In the work described here, an advance in methodology led to the conclusion that previous results about the attachment of chitin to other cell wall components were partially incorrect in a crh1Δ crh2Δ
strain. Based on this result, Crh1p and Crh2p were found to be responsible for the linkage of chitin not only to β(1-6)glucan, as previously reported (7
) but also to the main structural component of the cell wall, β(1-3)glucan. As part of this investigation, I developed two new procedures for the quantitative determination of chitin cross-links to other polysaccharides in the yeast cell wall, which may be useful for future research, both in yeast and other fungi. These methods are based on different principles from each other and from the previously devised carboxymethylation-chromatography technique; therefore, they may provide critical information in difficult cases. After the curdlan procedure was devised and used, a literature search showed that the same principle, adhesion between different β(1-3)-linked polysaccharides, had been used for a different purpose, i.e., to study the effect of molecular weight on the action of schizophyllan, a fungal polysaccharide, on the regeneration of yeast protoplasts (10
). This confirms the specificity of the polysaccharide interaction. In their work, Hisamatsu et al. (10
) used curdlan hard gel, obtained at 120°C, which I found less effective than the soft gel formed at a lower temperature.
Both the curdlan and the chitosan procedures are simpler than the older one because they do not require a chromatographic step. The chitosan method does not even entail carboxymethylation. The whole operation is carried out in a microcentrifuge tube, from which the different extracts are withdrawn, thus minimizing losses. The procedure is much faster than the carboxymethylation-chromatography method, lasting 2 to 3 days instead of about 10. In addition, several samples can be run simultaneously. The correction described above for the small chitin linked to β(1-3)glucan does require one chromatographic separation, but three such chromatographies can be run in 1 day.
Because in the chitosan method each type of chitin (now chitosan) is extracted separately, it is possible to determine the size of the individual fractions (Fig. ). Interestingly, the chitins linked to β(1-3)- and β(1-6)glucan show a similar size distribution and are smaller than the free chitin, which suggests that a relatively short fragment of the nascent chitin is transferred by Crh1p and Crh2p to the glucans. This type of experiment also shows that the chitin of the double mutant crh1Δ crh2Δ has a higher molecular weight than any of the chitins from the wild type (Fig. ), a finding which is in agreement with the expectation that no part of that chitin is transferred to an acceptor.
There is fairly good agreement among the three procedures now available (Table , Fig. ). Some of the differences may arise from the harsh alkali treatment in the chitosan method. It would be desirable to find a milder way to deacetylate chitin, but none is currently available, since the enzymatic treatment with deacetylase proved ineffective, and acid hydrolysis would break polysaccharide bonds. Where all three methods now agree is in the absence of any bound chitin in the cell wall of the crh1Δ crh2Δ
mutant. The two new procedures were instrumental in forcing a review of the older one, which resulted in the finding of chitinase contamination in zymolyase. It may be asked whether the possibility of that contamination was entertained earlier. Surprisingly, this was indeed the case. When it was found that zymolyase solubilized in water a large amount of radioactivity from [14
C]glucosamine-labeled cell walls (4
), it was first suspected that chitinase was responsible. However, Quantazyme, which contains no chitinase, had a similar effect (4
) and the solubilized material turned out to be a mixture of a chitin-β(1-6)glucan complex plus “small chitin,” previously attached to β(1-3)glucan. Paradoxically, this result put to rest our suspicion about the presence of chitinase in the zymolyase preparation.
For a β(1-3)glucanase preparation free from chitinase, purification of zymolyase was preferred over use of Quantazyme, for two reasons: first, from previous experience it seemed that zymolyase digests β(1-3)glucan faster and more completely than Quantazyme, perhaps because it contains more than one glucanase. Second, Quantazyme is no longer available commercially, and preparation of the recombinant enzyme would be laborious, whereas purification of zymolyase with a chitin column is very simple and fast. Because the purification is based on substrate affinity, it is very specific. The lack of effect of the purified zymolyase on the chitin of the crh1Δ crh2Δ mutant confirms that the chitinase was responsible for the previous spurious results.
Except for the case of the crh1Δ crh2Δ double mutant, results with the purified β(1-3)glucanase do not differ greatly from those obtained previously. The exception was in the sharp decrease in the small chitin-β(1-3)glucan fraction. Thus, part of that small chitin must come from degradation of chitin by chitinase. It may be asked whether the small chitin-β(1-3)glucan is a separate entity or just represents the tail end of a wide range of fragments of different lengths transferred from nascent chitin to glucan. Analysis of the material generated by the chitosan procedure suggests a separate peak for the small chitin (Fig. ), but with the evidence at hand this point cannot be settled.
Note that the results described here do not invalidate our findings on transfer of chitin to β(1-3)-linked fluorescent oligosaccharides (8
). Rather, it may be concluded that the reactions studied there serve as models for chitin transfer not only to β(1-3)-linked side chains of β(1-6)glucan but also to the main chain of β(1-3)glucan.
What is not in doubt is that the glycosyltransferases Crh1p and Crh2p are responsible for the attachment of chitin to both β(1-6)- and β(1-3)glucan, i.e., for all of the wall cross-links in which chitin has been found to be involved. This finding simplifies the situation with regard to chitin cross-links because a single system gives rise to all of them. On the other hand, it is not clear how the system is regulated to favor transfer to one or the other of the acceptors. Previous work, confirmed here, indicated that most of the bound chitin is linked to β(1-3)glucan at the mother-daughter neck, whereas it is attached to β(1-6)glucan in the lateral walls. How this differential localization is achieved remains to be explored. Another consequence of the results reported here is that we know how to abolish all chitin cross-links. This should facilitate studies of the physiological function of the cross-links. Work toward that goal is in progress.