Herman van den Ende

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.

In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae

Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N‐terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl‐phosphatidylinositol (GPI)‐attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI‐attachment sites, it was found that in yeast the most probable amino acids for GPI‐attachment are asparagine and glycine.

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.

Differential regulation of cell wall biogenesis during growth and development in yeast

A connector having an improved mechanism for its lock onto a panel. A housing of the connector has a wall on which a sliding rail capable of slidingly fitting onto guide rails of the panel is provided. A flexible engagement member having an engagement projection engageable with an engagement projection of the panel is formed in a portion of the wall by cutting a part of the wall. When the sliding rail is slid on the guide rail to fit thereon, the flexible engagement member is deformed and has its position changed toward the inside of the housing while its engagement projection abuts on the engagement projection of the panel until the member recovers its original position upon the engagement between the projections. The arrangement of the flexible engagement member is advantageous in that it does not require any deformation distance to be provided on the outside of the housing, thereby enabling the entire connector to be compact. The member may be provided with lock release portions engageable with a lock release instrument inserting into the inside of the housing. With this arrangement, the connector can be easily released from its state of being locked.

A comparative study of fossil- and extant algaenans using ruthenium tetroxide degradation

In this study the chemical structure of algaenans isolated from the freshwater algae Tetraedron minimum, Pediastrum boryanum and Botryococcus braunii are compared with their fossil counterparts by means of RuO4 oxidation. The results show that the algaenans investigated are preserved in sediments with only minor structural alterations. However, product mixtures from RuO4 degradation of the fossil algaenans exhibit a broader distribution of oxidation products than freshly isolated algaenans indicating that the fossil biopolymers contain a greater proportion of ether cross-links, which maybe an effect of diagenetic alteration or different algal strains. Despite these differences, fossil algaenans can still be recognised chemically on the basis of the specific RuO4 oxidation products, even after 50 Ma of sediment burial.

Molecular structure of the resistant biopolymer in zygospore cell walls of Chlamydomonas monoica

Abstract. The unicellular green alga Chlamydomonas monoica Strehlow is known to produce zygospores with a cell wall that is resistant against microbial and chemical attack. This resistance is thought to be due to the presence of a sporopollenin-like material. However, the resistant nature of sporopollenin-like materials seriously hampers their structural analysis. With complementary techniques such as 13C-nuclear magnetic resonance spectroscopy, Curie-point pyrolysis-gas chromatography/mass spectroscopy and RuO4 chemical degradation, the chemical composition of resistant biopolymer in the isolated cell walls of C. monoica zygospores was determined. This material is composed of C22–C30 linear alcohols and carboxylic acids, intermolecularly linked via ester and ether-linkages similar to the resistant aliphatic biopolymers encountered in the walls of the vegetative cells of the algae Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum.

Polar glycerolipids of Chlamydomonas moewusii

The fatty acid and polar lipid compositions of the unicellular green alga Chlamydomonas moewusii were characterized. Since this organism is an important plant model for phospholipid-based signal transduction, interest was focused on the lipids phosphatidic acid, phosphatidylinositolphosphate and phosphatidylinositolbisphosphate. A phosphatidylinositol:phosphatidylinositolphosphate: phosphatidylinositolbisphosphate ratio of 100:1.7:1.3 was found. The polyphosphoinositides accounted for 0.8 mol% of the total phospholipids and their fatty acid compositions were similar to that of phosphatidylinositol except for the enrichment of linolenic acid in phosphatidylinositol phosphate. Phosphatidic acid accounted for 0.67 mol% of the phospholipids. Major structural glycerolipids were monogalactosyldiacylglycerol (35 mol%), digalactosyldiacylglycerol (15 mol%), sulfoquinovosyldiacylglycerol (10 mol%), diacylglyceryltrimethylhomoserine (16 mol%), phosphatidylglycerol (9 mol%), phosphatidylethanolamine (8 mol%) and phosphatidylinositol (6 mol%). Relative changes in the total fatty acid compositions found during growth on nutrient-limited medium reflected mainly alterations in the compositions of the chloroplast lipids phosphatidylglycerol and monogalactosyldiacylglycerol. [32P]Pi-incorporation studies revealed that it took 6 days before the amount of label in the major phospholipids was proportional to their abundance.

Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos

Previously, we have shown that the monomeric-sugar composition of cell-surface-associated glycoconjugates of two strains of Chlamydomonas eugametos, of different mating type, differs strikingly (Gerwig et al. 1984, Carbohydr. Res. 127, 245–251). Besides the common occurrence of various pentoses and hexoses, the glycoconjugates of one strain contain 4-O-methyl xylose, a 2-O-methyl pentose (probably 2-O-methyl arabinose) and 3-O-methyl galactose, whereas those of the other strain contain 6-O-methyl mannose and 3-O-methyl glucose. In order to investigate whether these differences are relevant to the mating process of this organism, the sugar composition of the sexual progeny of these strains was analyzed. The ability to produce 4-O-methyl xylose, 2-O-methyl pentose and 3-O-methyl galactose on the one hand, and the ability to produce 6-O-methyl mannose and 3-O-methyl glucose on the other hand, appear to be genetically linked. However, the ability to produce either set of O-methyl sugars was inherited independently of mating type. O-Methylated sugars do not occur in the cell wall of C. eugametos, or in the cell-free medium, but only in surface-membrane-associated glycoconjugates, extractable with salt or detergent solutions.

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

Cell wall-specific ω-hydroxy fatty acids in some freshwater green microalgae

Abstract Ester-bound lipids from the cell walls of the green algae, Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum , were analysed by gas chromatography-mass spectrometry. The double bond positions of the C 30 , C 32 and C 34 mono- and C 30 and C 32 diunsaturated ω-hydroxy fatty acids obtained were determined by derivatisation with dimethyl disulphide (DMDS) prior to mass spectrometric identification. All unsaturated ω-hydroxy fatty acids have a double bond located at the ω9-position, whilst the C 30 and C 32 diunsaturated acids have a second double bond at the ω19 and ω18 positions, respectively. Unsaturated ω-hydroxy fatty acids are released upon saponification of the isolated cell walls in all three algae, but the distribution patterns of these compounds differ between species. ω-Hydroxy fatty acids are the main building blocks of the highly cross-linked constituent of the cell walls, herein termed algaenan, in which linear chains of esterified monomers are ether cross-linked at the position of the double bonds. Because none of these monomeric compounds were observed in the cytosolic fraction of the cells, it is believed that they are rapidly secreted and incorporated in the aliphatic cell walls of these algae.