Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall

Abstract

Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with beta(1-3)- or beta(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to beta(1-6)glucan. With this technique, crh1Delta crh2Delta mutants still appeared to contain a substantial percentage of chitin linked to beta(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a beta(1-3)glucan, for beta(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1Delta crh2Delta strains is free. Reexamination of the previously used procedure revealed that the beta(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both beta(1-3)- and beta(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.

FIG. 1.

Outline of the two novel procedures for determination of chitin cross-links. In both cases chitin was specifically labeled in vivo with [¹⁴C]glucosamine and estimated in the various fractions by radioactivity measurement. For details of the procedures, see Materials and Methods. (A) Curdlan method. Free chitin was determined from the unbound radioactivity (step 1). This value was subtracted from the unbound radioactivity (step 2) to obtain the chitin attached to β(1-6)glucan. The chitin bound to β(1-3)glucan can be calculated by subtracting the unbound value (step 2) from the total or by determining the bound radioactivity (step 2). (B) Chitosan method. Each step 1 to 4 was performed on the insoluble residue remaining from the preceding step. The radioactivity of each extract was measured.

FIG. 2.

(A) Chromatography of crh1Δ crh2Δ carboxymethylated cell walls on a Sephacryl S-300 column, either untreated or after digestion with zymolyase or Quantazyme. The void volume in this and all subsequent Sephacryl columns is at fractions 26 to 27. Also, in all Sephacryl columns the radioactivity represents chitin labeled in vivo with [¹⁴C]glucosamine. (B) Determination of chitinase in zymolyase by viscosimetry. Travel time is the time taken by a glass bead to travel the length of a 0.2-ml pipette filled with the solution to be tested. Incubation time is the time of incubation with zymolyase previous to the measurement of viscosity. Symbols: •, 7.5 μl of zymolyase, 10 mg/ml; □, 15 μl of zymolyase, 10 mg/ml; ○, 60 μl of purified zymolyase [contains an amount of β(1-3)glucanase activity similar to that of 15 μl of crude zymolyase]. The broken line indicates the travel time with water as the test solution.

FIG. 3.

Chromatography on Sephacryl S-300 of crh1Δ crh2Δ carboxymethylated cell walls after the original walls were either left untreated or digested with different enzymes. (A) Cell walls, either untreated or digested with either purified zymolyase or recombinant β(1-6)glucanase. (B) Cell walls, either untreated or after digestion with crude zymolyase.

FIG. 4.

Chromatography on Sephacryl S-300 of different cell wall fractions from a wild-type strain (FY001). (A) Carboxymethylated cell walls, either undigested or after incubation with different enzymes, including crude zymolyase. (B) Same as panel A, but purified zymolyase was used. (C) Material solubilized in water by digestion of FY001 cell walls with either crude or purified zymolyase. The first peak corresponds to a chitin-β(1-6)glucan complex (4), and the second peak corresponds to small chitin that was previously linked to β(1-3)glucan (4).

FIG. 5.

(A) Chromatography on Sephacryl S-300 of different chitosan fractions extracted from FY001 (wild type) or RCA003 (crh1Δ crh2Δ mutant) cell walls with the chitosan procedure. (B) The second peak (yellow) is obtained by drawing a curve symmetrical to its second half, taking fraction 58 as the maximum. The first peak (red) can then be drawn by subtracting the second peak calculated values from the experimental measurements.

FIG. 6.

Distribution of chitin in the wild type and in different crh mutants, measured by either carboxymethylation-chromatography (A) or the chitosan procedure (B). In both cases, purified zymolyase was used.