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Originally published In Press as doi:10.1074/jbc.M414005200 on January 6, 2005

J. Biol. Chem., Vol. 280, Issue 10, 9170-9179, March 11, 2005
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Synthase III-dependent Chitin Is Bound to Different Acceptors Depending on Location on the Cell Wall of Budding Yeast*{diamondsuit}

Enrico Cabib{ddagger}§ and Angel Durán§

From the {ddagger}Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20852 and the §Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, 37007 Salamanca, Spain

Received for publication, December 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to {beta}(1–3)glucan or {beta}(1–6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to {beta}(1–3)glucan at that site. Here, we devised a novel strategy for the analysis of chitin cross-links in [14C]glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation. Intact cell walls or their digestion products with {beta}(1–3)glucanase or {beta}(1–6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated. Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells. The former was mostly attached to {beta}(1–6)glucan and the latter to {beta}(1–3)glucan. This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location. In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to {beta}(1–3)glucan.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell wall of budding yeast, Saccharomyces cerevisiae, is essential, as in all fungi, for protection of the cell from osmotic shock. It also imparts shape to the cell during growth. A specialized structure of the cell wall, the septum, forms the partition between mother and daughter cell at cytokinesis and ensures that cell separation will occur without lysis (for reviews, see Refs. 1 and 2). The cell wall and septum consist mainly of carbohydrates. {beta}(1–3)Glucan and mannoproteins are the main components, followed by {beta}(1–6)glucan and chitin. The latter accounts for only a few percent of the wall but is essential for normal cell division. The deposition of chitin in the yeast cell wall occurs in several steps (Fig. 1A). Concomitant with bud emergence, a chitin ring is laid down in the cell wall at the neck between mother and daughter cell (Fig. 1A, cell b). Later, at cytokinesis, a chitin primary septum is built concurrently with contraction of an actomyosin ring (Fig. 1A, cell c; Refs. 3–6). Finally, after septation, more chitin is deposited throughout the daughter cell wall (Fig. 1A, cells d and e; Ref. 7). The enzyme responsible for synthesis of the chitin ring and of the chitin dispersed in the wall is chitin synthase III (CSIII),1 whereas chitin synthase II (CSII) is involved in primary septum construction (7). A third enzyme, chitin synthase I, has a repair role during cell separation (8, 9).



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  		FIG. 1.
Scheme for the deposition of chitin in the yeast cell wall during the cell cycle. A, in image a, a daughter cell ready for budding has chitin (CWC, green spots) dispersed in the cell wall. A chitin ring (CR) is formed at the base of an emerging bud (b), as a result of CSIII activity. In image c, the chitin primary septum (PS) is formed with the participation of CSII (for review of this complex process, which includes contraction of an actomyosin ring, see Ref. 6). In image d, secondary septa (SS, yellow) are laid down, and chitin starts appearing in the bud wall, a process that continues as the daughter cell separates from the mother cell and matures (images e and a). Note in image e the mother cell with the bud scar (BS) that still contains most of the primary septum, some of which was digested by a chitinase to facilitate cell separation. One of the secondary septa remains with the daughter cell to form the birth scar, which is not shown. B, expected products from the action of different glucanases on alkali-extracted cell walls. Note that the only detectable material, free or combined, is chitin because of its radioactive label.

 

Whereas the role of the primary septum is fairly obvious and has been well established, that of the chitin ring remained unknown for many years. Recently, we presented evidence that, together with the septin ring, the chitin ring acts to maintain integrity of the mother-daughter neck by preventing cell wall growth at that location (10). We also suggested a possible mechanism for chitin ring function, based on the cell wall architecture. The components of the yeast cell wall are linked to each other (11, 12). Thus, although part of the chitin is free, another portion is attached to the non-reducing end of {beta}(1–3)glucan (13). Some of the chitin is linked to {beta}(1–6)glucan, which in turn is attached to mannoprotein and to {beta}(1–3)glucan, again at the non-reducing end (12). Our hypothesis (10) was that the large amount of chitin at the ring would be predominantly linked to {beta}(1–3)glucan, thus competing with and preventing the attachment to the same site of the {beta}(1–6)glucan, followed by mannoprotein (14). In this way, growth of the cell wall at the neck would be prevented. This hypothesis also implies that the chitin dispersed through the wall should be mainly bound to {beta}(1–6)glucan.

We were interested in finding out whether our guess was correct because of the information this would yield on chitin ring function but also because, if the hypothesis turned out to be accurate, it would tell us that the cell is endowed with systems to attach chitin to different acceptors, depending on location. This feat would be all the more remarkable, because chitin is extremely insoluble in water and it is produced through the same enzyme, CSIII, at the two locations, mother-bud neck and lateral cell wall. It seemed that further investigation of this topic may lead to the understanding of fundamental mechanisms for processes of cellular localization. Since, however, no method was available to determine the differential attachment of a cell wall component to others, it was necessary to develop a new approach to cell wall analysis, involving dissolution of the entire wall. By coupling this methodology to the specific labeling of chitin at specific stages of the cell cycle, we were able to study the distribution of chitin linkages to other polysaccharides at different locations of the cell wall.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains—The yeast strains used were YPH499 (Mata ura3–52 lys2–801 ade2–101 trp1{Delta}63 his3{Delta}200 leu2{Delta}1) (15), 4250-3B (Mata his3 ura3 trp1 GAL1 cdc24-5) (from R. Wickner), JDY6-7A [pRS316: rho1E45I] (Mata ura3 ade2 leu2 his3 trp1 rho1::HIS3) (16), and ECY46-4-1B (Mata ura3–52 lys2–801 ade2–101 trp1{Delta}63 his3{Delta}200 leu2{Delta}1 chs3::LEU2) (17). All strains were grown in YEPGal/Raf (2% peptone, 1% yeast extract, 2% each of galactose and raffinose) at 30 °C, except for 4250-3B and JDY6-7A [pRS316:rho1E45I], which were grown at 25 °C.

Labeling of Cells and Preparation of Cell Walls—For random culture labeling, cells were grown to a density of ~3 x 107 cells/ml. To 20 ml of culture, 100 µl of [1-14C]glucosamine (10 µCi; 55 mCi/mmol) were added, and the culture was incubated at 30 °C or 25 °C (see preceding section) until the OD660 was doubled. The cells were collected by centrifugation, and cell walls were prepared by disruption with glass beads as described (16). The washed cell walls were suspended in 1 ml of water in screw-capped microcentrifuge tubes and 0.25 ml of 5 N NaOH was added. After heating in a dry bath for 1 h at 80 °C, the tubes were centrifuged for 5 min at 16,000 x g (the same conditions were used in subsequent centrifugations). The pellet was suspended in 0.5 ml of 1 M NaOH, and the heating at 80 °C was repeated. After centrifugation, the alkali-treated cell walls were washed twice with 1 ml of water and suspended in 1 ml of water containing 0.02% sodium azide. The walls were stored at 4 °C, because freezing resulted in aggregation.

For {beta}(1–3)glucanase digestion, the reaction mixture, in a cylindrical 2-ml microcentrifuge screw-cap tube, contained 150 µl of alkali-treated cell wall suspension, 7.5 µlof1 M potassium phosphate, pH 6.3, and 7.5 µl of Zymolyase 100T (Seikagaku America) at 10 mg/ml. For {beta}(1–6)glucanase, to 150 µl of cell wall suspension, 7.5 µl of 1 M sodium acetate, pH 5, and 7.5 µl of recombinant {beta}(1–6)glucanase (18), corresponding to ~0.2 µg of protein, were added. Both tubes were incubated for 3 h at 37 °C on a rotator. In Stage I of some of the two-stage experiments, where the percentage of chitin attached to {beta}(1–6)glucan was very high, an overnight incubation with {beta}(1–6)glucanase was necessary for complete liberation of the chitin. When a double incubation with {beta}(1–3) and {beta}(1–6)glucanase was needed, the cell walls were first incubated with {beta}(1–3)glucanase as described above, then the pH was brought to ~5 with 1 M acetic acid, and {beta}(1–6)glucanase was added, followed by a 3-h incubation at 37 °C.

The incubated mixtures as well as an untreated cell wall suspension were placed on ice for 10 min, followed by addition of 25 µl of a suspension of unlabeled alkali-treated cell walls from strain YPH499, containing 15 µmol/ml glucose equivalents, as measured with anthrone (19). The tubes were centrifuged for 5 min at 16,000 x g, and the supernatants were saved for measurement of solubilized radioactivity and, in the case of the {beta}(1–3)glucanase digestion, for further manipulation. The pellets were used for carboxymethylation, as outlined below.

Carboxymethylation—The method used for carboxymethylation was essentially that of Hirano (20), with some modifications to adapt it to the small amounts used here and to maximize recovery. To avoid losses, it was important to carry out the whole operation in a single tube.

The pellets from either the untreated walls or those digested with enzymes were suspended in 0.3 ml of 60% NaOH containing 0.2% SDS and placed on ice for 1 h, then stored at –20 °C overnight. After thawing, 200 µl of cold monochloroacetic acid (135 mg/ml in isopropanol) were added, followed by shaking in a Mini-BeadBeater (Biospec Products, Bartlesville, OK) for 20 s. A total of five such additions and agitations were made every 5 min, returning the tube to the ice bath each time. After a total time of 30 min on ice, the tubes were rotated at room temperature for 5 h, with an agitation in the Mini-BeadBeater every hour. The reaction mixture had the consistency of a slurry in the beginning, but liquefied after about 1 h of rotation. Tubes were centrifuged 5 min at 16,000 x g. A trilayered pellet formed, with a transparent layer in the middle. The supernatant was carefully withdrawn with a pipettor. The pellet was suspended in 1 ml of absolute ethanol by shaking in the Mini-BeadBeater. After centrifugation as above, the pellet, now compact, was washed twice in the same fashion with 1 ml of ethanol each time and finally dried by evaporation in a SpeedVac. The dried pellet was dissolved in 0.4 ml of water by first vortexing for 1 min, then shaking in the Mini-BeatBeater for 30 s, followed by heating at 80 °C for 5 min and new vortexing. The cooled tube was centrifuged as above. The supernatant was saved, and the pellet (usually invisible) was suspended in 0.4 ml of water.

By measuring the radioactivity of the two aqueous solutions, it was usually found that most of the counts were in the first fraction, but a variable amount, which could reach almost 50% of the total, was in the second fraction. Recentrifugation of the latter by itself or mixed with the first fraction did not bring down any radioactivity. Our interpretation of these results is that the large amount of salts present in the dried pellet prevents some of the high molecular weight material from dissolving in the first fraction. In fact, when both fractions were run separately in a column, the first fraction yielded a profile similar to that of Fig. 2A, whereas all the radioactivity of the second fraction was in the void volume peak. Furthermore, the radioactivity of the {beta}(1–3)glucanase digest, which corresponds to lower size material, was invariably found only in the first fraction. In general, both fractions were mixed before chromatography when there was a significant amount of radioactivity in the second fraction. The average recovery of radioactivity in 133 carboxymethylations was 83 ± 12.6%.



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  		FIG. 2.
Chromatography of different carboxymethylated fractions on a Sephacryl S-300 column. A, alkali-treated but undigested carboxymethylated cell walls of strain YPH499. V0, void volume, as determined with Dextran 2000. D70 and D40, positions of peaks of dextran 70 and dextran 40, respectively. The average molecular weight of each dextran is 1000-fold the corresponding number. B, water-insoluble fraction after digestion of cell walls with {beta}(1–3)glucanase (filled circles). Values were normalized as described under "Experimental Procedures." The open circles represent the subtraction of fractions 29–46 of A from the corresponding ones of B. C, same as described for B but after digestion of cell walls with {beta}(1–6)glucanase. D, filled circles, same as described for B and C but after sequential digestion with {beta}(1–3) and {beta}(1–6)glucanase. Continuous line is the same as that represented by the filled circles in B, although with a different scale.

 
Chromatography—Carboxymethylated material was applied to a Sephacryl S-300 column, 75 x 1 cm, and eluted with 50 mM Tris chloride, pH 7.5, containing 150 mM sodium chloride and 0.02% sodium azide. A flow rate of 1.35 ml/min was achieved by applying pressure with a peristaltic pump, and fractions of 1 ml were collected. Radioactivity was measured in a portion of each fraction. The column is very stable: more than 150 runs were done with the same column without change in flow rate or efficiency of separation.

For chromatography of the "small chitin," a Bio-Gel P-4 column (85 x 1 cm) was used. The supernatant of a centrifuged {beta}(1–3)glucanase/{beta}(1–6)glucanase digest, like that used in the Sephacryl column of Fig. 4B (see below), was applied to the Bio-Gel column. The eluting solution was 0.1 M acetic acid, and the column was run by gravity with some hydrostatic pressure, with a flow rate of ~0.17 ml/min. Fractions of 1 ml were collected.



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  		FIG. 4.
Chromatography on Sephacryl S-300 of water-soluble fraction after digestion of cell walls with {beta}(1–3)glucanase. A, without further treatment. B, supernatant after further digestion with {beta}(1–6)glucanase and centrifugation.

 
Calculations—Because the recoveries in each operation showed some variation, before making calculations it was necessary to normalize the results and also to account for the material solubilized in water and therefore missing after some of the enzymatic digestions. Thus, the radioactivity of all fractions in each chromatographic run (such as those shown in Fig. 2) was added up. From the sum of the cell wall column (in Fig. 2, column A) a percentage, corresponding to the amount of radioactivity solubilized in water, if any, by the pertinent enzyme, was subtracted. The result was divided by the total for the enzymatic digest to be normalized, and each of the fractions of the enzymatic digest was multiplied by the obtained ratio. These are the normalized curves with filled circles in Fig. 2, B and C. The values for the cell wall column, between the first fraction after the void volume peak (around fraction 30) and the last fraction with significant radioactivity (usually fraction 46), were subtracted from the corresponding ones of each corrected enzymatic digest curve, generating the curves with open circles in Fig. 2, B and C. The sum of the values of those curves represent the chitin liberated by {beta}(1–3)glucanase and {beta}(1–6)glucanase, respectively.

As explained under "Results," the value for large chitin attached to {beta}(1–3)glucan needed a correction because of the presence of some chitin-{beta}(1–6)glucan complex in the early portion of the peak resulting from {beta}(1–3)glucanase digestion (Fig. 2B). The correction was done by adding a double incubation with both {beta}(1–3) and {beta}(1–6)glucanase. The area between the profiles yielded by the double incubation and the single incubation with {beta}(1–3)glucanase (fractions 24–35 in Fig. 2D) represents the radioactivity to be subtracted from the open circle curve of Fig. 2B.

Reciprocally, this amount of radioactivity should be added to the void volume peak of Fig. 4A (see below), to calculate by an alternative method the amount of chitin bound to {beta}(1–6)glucan.

As mentioned under "Results," the amount of large chitin linked to {beta}(1–3)glucan may also be calculated by subtracting the sum of the fractions after the void volume (Fig. 2C, filled circle profile) from the sum of the corresponding fractions in the chromatography of the double incubation digest (curve with filled circles, Fig. 2D).

For the free chitin, the sum of fractions after the void volumen in the cell wall chromatography (Fig. 2A) was used. A small portion of the free chitin was still under the void volume peak and that was calculated either by extrapolation or as the sum of fractions 22–30 in the double incubation chromatography (Fig. 2D).

When all these corrections were applied, the sum of percentages came usually close to 100.

Two-stage Experiments—The isolation of unbudded cells was carried out essentially as described (16). However, the yield of single cells was lower when the yeast was grown with galactose as carbon source, probably because of changes in the density of the cells, and it was necessary to use larger amounts of culture. Six sucrose gradients (16) were used in each experiment, each one loaded with cells from an 80-ml culture containing about 5–5.5 x 107 cells/ml. In experiments with the rho1E45I mutant, sonication of the cell suspension in 0.75 M {alpha}-methyl-mannoside (16) was substituted with 30-s shaking in the Mini-BeadBeater to prevent lysis of the rather fragile cells.

In each case, two 20-ml cultures containing unbudded cells at a concentration of 1.5 x 107 cells/ml were prepared (in some instances the total volume was somewhat smaller because of a lower yield of unbudded cells). For the cdc24-5 mutant (strain 4250-3B) temperature shift experiment (Figs. 7A and 8A), 20 µCi of [1-14C]glucosamine was added to one of the flasks, and both flasks were incubated at 37 °C for 2 h. At that point, the flask containing the label was placed on ice for cell harvesting and cell wall preparation. The other flask was shifted to 25 °C, and 15 min later 20 µCi of [1-14C]glucosamine was added. Budding was monitored, and when it reached 80% of the cells (about 1 h incubation) this culture was also cooled on ice for further processing.



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  		FIG. 7.
Budding of single cells isolated in a sucrose density gradient. A, cells of cdc24-5 (strain 4250-3B) incubated at 25 °C (open circles)orat37 °C(filled circles). At the time indicated by the arrow, the 37 °C culture was shifted to 25 °C. The starting point for the 25 °C control is higher, because the cells were from a different gradient. B, cells of strain JDY6-7A [pRS316:rho1E45I] were incubated at 25 °C (open circles) or 37 °C (filled circles). At the time indicated with the arrow, the latter cells were transferred to 25 °C.

 



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  		FIG. 8.
Chitin links distribution in cdc24-5 (strain 4250-3B) under different conditions. A, two-stage experiment with temperature shift. Cross-hatched bars represent the values for the first stage, and solid bars represent the values for the second stage. B, random culture labeled at 25 °C.

 
For rho1E45I (strain JDY6-7A[pRS316:rho1E45I]; Figs. 7B and 9A) the conditions were as in the temperature shift experiment of cdc24-5, except that the incubations at both temperatures were somewhat longer in this case, because both the increase in cell size at 37 °C and the budding at 25 °C were slower with this strain.



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  		FIG. 9.
Distribution of chitin cross-links in strain JDY6-7A [pRS316:rho1E45I] under different conditions. A, two-stage temperature shift experiment. Designation of bars as in Fig. 8A. B, random culture labeled at 25 °C.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A New Strategy for the Study of Cross-links in the Fungal Cell Wall—Theoretically, one could evaluate how much chitin is free or linked to other polysaccharides by determining first the total amount and then how much is set free by digestion with specific hydrolases for each polysaccharide. However, chitin, whether free or linked to the cell wall, is insoluble in water, which prevents discrimination between different fractions. Therefore, we concluded that a successful procedure would have to involve solubilization of the cell wall. We devised a scheme including the following steps: 1) label specifically chitin in vivo with a radioactive precursor and prepare cell walls from the culture; 2) solubilize the cell wall in a suitable solvent; 3) fractionate the solution, either intact or after treatment with appropriate glucanases, on size-exclusion columns and compare the elution profiles. To ascertain the distribution of chitin at different locations, it was also necessary to find conditions in which either the chitin ring or the daughter cell wall would be specifically labeled.

For the first step, chitin labeling, we used [14C]glucosamine with galactose-raffinose as carbon source in a rich medium (21). Cell walls were prepared after breakage of the cells with glass beads and treated with alkali to eliminate mannoproteins, which contain two N-acetylglucosamine residues at the junction between the mannose polysaccharide and the protein. Some of the unlinked {beta}(1–3)glucan is also solubilized by alkali (22). The incorporation of radioactive material in cells varied among strains, from 3–5% of the total label in strain YPH499 to 15–16% in JDY6-7A[pRS316(rhoIE45I). In all cases, about one-third of the radioactivity was recovered in the alkali-treated cell walls. All of the radioactivity in the alkali-treated cell walls was incorporated into chitin, as indicated by its total solubilization upon incubation with Serratia marcescens chitinase (data not shown).

In early experiments, we were able to solubilize alkali-extracted cell walls in N,N-dimethylamide-lithium chloride, a solvent used for cellulose, chitin, and other polysaccharides (23, 24). However, many attempts to fractionate the material, before or after enzymatic treatment, on divinylbenzene copolymer gel (25) or other columns were unsuccessful. We finally opted for a different approach, i.e. a chemical modification of the cell walls to make them soluble in water, which would allow the use of conventional size-exclusion columns. The chosen procedure was carboxymethylation of the polysaccharides, which has been widely used to obtain water-soluble derivatives of cellulose (26) and chitin (20) derivatives. By use of Hirano's method, with some modifications to adapt it to the small amounts of available material, we were able to solubilize all of the radioactivity in water with a recovery usually better than 80% (see "Experimental Procedures").

A portion of the cell walls was left untreated, whereas other portions were digested with either endo-{beta}(1–3)glucanase (zymolyase) or recombinant endo-{beta}(1–6)glucanase. Then, all three preparations were carboxymethylated, and the products were subjected to size-exclusion chromatography on a Sephacryl S-300 column (Fig. 2).

At this point it is useful to mention what kind of products would be expected in each case (Fig. 1B). As mentioned above, three forms of chitin have been found in the cell wall: free chitin, chitin linked to {beta}(1–3)glucan, and chitin linked to {beta}(1–6)glucan, the latter being attached to both mannoprotein and {beta}(1–3)glucan. The complexes in which chitin is bound to other polysaccharides should be large, compared with the free chitin, for which an average size of about 100 monosaccharide residues, corresponding to a molecular weight of ~20,000, was estimated (27). By comparison, {beta}(1–6)glucan appears to be polydisperse with chains between 60 and 600 glucose residues (12), whereas a degree of polymerization of 1500 was assigned to {beta}(1–3)glucan (28). Therefore, the untreated cell wall should show some very large material (bound chitin) and some smaller one (free chitin). In fact, a large portion of the radioactivity emerged as a sharp peak at the void volume, whereas the remainder eluted later as a rather flat plateau, which we provisionally attributed to the free chitin (Fig. 2A). There was some concern that this plateau would be just a tail of the main peak, resulting from transient adsorption of the radioactive material to the column matrix. This possibility was eliminated when fractions from different parts of the eluate were pooled and run again on the same column (Fig. 3). Each pool eluted approximately in the original position, showing that the material in each fraction was different in size from the others. This also indicated that the putative free chitin is surprisingly polydisperse.



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  		FIG. 3.
Rechromatography of different fractions from Sephacryl S-300 column eluate. A, chromatography of carboxymethylated cell walls, similar to Fig. 2A. Fractions under segments 1, 2, and 3 were pooled and concentrated, followed by chromatography on the same column (B, C, and D, respectively). Note the different scales in C and D.

 
For the {beta}(1–3)glucanase digest, one would expect an increase in free chitin, liberated from its complex with the {beta}(1–3)-linked polysaccharide (Fig. 1B), and a correspondingly diminished void volume peak. The first prediction was borne out: a higher but equally wide peak appeared in the same elution area of the putative free chitin (Fig. 2B), strengthening the attribution of the plateau in Fig. 2A to free chitin. However, very little material was eluted in the void volume area. On the other hand, a substantial portion of the radioactivity, amounting to between 25 and 35%, depending on the strain used, was found in the water-soluble fraction after centrifugation of the {beta}(1–3)glucanase digest. The same amount of radioactivity was solubilized by a recombinant {beta}(1–3)glucanase ("Quantazyme," results not shown). This soluble fraction, subjected to chromatography on the same Sephacryl S-300 column, gave rise to two peaks, one at the void volume and the other of apparently small size material at the included volume (Fig. 4A). When the water-soluble fraction was incubated with {beta}(1–6)glucanase, part of the radioactivity sedimented upon centrifugation. Chromatography of the supernatant showed a total disappearance of the void volume peak, whereas the second peak remained intact (Fig. 4B). We infer that, after removal of {beta}(1–3)glucan by the glucanase, most or all of the chitin now bound only to {beta}(1–6)glucan formed a water-soluble complex with this polysaccharide (see "Discussion").

In conclusion, the disappearance of the void volume peak upon digestion with {beta}(1–3)glucanase is accounted for by the liberation of three forms of chitin: an insoluble fraction, later carboxymethylated (Fig. 2B), and two water-soluble fractions, one containing a large {beta}(1–6)glucan-chitin complex and the other a "small chitin" component (Fig. 4A; see also below).

Subtraction of the free chitin data (fractions 30–45 of Fig. 2A) from the corresponding ones of the {beta}(1–3)glucanase digest yields a curve for the chitin bound to {beta}(1–3)glucan and liberated by {beta}(1–3)glucanase (Fig. 2B, open circles).

The pattern after {beta}(1–6)glucanase digestion of the walls was as expected (Fig. 2C), i.e. some decrease in the void volume peak and a corresponding increase in the free chitin area.

From the three chromatographic runs (Fig. 2, A–C) it is possible to calculate the percentage of free chitin (sum of fractions 30–45 of Fig. 2A) and that of chitin bound to {beta}(1–3)glucan and {beta}(1–6)glucan (sum of the same fractions in Fig. 2, B and C, respectively, from which the free chitin of Fig. 2A had been subtracted). There remains some uncertainty, because a small portion of the free chitin may be hidden under the void volume peak. We tried to apply corrections for that amount (see "Experimental Procedures"). Note also that the small chitin solubilized by {beta}(1–3)glucanase (second peak in Fig. 4A) must be included in the calculations. When all the calculated values for chitin bound to the different components or free were added together, often the sum somewhat exceeded 100% (by 3–8%, depending on the strain), which suggested that some of the components had been partially counted more than once. A sequential digestion with both {beta}(1–3)- and {beta}(1–6)glucanase pointed at the source of the problem. When the elution profile was compared with that yielded by {beta}(1–3)glucanase alone, it could be seen that some radioactive material moved to the right, i.e. toward lower molecular weights after the double incubation (Fig. 2D). This was presumably some chitin attached to {beta}(1–6)glucan that did not become soluble in water upon {beta}(1–3)glucanase treatment and therefore was counted as of it were linked to {beta}(1–3)glucan (Fig. 2B). When the area between the two curves of Fig. 3D was subtracted from the calculated percentage of chitin attached to {beta}(1–3)glucan, the sum of all the values came to ~100%. The correction was small for strain YPH499 but somewhat larger for other strains.

The values obtained for two independent preparations of YPH499 cell walls are shown in Fig. 5, to illustrate the kind of reproducibility that may be expected. It should be noted that some values can be calculated in more than one way from different sets of data. Thus, the percentage of chitin linked to {beta}(1–6)glucan can be calculated by the subtraction method illustrated in Fig. 2C or from the first peak of the material solubilized by {beta}(1–3)glucanase (Fig. 4A) plus the correction afforded from the double incubation data (Fig. 2D). For the first preparation shown in Fig. 5, the values, calculated in the two different ways, are 9.9 and 10.9%, whereas for the second preparation they are 15.5 and 14%.



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  		FIG. 5.
Distribution of chitin in different cross-links in the cell wall of strain YPH499. For each kind of chitin, the results of two completely independent experiments are shown. For calculations, see "Experimental Procedures."

 
Similarly, the percentage of large chitin attached to {beta}(1–3)glucan may be calculated as shown in Fig. 2B (with the correction based in Fig. 2D) or by subtracting fractions 30 to 45 of the {beta}(1–6)glucanase digest (Fig. 2C) from the corresponding fractions of the {beta}(1–3)/{beta}(1–6)glucanase digestion profile (Fig. 2D). Here, the results for the first preparation are 40.8 and 41.6% and for the second 40.4 and 36.2. The internal consistency of the data inspires confidence in the general reliability of the results.

Short N-Acetylglucosamine Chains Are Attached to {beta}(1–3)Glucan—Of all the fractions isolated in this study, the only one that was never encountered before is the material of small molecular weight solubilized by {beta}(1–3)glucanase (second peak of Fig. 4A). This is understandable, because in previous work the {beta}(1–3)glucanase-soluble fraction was discarded (12, 13, 27). This material must contain N-acetylglucosamine groups linked to each other, because practically all of the radioactivity solubilized by {beta}(1–3)glucanase was retained by a wheat germ agglutinin-agarose column (92–93% retained in two experiments). The fraction was further analyzed by chromatography on Bio-Gel P-4. Here, a jagged profile consisting of several peaks was observed, corresponding in position to standards from diacetylchitobiose to hexaacetylchitohexaose, with some radioactivity moving even more slowly (Fig. 6A). It should be kept in mind that, because zymolyase has endo-{beta}(1–3)glucanase activity, preferentially cutting glucose oligosaccharides of 5 units, the compounds seen here probably consist of chains including both N-acetylglucosamine and glucose. Furthermore, in these columns each N-acetylglucosamine residue counts as two hexose residues in determining elution position (13, 29). Incubation of the fraction with S. marcescens purified chitinase led to the partial or total disappearance of most of the peaks, with the bulk of the radioactivity now emerging at the position of N-acetylglucosamine (Fig. 6B) instead of the expected diacetylchitobiose. This can be ascribed to some contaminant chitobiase in the chitinase preparation (30), because when unlabeled diacetylchitobiose was added to the reaction mixture to trap the radioactive intermediate, most of the radioactivity emerged at the position of the disaccharide standard (Fig. 6C). We conclude that the small chitin fraction consists of N-acetylglucosamine oligosaccharides linked to glucose, with a total length between about 3 and 7 total units.



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  		FIG. 6.
Chromatography of small chitin on a Bio-Gel P-4 column. A, the supernatant from {beta}(1–3)glucanase digestion of cell walls was further incubated with {beta}(1–6)glucanase and centrifuged, as described for Fig. 4B. The supernatant was applied to the Bio-Gel P-4 column. Standards are as follows (in all panels): 1, dextran 50; 2, hexaacetylchitohexaose; 3, tetraacetylchitotetraose; 4, laminarihexaose; 5, diacetylchitobiose; 6, laminaritetraose; 7, N-acetylglucosamine; 8, laminaribiose; 9, glucose. B, same material as in A but after digestion with S. marcescens chitinase (30). C, same material as in A but after digestion with chitinase in the presence of 10 mM diacetylchitobiose. D, material prepared as described for A but from strain JDY6-7A [pRS316:rho1E45I].

 
Chromatography of the small chitin fraction from a genetically unrelated strain, JDY6-7A[pRS316(rhoIE45I), yielded an almost identical profile (Fig. 6D).

Labeling of Chitin Either in the Neck Ring or Dispersed in the Cell Wall and Analysis of Its Linkages—Having developed a procedure for the determination of the proportion of chitin linked to different polysaccharides, we now needed to find conditions in which either the neck ring chitin or that dispersed in the cell wall would be labeled. Previous work has shown that incorporation of chitin in the cell wall of the bud takes place after septation has been executed (7) and continues during growth of the daughter cell until it has completed its maturation and is ready to bud (Fig. 1A, sequence d, e, and a). On the other hand, synthesis of the chitin ring is concomitant with bud emergence (Fig. 1A, cell b). Small unbudded cells (Fig. 1A, image e) can be isolated by centrifugation in sucrose gradients (16). In the presence of a reversible budding block, provided by a conditional mutation or by an inhibitor, these cells can be labeled in the cell wall with [14C]glucosamine. In another aliquot of the culture, treated in the same way but left unlabeled, the block may now be removed and labeling started, which should result in tagging of the chitin ring.

To try this scheme we first used cdc24-5, a temperature-sensitive mutant (strain 4250-3B). As the original cdc24-1 mutant (31), this strain is blocked at budding at the nonpermissive temperature of 37 °C. Accordingly, no increase in the percentage of budded cells was observed by incubating isolated unbudded cells at 37 °C (Fig. 7A). Once the culture was shifted to 26 °C, all the cells acquired buds over a period of about 1 h (Fig. 7A). Unbudded cells were labeled with [14C]glucosamine for 2 h at 37 °C, then harvested (Stage I; Fig. 1A, sequence e-a). Another portion of the cells went through the same treatment, but labeling was started after the culture was shifted to 25 °C, and the cells were harvested when about 80% of them presented buds (Stage II; Fig. 1A, sequence a-b). In both cases, cell walls were prepared and treated with alkali, followed by analysis of the chitin linkage distribution as described above (Fig. 8A). The most striking change was that of the large chitin attached to {beta}(1–3)glucan. Only 3% of the total was labeled in Stage I, a value that climbed to 47.5% in Stage II (Fig. 8A). Reciprocally, a large, although not as pronounced, decline was observed in the chitin linked to {beta}(1–6)glucan, from 55 to 11%. It should be mentioned that in these and subsequent experiments, when the amount of chitin bound to {beta}(1–6)glucan was very high, some radioactivity, usually not more than 5% of the total, was solubilized in water by incubation with {beta}(1–6)glucanase. Thus, in those cases some small chitin may be attached to {beta}(1–6)glucan as well. Because of the small amounts available, this fraction was not analyzed further and the corresponding values were added to those calculated for {beta}(1–6)-linked chitin.

Compared with those described above, lesser changes occurred in the proportion of free chitin and {beta}(1–3)glucan-linked small chitin. The results were in agreement with the notion that the bound chitin is mostly attached to {beta}(1–6)glucan in the lateral cell wall (Stage I) and to {beta}(1–3)glucan in the neck ring (Stage II), as suggested previously (10). The distribution of linkages in randomly labeled cells (Fig. 8B) is rather similar to that of Stage II, which is not surprising if one assumes that most of the chitin is in the neck area, as suggested from earlier results of our laboratory (21).

Although the percentage values yielded the expected pattern, the absolute numbers for incorporation were a reason of some concern. It has been observed before that blockage of the cell cycle in several temperature-sensitive mutants leads to deposition of chitin all over the cell wall with a substantial increase in chitin content (32, 33), which in the case of cdc24-1 reaches six times that measured at permissive temperature (33). In fact, the total incorporation in Stage I with strain 4240-3B was between three and seven times that of Stage II. Although, as mentioned above, the extra chitin appears to be dispersed all over the wall, like that formed normally in maturing cells, and the values we measured were in percentage rather than absolute numbers, we feared that behind the high incorporation there might be abnormal events that would compromise the results. Therefore, we looked for other ways to stop budding. We formerly reported that a temperature-sensitive mutant of the GTP-binding protein Rho1p, rho1E45I, is blocked at budding at 37 °C (16). In that case we did not observe an increase in chitin in the cell wall, as judged by Calcofluor staining. The arrest before budding at 37 °C is very effective and completely reversed by a shift to 25° (Fig. 7B). The experiment with strain JDY6-7A[pRS316(rhoIE45I) was carried out in the same way as that involving temperature shifts with the cdc24-5 strain. Despite the apparent lack of Calcofluor staining, the total incorporation was higher at 37 °C (Stage I) than in Stage II at 25 °C, although much less so than with cdc24-5 (incorporation in Stage I 75% higher than in Stage II, average of two experiments). On the other hand, the results for chitin distribution were similar to those of the cdc24-5 mutant (Fig. 9A). As in that case, the incorporation in large chitin attached to {beta}(1–3)glucan was very low in Stage I and increased sharply in Stage II. The reciprocal effect was observed for chitin linked to {beta}(1–6)glucan, whereas free chitin and small chitin bound to {beta}(1–3)glucan showed little change. Here too, the general pattern at Stage II resembled that of a random culture (Fig. 9B).

As shown above, for a random culture of wild type the free chitin peak only increased slightly after {beta}(1–6)glucanase digestion (Fig. 2, A and C). In contrast, the corresponding peak for Stage I of the two-stage experiments, where the incorporation in {beta}(1–6)glucan-linked chitin was greatly increased, was in some cases as high or even higher than the remaining void volume peak (data not shown), but the width of the peak and the position of the maximum were unchanged, indicating that the distribution of chitin chain lengths had not been perturbed.

The two-stage experiments were carried out twice, and the duplicates yielded similar results.

The Primary Septum Chitin Is Different from the CSIII-dependent Chitin—Whereas the above results describe the different forms and linkages of the chitin synthesized through CSIII, they give no information about the primary septum chitin (Fig. 1A, cell c), whose formation is catalyzed by CSII (7). This chitin was not detected in the single cell experiments of the preceding section, because the part of the cell cycle used did not include septation. It would contribute to the data for random cultures, but in a very minor way, because it accounts for only about 10% of the total chitin (7, 34). To look specifically at the primary septum chitin, we used a chs3{Delta} mutant. As expected, the incorporation of [14C]glucosamine in alkali-treated cell walls of the mutant was very small, only 6–7% of wild type. However, by doubling the amount of radioactive precursor and using larger portions of the isolated cell walls it was possible to obtain enough material for the determinations. The pattern shown by carboxymethylated cell walls was quite different from that of the wild type (compare Fig. 10 with Fig. 2A). Although the radioactive material started to emerge from the column at the void volume in both cases, the shoulder of the peak was much higher here. Treatment of the walls with {beta}(1–6)glucanase did not affect significantly the elution profile (data not shown), whereas incubation with {beta}(1–3)glucanase decreased somewhat the height of the void volume peak and caused a small but reproducible shift of the shoulder radioactivity toward the smaller sizes (Fig. 10), whereas no radioactivity was solubilized in water. Thus, most of the chitin of the primary septum appears to be free and larger in average size than that made through CSIII, although still quite polydisperse. None of it is attached to {beta}(1–6)glucan and only a small amount to {beta}(1–3)glucan (about 14%, average of two determinations).



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  		FIG. 10.
Chromatography on Sephacryl S-300 of carboxymethylated fractions from strain ECY46-4-1B (chs3{Delta}). To compensate for the low incorporation of [14C]glucosamine in this strain, twice as much label was used, and the amount of carboxymethylated material used for chromatography was doubled. Filled circles, alkali-treated cell walls. Open squares, {beta}(1–3)glucanase digest of cell walls. The elution profile after incubation with {beta}(1–6)glucanase was not significantly different from that of the untreated cell walls (data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cell Wall Solubilization to Study the Distribution of Cross-links—As mentioned above, it was imperative to find a way of solubilizing the cell wall before or after enzymatic digestion, to separate and quantify the linkages of its components to chitin. Carboxymethylation was an effective way of accomplishing this objective. The good yield obtained with this derivatization was essential for the quantitation of the results.

One quite surprising result was the polydisperse nature of the chitin, either free or attached to other polysaccharides. Unfortunately, the abnormal behavior of N-acetylglucosamine and its polymers on size-exclusion columns and the lack of convenient standards do not allow good estimates of the chain length. However, despite the belief that chitin synthesis is a processive reaction, there is clearly little control of the length at which chains are released from the synthases, whether the acceptor is water or a transferase that attaches chitin to another polysaccharide.

The very short, water-soluble, chitin chains released by {beta}(1–3)glucanase are probably at least 1 order of magnitude smaller than those remaining in the insoluble portion. Their proportion as percent of the total chitin varied relatively little between Stage I and Stage II of the single cell experiments. Furthermore, their size distribution, as measured by fractionation on a Bio-Gel P-4 column, was amazingly similar in two unrelated strains (Fig. 6). These findings suggest that the small chitin attached to {beta}(1–3)glucan, although synthesized through the action of the same CSIII, is functionally different from the remainder.

The solubility in water of chitin attached to {beta}(1–6)glucan after digestion of the {beta}(1–3)glucan linked to the complex was a surprising and, to our knowledge, unique finding. Presumably, interactions between the two polysaccharides prevent the chitin chains from coming together and forming the hydrogen bonds that determine their insolubility. In fact, elimination of the {beta}(1–6)glucan by enzymatic digestion led to precipitation of the chitin (Fig. 4).

It would be desirable to compare our results with those obtained by some other method, but this is not possible because the procedure presented here is the first that measures quantitatively the distribution of chitin in different linkages. However, it is reassuring that the results are internally consistent, since fairly close values were obtained for chitin attached to either {beta}(1–3) or {beta}(1–6)glucan from different sets of independent data (see "Results"). Furthermore, the present findings agree qualitatively with those of our previous studies of cell wall cross-links (12, 13). As predicted from the earlier results, part of the chitin is free, {beta}(1–3)glucanase liberates more chitin and a {beta}(1–6)glucan-chitin complex from the alkali-treated (protein-free) cell walls, whereas {beta}(1–6)glucanase also sets free some chitin, leaving a large {beta}(1–3)glucan-chitin complex undisturbed.

Chitin Is Attached to Different Polysaccharides Depending on Location in the Cell Wall—The isolation of unbudded cells, coupled to the presence of a reversible block in bud emergence, enabled us to label the cell wall chitin during cell maturation (Stage I). Subsequent removal of the block made it possible to label the chitin in the neck ring (Stage II). Analysis of the cell walls from both stages by our novel procedure showed that very little large chitin was bound to {beta}(1–3)glucan during cell wall growth, with a dramatic increase during chitin ring formation. The chitin attached to {beta}(1–6)glucan exhibited a reciprocal behavior, although relatively high levels of incorporation remained in Stage II. The latter result may be due to the fact that, although synthesis of the chitin ring and random deposition of chitin in the cell wall may seem distant events of the cell cycle when one starts with budding (Fig. 1A, sequence b–e), they are very close in the portion of the cycle that we had to use for our experiments (Fig. 1A, sequence e, a, and b). Thus, it is possible that addition of chitin to the cell wall does not stop immediately at budding but continues in the mother cell for some time. This may be especially true of cells coming out of a budding block, where chitin synthesis and attachment to {beta}(1–6)glucan had been greatly activated.

The high total incorporation in Stage I of the temperature shift experiment with the cdc24-5 mutant was of concern to us, because it might have caused some artifact. However, a strain from a different genetic background, with a different mutational block giving rise to a much smaller increase in chitin deposition, yielded very similar results. Together, the two experiments strongly suggest that the large bound chitin is mainly attached to {beta}(1–3)glucan in the neck ring and to {beta}(1–6)glucan in the lateral wall. This supports our hypothesis about the mechanism by which the ring chitin helps to maintain neck integrity. It also raises the question, how does the cell carry out this differential binding, depending on location but always with the same chitin synthase?

Due to its insolubility in water, it is unlikely that chitin would be made at one location and then transported to another one. Our finding that chitin attached to {beta}(1–6)glucan is soluble in water suggests that this complex might be a way of moving chitin around. However, our previous evidence showed that the order of arrival to the cell wall is {beta}(1–3)glucan, followed by {beta}(1–6)glucan, mannoprotein, and finally chitin (7, 14). Thus, by the time chitin is synthesized, no free {beta}(1–6)glucan would be present. It appears more probable that it is Chs3p and auxiliary proteins that are localized. It is already known that Chs3p is localized at the mother-bud neck through a bridge of two proteins, Chs4p and Bni4p, that attach it to the septin ring (35). This must be the Chs3p involved in synthesis of the chitin ring. Little is known about Chs3p in the remainder of the plasma membrane, except that there is a complex traffic pattern between membrane and vesicular compartments (see Roncero (36) for review). The results of Schorr et al. (37) with latrunculin suggest that Chs3p under certain conditions may be localized all over the plasma membrane. Thus, there is evidence in favor of the presence of Chs3p at both locations where chitin deposition occurs. What confers specificity to the transfer of the synthesized chitin is probably the presence, at each location, of an appropriate transglucosidase that transfers the chitin to {beta}(1–3)glucan or {beta}(1–6)glucan, respectively.

The Primary Septum Chitin Is Different—The primary septum appears to consist exclusively of chitin and has a structural function, because it must stand alone until supported by secondary septa. It is also synthesized through a special enzyme, CSII. Thus, it is not surprising that the septum chitin should be different from that found at other locations. Although this chitin is also polydisperse, its average size is considerably larger than that of the chitin dependent on CSIII, and most of it is free, except for some linked to {beta}(1–3)glucan. There is no experimental evidence on the function of the glucan cross-link, but an attractive hypothesis is that it may serve to attach the septum to the lateral cell wall, whereas the longer chains would be more appropriate for a structural role.

Future Prospects—The procedure presented here for the analysis of cross-links in the fungal cell wall opens a new window on cell wall structure, in particular on the different bonds by which chitin attaches to other components of the wall and their dynamics during the cell cycle. It will be interesting to extend the quantitative analysis of the different linkages to mutants in which cell wall structure and often chitin content are altered (38). There is also no apparent reason why this methodology could not be extended to other fungi. As mentioned above, it is shown here that the chitin cross-links vary depending on localization and phase of the cell cycle. Thus, our findings open the way to study how the cell remodels a structure at defined locations. Work on this subject, with the use of the new approach, is presently under way.


   FOOTNOTES
 
* This work was supported in part by Grant BIO2001-2048 of the Comisión Interministerial de Ciencia y Tecnología. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{diamondsuit} This article was selected as a paper of the week. Back

E. C. carried out part of this work during a sabbatical sponsored by the Spanish Ministerio de Educación, Cultura y Deporte (SAB 2000-0336), which was also part of a PHS Foreign Work/Study program. To whom correspondence should be addressed: NIDDK, National Institutes of Health, Bldg. 8, Rm. 403, Bethesda, MD 20854. Tel.: 301-496-1008; Fax: 301-496-9431; E-mail: enricoc{at}bdg10.niddk.nih.gov.

1 The abbreviations used are: CSIII, chitin synthase III; CSII, chitin synthase II. Back


   ACKNOWLEDGMENTS
 
We thank all the members of the Instituto de Microbiología Bioquímica for their unfailing support and help. We also thank S. González for help with the HPLC, R. Wickner for strains, and J. Arroyo and G. Ashwell for a critical reading of the manuscript



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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