Presence of a large β(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control

Abstract

Previous results suggested that the chitin ring present at the yeast mother-bud neck, which is linked specifically to the nonreducing ends of β(1-3)glucan, may help to suppress cell wall growth at the neck by competing with β(1-6)glucan and thereby with mannoproteins for their attachment to the same sites. Here we explored whether the linkage of chitin to β(1-3)glucan may also prevent the remodeling of this polysaccharide that would be necessary for cell wall growth. By a novel mild procedure, β(1-3)glucan was isolated from cell walls, solubilized by carboxymethylation, and fractionated by size exclusion chromatography, giving rise to a very high-molecular-weight peak and to highly polydisperse material. The latter material, soluble in alkali, may correspond to glucan being remodeled, whereas the large-size fraction would be the final cross-linked structural product. In fact, the β(1-3)glucan of buds, where growth occurs, is solubilized by alkali. A gas1 mutant with an expected defect in glucan elongation showed a large increase in the polydisperse fraction. By a procedure involving sodium hydroxide treatment, carboxymethylation, fractionation by affinity chromatography on wheat germ agglutinin-agarose, and fractionation by size chromatography on Sephacryl columns, it was shown that the β(1-3)glucan attached to chitin consists mostly of high-molecular-weight material. Therefore, it appears that linkage to chitin results in a polysaccharide that cannot be further remodeled and does not contribute to growth at the neck. In the course of these experiments, the new finding was made that part of the chitin forms a noncovalent complex with β(1-3)glucan.

Fig 1

Schematic representation of the cell wall structure of Saccharomyces cerevisiae (13). The arrowheads indicate the reducing ends of the different polysaccharides. MP is mannoprotein, where the amino acid chain is brown and the branched mannosyl chains are white. The connection between mannoprotein and β(1-6)glucan consists of the remnant of a glycosylphosphatidylinositol anchor, where Et stands for ethanolamine, P stands for phosphate, and the five white dots represent mannose residues (13). Chitin is attached to β(1-6)glucan through a β(1-3)-linked glucose branch, which is not shown here. Part of the chitin that is free is not shown.

Fig 2

Isolation and fractionation of β(1-3)glucan. (A) Scheme for isolation of β(1-3)glucan from yeast cell walls. All carboxymethylated (CM) fractions are soluble in water. (B) Chemical linkage of acetic acid to hexose in a carboxymethylated sugar. The open structure of the sugar is shown for simplicity. Monochloroacetic acid is the reactant, and an ether linkage is formed as shown. The acetic acid residue may be attached to any of the free hydroxyl groups in a polysaccharide and will be negatively charged at neutral pH. (C) Chromatography of total (no NaOH), alkali-soluble, and alkali-insoluble β(1-3)glucan on Sephacryl S-500. Here as in subsequent columns, fractions of 1 ml were collected. The void volume of the column is at fractions 24 to 26. For glucose, the position of the peak maximum was at fraction 57, as determined with [¹⁴C]glucose as the standard. (D) Chromatography of total β(1-3)glucan on Sephacryl S-500 before and after filtration through WGA-agarose.

Fig 3

Chromatography of total, NaOH-soluble, and NaOH-insoluble fractions of different strains on Sephacryl S-500. (A) Strain BY4741 (wild type of the gas1Δ mutant). (B) Strain YHR307W (gas1Δ). (C) Strain ECY46-4-1B (chs3Δ). (D) Strain NBT014 (crh1Δ crh2Δ). The two latter mutations are in the YPH499 background. In all cases, the procedure was as that used for Fig. 2A.

Fig 4

Cell walls before and after different treatments. (A) Untreated cell walls. Note that bud cell walls are in several cases attached to mother walls (arrows). (B) Cell walls after incubation with β(1-6)glucanase. (C) Cell walls after further treatment with 1 M sodium hydroxide at room temperature. No bud cell walls are visible here. Scale bar represents 10 μm.

Fig 5

Isolation of the covalent chitin–β(1-3)glucan complex. (A) Left, components of the sodium hydroxide-insoluble fractions. The covalent complex of chitin and β(1-3)glucan is symbolized by a hyphen between the two, whereas the noncovalent complex has a dot. Right, components of the fraction solubilized by carboxymethylation. Note that the number of components decreased from 4 to 3 after carboxymethylation. The carboxymethylated mixtures from the wild type and the crh1Δ crh2Δ mutant were applied to WGA-agarose columns (B) Sephacryl S-500 chromatography of material not retained by WGA-agarose for YPH499 (wild type) and NBT014 (crh1Δ crh2Δ). This represents free CM–β(1-3)glucan (C) fractionation of 0.1 NaOH eluates of WGA-agarose columns on Sephacryl S-500. For the wild type, the first peak contains the CM–chitin–β(1-3)glucan covalent complex, while the second peak corresponds to free chitin. Only the free chitin peak is present in the mutant fraction. See text and Materials and Methods for details.

Fig 6

Chromatography of WGA-agarose fractions on Sephacryl S-500 after treatment with Zymolyase [β(1-3)glucanase]. (A) Percolates of strain YPH499 (wild type). (B) Percolates of NBT014 (crh1Δ crh2Δ). (C) NaOH fractions of the wild type. (D) NaOH fractions of the crh1Δ crh2Δ mutant.

Fig 7

Chromatography of WGA-agarose fractions on Sephacryl S-500 after treatment with chitinase. (A) Chromatography of [¹⁴C]CM-chitin before and after treatment with untreated or curdlan-treated chitinase. (B) Percolates of the wild type. (C) Percolates of the crh1Δ crh2Δ mutant. (D) NaOH fractions of the wild type. (E) NaOH fractions of the crh1Δ crh2Δ mutant.

Fig 8

Chromatography on Sephacryl S-500 of WGA-agarose fractions from cells labeled with [¹⁴C]glucosamine. (A) NaOH fraction of the wild type. (B) NaOH fraction of the crh1Δ crh2Δ mutant. The positions of peaks in these graphs should be compared to those in Fig. 5C.