Johan C. Kapteyn

The contribution of cell wall proteins to the organization of the yeast cell wall.

Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild‐type cells, the β1,6‐glucanase‐extractable glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI‐CWPs, including Als1p and Als3p, are attached via β1,6‐glucan to β1,3‐glucan. The remaining GPI‐CWPs are linked through β1,6‐glucan to chitin. The β1,6‐glucanase‐resistant protein fraction is small and consists of Pir‐related CWPs, which are attached to β1,3‐glucan through an alkali‐labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N‐ and O‐glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI‐CWPs through β1,6‐glucan to chitin. In these cells, the level of Pir‐CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6‐glucan‐deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Cell wall dynamics in yeast

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

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.

The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and β1,6-glucan-deficient mutants

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)‐derived structure to β1,6‐glucan, which is linked in turn to β1,3‐glucan. When isolated cell walls were digested with β1,6‐glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve β1,6‐glucan. The β1,6‐glucanase‐resistant protein fraction contained the recently identified GPI‐lacking, O‐glycosylated Pir‐CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to β1,3‐glucan through an alkali‐sensitive linkage, without β1,6‐glucan as an interconnecting moiety. In β1,6‐glucan‐deficient mutants, the β1,6‐glucanase‐resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of β1,6‐glucan.

Posttranslational modifications of secretory proteins

Publisher Summary This chapter focuses on post-translational modifications of secretory proteins. Proteins that traverse the secretory pathway and reach the plasma membrane may be targeted to various locations. One final destination is the plasma membrane itself. The plasma membrane contains not only many transmembrane proteins, but also a special group of proteins, which are C-terminally linked to the outer leaflet of the plasma membrane through a so-called “glycosylphosphatidylinositol” (GPI) anchor. Between the plasma membrane and the cell wall, periplasmic proteins, such as invertase and acid phosphatase, may accumulate. A limited number of proteins, such as chitinase, and several heat shock proteins are secreted into the medium. Secretory proteins are modified in various ways during their passage through the secretory pathway. The chapter discusses techniques to identify and characterize the post-translational modifications of secretory proteins. It is important to realize that these techniques, although primarily developed for Succhuromyces cereuisiae , are in many cases also valid for other Ascomycetes, including the filamentous fungi belonging to this taxonomic group. The chapter discusses the glycosylation of secretory proteins and the detection of N - and O -glycosylation in secretory proteins.