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Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.

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 β-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 β-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 β-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.

1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.

SUMMARY: Vesicles accumulate at the site of bud formation in the mother cell and are also found in the growing bud during the growth of its wall. The bud is in direct communication with the mother cell until maturity when a septum which is formed across the cytoplasm grows inwards from the wall and is subsequently thickened on both sides. During the thickening process vesicles are present at both surfaces of the cross-wall which finally has two layers one of which becomes continuous with the bud cell wall. It is suggested that the vesicles carry material both into the growing bud wall and into the septum.