The structure of cell wall alpha-glucan from fission yeast

Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.     

The structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

Correlative studies of cell wall enzymes and growth

If cell wall hydrolytic enzymes are involved in extension growth, a correlation may be expected between hydrolytic activity of the cell walls and growth rate of the tissue from which the walls are prepared. Epicotyl sections from 0 to 5 mm, 6 to 10 mm, and 11 to 15 mm below the apical hook of pea seedlings (Pisum sativum var. Alaska) have relative growth rates of 100:15:2, respectively. The relative β-glucosidase activities (units/mg wall) of cell walls from these sections are respectively, 100:24:23, for walls prepared in glycerol and 100:42:23 for walls prepared in aqueous solution. Thus, there is a correlation between growth rate of the tissue and specific activity of the wall-associated β-glucosidase. Similar correlations were found for other cell wall-associated hydrolases.     

Pieiotropic effect of two cell wall mutants on the activity of some cell wall enzymes in the yeast Saccharomyces cerevisiae.

Summary The two mutants (abs) and (wal) affecting the cell morphology of yeast lead also to higher in vivo activities of the cell wall enzymes acid phosphatase. invertase and melibiase.The investigations were supported by a fellowship from the Max Kade Foundation New York.     

Relative effectiveness of yeast cell wall digesting enzymes on Yarrowia lipolytica

Novozym 234 at a concentration of 1.0 mg/ml yielded 95.5% spheroplasts within 30 min at 37 degrees C, pH 7.0, with 36% regeneration which was the highest level of regeneration observed. Yeast lytic enzyme at a concentration of 1.0 mg/ml yielded 99.8% spheroplasts with only 1.5% regeneration. Glusulase was significantly less active in producing osmotically sensitive cells. All three enzymes yielded significantly higher levels of osmotically sensitive cells when cells were harvested from the mid-logarithmic phase of growth compared with later growth phases. beta-Glucuronidase failed to produce osmotically sensitive cells regardless of the phase of growth from which cells were harvested.     

Integrative studies put cell wall synthesis on the yeast functional map

The fungal cell wall field, traditionally focused on polysaccharide composition and synthesis, retains a certain static architectural imagery of structural rigidity and integrity, with the wall offering protection from a harsh environment. This picture of the wall is increasingly changing to that of a bustling construction site, as research uncovers the organizational complexity of its assembly. With recent molecular and genomic studies on Saccharomyces cerevisiae, cell wall synthesis and biology appear increasingly to be dynamic and adaptable processes that are fully integrated with the underlying cytoskeletal and polarity machinery that drive cell cycle progression.     

Further studies on the quaternary structure of yeast casein kinase II

Casein kinase type II were isolated by the same procedure, from rat liver, human placenta, Querin carcinoma and yeast, and characterized. The mammalian enzymes were composed of three subunits alpha, alpha' and beta, whereas yeast kinase was composed of two subunits alpha and alpha'. It was shown that the catalytic activity, substrate and phosphate donor specificity, sensitivity to heparin and spermine were the same for all the kinases tested. The results give additional support to the suggestion [1] that the beta subunit is not required for optimal activity and specificity of yeast casein kinase II. The quaternary structure of the yeast enzyme of a molecular weight of approximately 150 000 is proposed as alpha2 alpha'2.     

Structure/function studies on enzymes in the diaminopimelate pathway of bacterial cell wall biosynthesis.

Within the past 18 months work has continued on the structure and mechanisms of enzymes involved in the diaminopimelic acid/lysine biosynthetic pathway. A novel structure has been determined for a PLP-independent epimerase, and structures with bound substrates have been solved for two other enzymes. Additionally, new studies have appeared describing the chemical mechanisms of three enzymes in the pathway.     

A cell wall proteo-heteroglycan from Piricularia oryzae: further studies of the structure.

The carbohydrate part of a proteo-heteroglycan from Piricularia oryzae was further studied by chemical and immunological methods. Acetolysis studies of the proteo-heteroglycan and exo-alpha-D-mannanase resistant core showed a (1 leads to 6) mannan back-bone structure with side chains composed of one to four mannose units, and some of which are terminated by D-glucose or D-galactofuranose. The mode of attachment of the terminal glucose was characterized to be alphaGlc(1 leads to 6)Man by inhibition reaction with oligosaccharides. Rabbit anti-serum formed against P. oryzae cells had three specificities, the first one for alphaGlc(1 leads to 6)alphamannosyl, the second one for alphaMan(1 leads to 3)alphamannosyl, and the last one for alphaGal-f(1 leads to 2)alphamannosyl residues. The most immunodominant side chain structure of the P. oryzae heteroglycan was shown to be alphaGlc(1 leads to 6)alphaMan(1 leads to 2)alphaMan(1 leads to 2)Man.     

Cementing the wall: cell wall polysaccharide synthesising enzymes

The year under review has seen the first molecular characterisation, with proof of functionality, of Golgi membrane-bound glycosyltransferase enzymes catalysing the synthesis of non-cellulosic plant cell-wall polysaccharides.