (HOG) pathway‐dependent genes, including four cell wall‐related genes, namely CWP1, HOR7, SPI1 and YGP1.

An in vitro assay for (1-->6)-beta-D-glucan synthesis in Saccharomyces cerevisiae.

(1 → 6)‐β‐D‐glucan is a key cell wall component of Saccharomyces cerevisiae and Candida albicans. Many genes are known to affect the levels or structure of this glucan, but their roles and a molecular description of the synthesis of (1 → 6)‐β‐D‐glucan remain to be established and a method to measure (1 → 6)‐β‐D‐glucan synthase activity in vitro would provide an enabling tool. Here, conditions for the detection of in vitro synthesis of this polymer are described. Crude membrane preparations from S. cerevisiae were isolated, and incubated in the presence of UDP‐glucose and GTP. With anti‐(1 → 6)‐β‐D‐glucan‐specific antibodies, a time‐dependent increase in the amount of this glucan was demonstrated in a dot–blot assay, or through an inhibition enzyme immunoassay. Antibody specificity was validated by competition experiments using pustulan, a (1 → 6)‐β‐D‐glucan, laminarin, a (1 → 3)‐β‐D‐glucan, yeast mannan and glycogen. The identity of the reaction product was also demonstrated by its sensitivity to a recombinant (1 → 6)‐β‐D‐glucanase. Extracts from mutants in 10 genes with a wide range of altered cell wall (1 → 6)‐β‐D‐glucan levels were assayed for in vitro synthesis of the polymer. A strong correlation of in vitro synthase activity with in vivo glucan levels was found, providing genetic support for the specificity of the assay. The basis for the GTP‐dependence of the synthase reaction was studied. Extracts from rho2, rho3, rho4 and rho5 null mutants had wild‐type in vitro activity. In contrast, Rho1p overproduction led to increased in vitro synthesis, implicating Rho1p in the regulation of (1 → 6)‐β‐D‐glucan synthesis. Copyright

Multi‐level Democracy: Deliberative or Agonistic? The Search for Appropriate Normative Standards

Abstract This article compares two contrasting conceptions of deliberative supranationalism, which together suggest a third alternative. All three draw on both the practical and idealizing aspects of communication theory, but each proposes a different view of the European constitutional model, the normative principles on which the European Union (EU) is to be based, and the Constitution for Europe. The first, by Joerges & Neyer, defends a ‘non‐majoritarian’ view in which democratic legitimacy is based primarily upon mutual recognition and Europe’s ‘multiple demoi’. The second, by Eriksen & Fossum, proposes a ‘majoritarian’ scheme based upon a European hierarchy of law, direct accountability to a ‘general public’ and ‘one constitutional demos’ as the ultimate source of European democratic legitimacy. The article concludes with a non‐consensual ‘complex majoritarian’ approach to multi‐level democracy aimed at combining the advantages of the first, polycentric and non‐hierarchical model with agonistic freedoms of citizens in a polity which finds its sources of democratic legitimacy in various modes of democratic representation as well as multiple access points and communication channels that open up EU decision making to European publics.

The protein kinase Kic1 affects 1,6-beta-glucan levels in the cell wall of Saccharomyces cerevisiae.

KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-beta-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-beta-glucan-deficient strain fks1delta, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-beta-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-beta-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-beta-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-beta-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-beta-glucan levels in the wall.