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Journal of General Microbiology (1984), 130, 1419-1428.
Printed in Great Britain
1419
Solubilization and Analysis of Mannoprotein Molecules from The Cell Wall
of Saccharomyces cerevisiae
By E U L O G I O V A L E N T I N , E N R I Q U E H E R R E R O ,
F. I. J A V I E R PASTOR A N D R AFAE L S E N T A N D R E U *
Departamento de Microbiologia, Facultad de Farmacia, Universidad de Valencia,
Avda. Blasco Ibafiez 13, Valencia 10, Spain
(Received 23 September 1983)
Purified walls from Saccharomyces cerevisiae were treated chemically to release intrinsic
mannoproteins. Boiling in 2%SDS gave the best results, although treatment in 6 M-urea at room
temperature also released significant amounts of mannoprotein radioactivity. Triton X-100,
sodium deoxycholate and EDTA were poor solubilizers. Electrophoretic patterns of SDS- or
urea-released mannoproteins in SDS-acrylamide gels indicated a great heterogeneity of
molecular species, with more than 60 bands. Zymolyase, a glucan-digesting complex, released
about half of the mannoproteins, but these species showed an altered mobility on SDSacrylamide gels and had a lowered capacity for precipitation by ethanol. Action of the enzyme
on isolated walls was favoured by dithiothreitol, as is the case with whole cells, and repeated
treatments with SDS and Zymolyase released all of the mannoproteins from the wall.
Solubilizing treatments other than SDS had a differential effect on recently or formerly
incorporated mannoproteins in the wall. The results suggest an asymmetrical arrangement of
molecules in the envelope and point to dynamic changes inside the wall as it thickens as a result
of cell aging.
INTRODUCTION
Yeast cell walls are made up of glucans [mostly (1,3)-P-~-polymersof glucose], mannoproteins
and chitin (for recent reviews see Farkaz, 1979; Wessels & Sietsma, 1981; Cabib et al., 1982).
While the location of chitin in the yeast cell is mainly restricted to the bud scars, glucan and
mannoprotein molecules are present all around the cell surface. From microscopical studies on
regenerating protoplasts (NecaH, 1971; Elorza et al., 1983), it seems that apposition of newly
formed chitin in a fibrillar fashion on the protoplast surface and, consequently, the capacity to
retain mannoprotein molecules in the regenerating wall is not restricted to specific zones of the
yeast cell.
On the other hand, several studies based on electron microscopy, cytochemistry or chemical
treatment of walls (Cassone, 1973; Kopecka et al., 1974; Linnemans et al., 1977; Poulain et al.,
1978; Tronchin et al., 1981) have revealed a stratification of the wall with an internal array of
fibrils, probably glucan in nature, and a more external amorphous layer of mannoprotein
molecules. There is no clear separation of the two layers and, in fact, the amorphous material
seems to be prolonged into the glucan fibril skeleton. Using cytochemical studies, Linnemans et
al. (1977) reported two extracytoplasmic locations for acid phosphatase of Saccharomyces
cerevisiae: while part of the enzyme is in the more external layers of the wall, other molecules are
in the zone proximal to the plasma membrane, known as the periplasmic space. This is not the
case for another extracytoplasmicenzyme of S. cerevisiae, invertase, which is almost exclusively
located in the periplasmic space since it does not co-sediment with repeatedly washed cell walls
Abbreviation: PMSF, phenylmethylsulphonyl fluoride.
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E . V A L E N T I N A N D OTHERS
(Nurminen et al., 1970; our unpublished results). Several other yeast enzymes, all of them
mannoproteins such as acid phosphatase and invertase, are to be found, at least in part, in the
periplasmic space : asparaginase (Dunlop et al., 1978), a-galactosidase (Lazo et al., 1977) and
chitinase (Elango et al., 1982).
The existence of an external layer of mannoproteins in the wall has also been suggested by the
fact that the action of a phosphomannanase (Lampen, 1968)and agents which reduce disulphide
bridges (Kidby & Davies, 1970) on whole cells of S . cerevisiue were able to release retained
periplasmic invertase molecules. Besides this, the fact that the activity of lytic enzymes on
glucan for the purpose of obtaining protoplasts is facilitated by previous treatment of intact cells
with proteases or reducing agents (Vrganskh et ul., 1977; Santos et ul., 1981)points to an internal
location of glucan in the wall relative to a more external proteinaceous layer.
In filamentous fungi, hyphal elongation occurs exclusively at the apex (Gooday, 1971, 1977;
Bartnicki-Garcia & Lippman, 1972), but the contribution of some wall material seems to be
continuous as time progresses at any region of the mycelium, thus resulting in increased
thickness and higher stratification of walls in older regions of hyphae than at the zones near the
apex (Trinci & Coolinge, 1975; Hunsley & Kay, 1976). Chemical rearrangements also occur in
mycelial walls once the macromolecules have reached their external locations. Sietsma &
Wessels (1979) and Sonnenberg et al. (1982) reported that an initially water-soluble (1,3)-pglucan becomes water- and alkali-insoluble due to its covalent linkage with chitin. Although
similar morphological and/or chemical changes in the yeast cell wall have not been reported, it is
clear that yeasts also show a polarized growth, with wall enlargement taking place almost
exclusively at the bud region (Cabib, 1975).
With the aim of furthering our knowledge of the wall structure in S. cerevisiae, as well as the
possible effect of aging on the relative distribution of wall constituents, we studied the effect of
several chemical and enzymic treatments on the solubilization of wall mannoproteins, and made
electrophoretic analyses of the solubilized molecules.
METHODS
Strain, medium and growth conditions. The strain used was Saccharomyces cerevisiae X-2 180-1A (Yeast Genetic
Stock Center, University of California, USA). Cells were cultured at 28 "C in Yeast Nitrogen Base medium
without amino acids (Difco), supplemented with 2% glucose. Liquid cultures were grown overnight up to early
exponential phase, 0 . 3 4 5 mg (dry weight) ml- I , before beginning the experiments.
Cell labelling and wall purification. In order to label cells uniformly they were diluted in growth medium to
0.2 mg ml- and incubated for 5 h in the presence of [U-14C]protein hydrolysate [Om2 pCi ml- ; sp. act. 56 mCi
per matom carbon (2.07 GBq per matom carbon)].
For double labelling of cells they were grown with ~~- [ G - ~ H ] t h r e o n[5
i npCi
e ml-l; sp. act. 213 mCi mmol-l
(7.81 GBq mmol- l)] for 5 h and in the last 15 min a third of the continuously labelled culture was pulsed with [U14C]proteinhydrolysate (0.4 pCi ml-l). The pulse was stopped by making the culture up to 2 mM in sodium azide
and chilling simultaneously. Tritiated and doubly labelled cells were mixed and processed as described below,
except that 2 mM-sodium azide was present during washing previous to cell disruption.
To purify walls, cells were washed twice with distilled water at 0°C and resuspended in 1 mM-PMSF at a
concentration of about 1 mg cells per ml solution. Glass beads were added at 5 mg per mg cells and the cells were
disrupted by shaking for 15 min in a Vibrogen Cell Mill at 0 "C. Walls were sedimented (12OOg, 8 min) and
washed four times in 20 mM-triethanolamine buffer pH 4.0 containing 0.4 M-KCl, 1 mM-MgC1, and 1 mMPMSF, and then four times more in 1 mM-PMSF. Purity of the walls was also assessed by observation of the
preparations in a Zeiss I11 photomicroscope.
The hot alkali-insoluble fraction consisting ofglucan and chitin was obtained as described previously (Elorza &
Sentandreu, 1969).
Solubilization of wall mannoproteins. The action of several chemicals was measured by resuspending labelled
walls in 2 ml 10 mM-Tris/HCl buffer pH 7.0 plus 1 mM-PMSF and the respective chemical. After treatment (time
and temperature conditions as indicated in each experiment), walls were sedimented and the radioactivity
measured by taking 100 pl samples from supernatant fluids and treated walls once resuspended in 2 ml of water.
The samples were dried on Whatman GF/C filters. Mannoproteins in the remaining supernatant fluids were
precipitated in 75% (v/v) ethanol at 4 "C for 16 h. The precipitates were recovered by filtration and repeated
washing using cold 75% ethanol, also on Whatman GF/C filters. Measurements were made in toluene-based fluid
for dried samples in a Beckman scintillation spectrometer, model 7500.
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Yeast cell wall mannoproteins
For Zymolyase digestion, in most experiments labelled walls were initially resuspended in the pretreatment
solution (100 mM-Tris/HCl pH 8.0, containing 5 mM-DL-dithiothreitoland 5 mM-EDTA) for 30 min at 28 "C. The
walls were then centrifuged and resuspended in a solution of Zymolyase in 1 M-sorbitol containing 1 mM-PMSF.
Incubation was carried out at 28 "C; 4 mg Zymolyase was used to treat the walls obtained from 10 mg cells.
Sorbitol(1 M)was used to make conditions identical to those used when Zymolyase acts on whole cells (Pastor et
a/., 1982), although similar results were obtained with distilled water. At different incubation times, samples were
taken and non-solubilized, solubilized, and solubilized and ethanol-precipitated radioactivity were determined as
indicated above.
Solubilization of mannan by autoclaving was carried out as described by Peat et a/. (1961).
Action ofZymolyase on whole cells. Doubly labelled cells were treated with Zymolyase (in the same proportion as
for isolated walls) in an osmotically stabilized medium at 28 "C as described by Pastor et al. (1982), except that the
pretreatment step was omitted and treatment with the enzyme was carried out in the presence of 1 mM-PMSF. All
solutions contained sodium azide at 2 m~ (final concentration).
SDS-acrylamide electrophoresis. Samples (80 pg) of protein were run in 11% (w/v) acrylamide slab gels. Details
of the procedure for running and staining of gels were described by Herrero et al. (1982).
Chromatographyon Sephadex G-ZOO. Samples (1 mg) of protein resuspended in 0.5 ml20 mM-Tris/HCl buffer
pH 7.0, containing 100 mM-KCl, 1 mM-EDTA and 0.2% N-lauroylsarcosine, were loaded on a Sephadex G-100
column (1.6 x 50 cm). In some experiments the buffer also contained 1 mM-dithiothreitol. The column was
developed with the above buffer at a flow rate of 4.5 ml h-I and 0.5 ml fractions were collected.
Chemicals. DL-Dithiothreitol, EDTA, urea, N-lauroylsarcosine and PMSF were from Sigma. Triton X-100,
sodium deoxycholate, toluene, PPO and POPOP were from Merck. SDS and other electrophoresis reagents were
purchased from Bio-Rad. Sephadex G-100 was purchased from Pharmacia. Zymolyase 5000 was from Kirin
Breweries Co., Takasaki, Japan. Radioactive products were purchased from Amersham.
a
5
RESULTS
Solubilization of wall mannoproteinsfrom S . cerevisiae
Walls uniformly labelled in the peptide moiety of mannoprotein molecules were obtained by
growing cells in the presence of a 14C-labelledmixture of amino acids, followed by mechanical
disruption of the cells and repeated washing of the walls as described in Methods. Under these
labelling conditions less than 8% of the incorporated radioactivity remained in the hot alkaliinsoluble wall material, that is, in glucan and chitin. Therefore, wall radioactivity corresponded
essentially to the mannoprotein fraction.
Different treatments were carried out on the purified walls in order to test the optimal
conditions for solubilization of mannoproteins (Table 1). Boiling in 2% SDS proved to be the
best, with a significant recovery of mannoproteins (almost half of the previously released
radioactivity) after ethanol precipitation. Treatment in an autoclave at 130 "Cfor 3 h released
most of the radioactivity but it remained ethanol non-precipitable. Denaturing agents such as
urea also released a significant amount of mannoprotein from the walls. On the other hand,
sodium deoxycholate or the non-ionic detergent Triton X-100, as well as EDTA, had only a
minor effect.
Table 1. Effect of different treatments on the solubilization of t4C-labelled wall mannoproteins of
S. cerevisiae
Results are expressed with respect to total radioactivity in purified walls before treatment. Where not
indicated, treatment was carried out at room temperature for 30 min.
Treatment
2% Triton X-100
1% Sodium deoxycholate
5 mM-EDTA
6 M-Urea
2% SDS
2% SDS (5 min, 100°C)
Tris buffer (5 min, 100 "C)
Autoclave (3 h, 130 "C)
C.p.m. solubilized
C.p.m. solubilized
and ethanol-precipitated
(%>
(%)
5.2
1.6
3.6
1.7
26.6
27.9
39.3
8.7
7.4
64.2
59.8
84.8
11.8
82.3
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5.6
3.7
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E . V A L E N T I N A N D OTHERS
60
50
t (4
n
40
.-x
.$ 30
Y
0
g
d
20
l
0
k
40
=
80
120
160
Time (min)
Fig. 1 . (a)Effect of Zymolyase on release of mannoprotein from purified walls. 14C-labelledwalls from
7.5 mg cells were treated with Zymolyase at 28 "C. Results are expressed as the percentage of released
c.p.m.relative to total radioactivity in the walls. @, Total c.p.m. released from dithiothreitol-pretreated
walls; I,
total c.p.m. released from non-pretreated walls; 0 ,ethanol-precipitablec.p.m. released from
pretreated walls; 0 , ethanol-precipitable c.p.m. released from non-pretreated walls. (b) Effect of
Zymolyase on release of mannoprotein from the insoluble residue resulting after treatment of purified
walls in 2% SDS at 100 "C for 5 min. The amount of radioactivity in this residue was given the value
100%. @, Total c.p.m. released; 0 , c.p.m. released and ethanol-precipitable.
Zymolyase is a lytic enzyme complex with activity against yeast walls, which is used for the
preparation of protoplasts from whole cells (Kitamura & Yamamoto, 1972). It contains mainly
(1,3)-P-glucanase activity. We checked the possibility that digestion of glucan in purified walls
by Zymolyase might release mannoproteins into the medium. When walls labelled with a
peptide precursor were pretreated with the reducing agent dithiothreitol and then incubated
with the enzyme about 50% of the radioactivity was released in 160 min (Fig. 1a). In control
experiments, dithiothreitol alone was shown not to release wall mannoproteins, and Zymolyase
at the concentration used gave no significant protein bands in electrophoresis after ethanol
treatment (data not shown). In contrast, when the pretreatment step was omitted, only 10% of
the radioactivity was released by Zymolyase. In contrast to the SDS- or urea-released
radioactivity, only about 10% of the Zymolyase-released radioactivity was ethanol-precipitable.
In our previous work (Pastor et al., 1982), wall purification involved treatment of the walls
with sodium deoxycholate. This step was omitted in the above experiments. However, when
deoxycholate-treated cells were used instead, pretreatment was observed not to be so crucial for
the release of radioactivity by Zymolyase: pretreated walls released up to 30% of the
radioactivity (data not shown in detail). This points to a partially alternative role of sodium
deoxycholate instead of dithiothreitol in favouring the action of Zymolyase, although the former
agent does not itself release mannoproteins.
Cumulative eflect of several treatments on the release of mannoproteinsfrom the walls
The action of Zymolyase on the wall mannoproteins not solubilized by boiling in SDS was
faster than that on walls not treated with the detergent (Fig. 1b). Up to 60% of the residual
radioactivity was released by the enzyme complex in about 1 h. Half of this radioactivity was
ethanol-precipitable, a higher proportion than from SDS-untreated walls (Fig. 1a). When
successive treatments with SDS and Zymolyase were applied, almost 98 % of the radioactivity
was released. The remaining insoluble radioactivity was not solubilized by hot acid (3 M-HCl,
3 h), and probably corresponds to residually labelled chitin.
Solubilization of newly incorporated and older mannoprotein molecules from the walls
To see whether Zymolyase discriminates between recently incorporated or older mannoprotein molecules in the walls, a double-labelling experiment was done in which cells were
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Yeast cell wall mannoproteins
Table 2. Release of doubly labelled wall mannoproteins by Zymolyase
Doubly labelled walls were obtained from cells continuously labelled for 5 h with [’Hlthreonine and
then pulsed for 15 min with [14C]proteinhydrolysate before cell breakage. Purified walls were divided
into two samples: one was pretreated with dithiothreitol while the other remained untreated. The ratio
14C: ’H at time 0, taken as unity, is that of purified walls at the beginning of the experiment. Those at
later times correspond to solubilized mannoproteins.
Duration of
Zymolyase
digestion (min)
’H (c.p.m.)
14C (c.p.m.)
14C:’H
3H (c.p.m.)
14C (c.p.m.)
14C:’H
0
30
60
90
120
150
4295
1600
1969
2383
2229
2387
2503
166
234
266
266
259
1 -00
0.18
0.20
0.19
0.20
0.18
4947
2758
3220
3429
3452
3699
2869
399
486
500
539
564
1 -00
0.27
0.28
0.27
0.29
0.28
Non-pretreated walls
I
A
Pretreated walls
I
f
A
3
Time (min)
Fig. 2. Effect of Zymolyase on doubly labelled whole cells. Cells were continuously labelled for 5 h with
[3H]threonine and pulsed for 15 rnin with [14C]protein hydrolysate immediately (a)or 90 rnin (m)
before being placed in the presence of Zymolyase in an osmotically stabilized medium. In the latter
case, incorporation of the 14C isotope was stopped by addition of an excess of a mixture of unlabelled
amino acids (Pastor et al., 1982). The walls corresponding to 15 mg cells were used in each digestion.
The figure shows the relative ratio 14C :3H in solubilized mannoproteins in samples taken at different
times of enzyme digestion. The 14C:3H ratio in purified non-digested walls obtained from the same
doubly labelled cells was taken as unity.
continuously grown for about two generations in the presence of a 3H-labelled precursor and
then given a short pulse (for about one-tenth of the generation time) of a 14C-labelledprecursor
just before cell disruption and wall purification. The results showed an initially preferential
release of older molecules from the walls, since the ratio 14C:3H in solubilized mannoproteins
decreased during the first 30 rnin of Zymolyase treatment (Table 2). However, the enrichment in
older material in the released fraction was higher in non-pretreated walls than in dithiothreitolpretreated ones, since the 14C:3Hratio decreased to a larger extent in the former case.
Action of Zymolyase was also measured on doubly labelled whole cells. In order to lower the
rate of protoplast formation, and consequently the release of periplasmic molecules, the cells
were not pretreated with dithiothreitol. When cells were placed in the presence of the digestive
complex immediately after the pulse, similar results were obtained as with isolated walls, that is,
a preferential release of the older mannoprotein molecules (Fig. 2). However, when cell
treatment was delayed for up to 90 min after the end of the pulse no preferential release of 3Hlabelled molecules over l4 C-labelled molecules was observed, indicating that a rearrangement
of pulsed molecules might have occurred during the chase period.
Similar experiments on purified walls were carried out using some of the solubilizing
conditions tested in Table 1. Poorly solubilizing treatments (sodium deoxycholate and EDTA)
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E . VALENTIN A N D OTHERS
Table 3 . Solubilization of doubly labelled walls by different chemical agents
Results are expressed as 14C:5Hratios in the solubilized materials and the remaining pellets, made
relative to the ratio I4C:3Hin the original untreated walls, which is taken as unity. Conditions of
treatment were the same as those in Table 1 for identical agents.
Relative 14C:3Hratio in:
r
Solubilizing agent
Sodium deoxycholate
EDTA
Urea
SDS (100 "C, 5 min)
>
Supernatant
Pellet
0.59
1-12
1.15
1-27
0.97
0.44
043
0-96
.
I
,,
\
I
Fig. 3. Electrophoretic patterns of wall mannoproteins in SDS-acrylamide gels. (a) Zymolyasesolubilized and ethanol-precipitated molecules ; (b) mannoproteins solubilized by boiling in SDS and
ethanol-precipitated; (c) SDS-solubilized mannoproteins before ethanol precipitation; (d) ureasolubilized mannoproteins after ethanol precipitation. The relative positions of standard proteins of
several sizes (expressed in kDal) are also indicated.
and also urea, preferentially released molecules that had been in the wall for some time, while
with SDS boiling the release was practically non-specific (Table 3). The latter is consistent with
the extensive action of SDS on wall mannoproteins.
Pattern of solubilized wall mannoproteins in SDS-acrylamide gels
Mannoproteins liberated from cell walls by treatment with Zymolyase (Fig. 3a), SDS (Fig. 3 b)
and urea (Fig. 3 d ) were precipitated with ethanol and run in 11% acrylamide gels. Zymolyasetreated samples gave a different pattern from those treated with urea or SDS. With Zymolyase,
major bands of 43, 40, 29, 28 and 27 kDal appeared. SDS- and urea-released mannoproteins
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Yeast cell wall mannoproteins
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Fig. 4. Chromatography of wall mannoproteins in Sephadex G-100.
Mannoproteins were solubilized
by boiling for 5 min in 2% SDS in 10 m-Tris/HCl buffer pH 7.0 with (a) or without (b) 5 mMdithiothreitol. They were then run in a column of Sephadex G-100 with 20 mM-Tris/HCl buffer pH 7.0
containing 100 mM-KC1, 1 mM-EDTA and 0.2% N-lauroylsarcosineas eluting buffer. In (a) the buffer
also contained 1 mM-dithiothreitol. Successive eluted fractions were then treated with mercaptoethanol
and analysed by SDS-acrylamide gel electrophoresis (Herrero et al., 1982). The lanes correspond to
fractions eluted at the following volumes: 1,31a 7 ml (corresponding to the void volume of the column);
2,33.3 ml; 3,34.9 ml; 4,36.5 ml;5 , 38.1 ml; 6,39.7 ml; 7,41.3 ml; 8,42-9ml. The positions of several
protein standards of known size (expressed in kDal) are also indicated.
gave similar patterns to each other, with major bands of 49,45,43,33,29 and 19.5 kDal. Ethanol
precipitation did not seem to discriminate between classes of molecules, since the
electrophoretic patterns of SDS-released mannoproteins before (Fig. 3c) and after (Fig. 3b)
precipitation were identical. Similar results were obtained with urea (data not shown).
Linkage between individual wall mannoproteins by disulphide bridges
To analyse the importance of disulphide bridges in the linkage between individual
mannoprotein molecules from the walls, these molecules were solubilized by treatment at 100 "C
in 2% SDS in the presence or absence of the reducing agent dithiothreitol (5 mM). The
solubilizedmolecules, after ethanol precipitation, were run in a Sephadex G-100 column with an
elution buffer with or without dithiothreitol. The eluted fractions were next analysed by
conventional SDS-acrylamide gel electrophoresis under reducing conditions (the samplesolubilizing buffer contained mercaptoethanol in addition to SDS).
A different electrophoretic pattern was obtained for cell wall mannoproteins prepared and
chromatographed in the presence (Fig. 4a) or absence (Fig. 4b) of dithiothreitol. Under
reducing conditions only high molecular weight species eluted with the initial fractions, whereas
when dithiothreitol was absent from the solubilizing steps low molecular weight species also
appeared in the electrophoretic patterns of the initially eluted fractions. These low molecular
weight species might thus be part of larger complexes present during the chromatographic
filtration. Quantification of the amount of mannoprotein appearing in lane 1 of Fig. 4(a)
(dithiothreitol present), and comparison with that in lane 1 of Fig. 4(b) (dithiothreitol absent)
indicated that about 30% of total wall mannoprotein may be interlinked by disulphide bonds,
without specificity for individual molecular species.
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E. V A L E N T I N A N D OTHERS
DISCUSSION
Although yeast cell walls are asymmetrical, there must be an interaction between the several
layers and, in particular, between the glucan microfibrils and the mannoproteins that form the
amorphous matrix of the wall. In regenerating protoplasts the synthesis of glucan begins
immediately after resuspension of the protoplasts in the regenerating medium, although at a
lower rate than chitin synthesis. The newly synthesized glucan and chitin form a network of
microfibrils (Villanueva, 1966; NecaH, 1971; Farkai & Svoboda, 1980); however, mannoproteins which are synthesized in these regenerating protoplasts are still excreted into the medium
over a considerable period of time, probably because the density of the fibrils is still not
sufficient to retain the mannoprotein molecules. This is consistent with the release of labelled
mannoproteins from isolated walls as a consequence of glucan digestion by Zymolyase.
However, the fact that only about 50% of the radioactivity is solubilized in these conditions
indicates that only part of the mannoprotein in the yeast wall interacts with glucan fibrils. In
addition, the mannoproteins that interact with the wall glucan seem to be the older molecules
present.
In the interactions involving mannoproteins, disulphide bonds must be important in
interlinking individual species. On the other hand, no specific molecules seem to be involved in
these interactions. Hydrogen bonds also must play an important role in the structure of the wall,
since powerful denaturing agents such as urea release a high proportion of molecules.
Nevertheless, some ionic interactions must also exist in order to explain why chelating agents
like EDTA release a minor but significant amount of mannoprotein. Cations might be acting to
stabilize negative charges, involving either the phosphate groups present in the outer core of the
mannan chains (Ballou, 1976) or the peptide moieties of the molecules. It should be noted that
the solubilizing conditions tested preserved the integrity of covalent linkages involving the
peptide moieties.
Purified walls, essentially free of contamination, were obtained by shaking cells with glass
beads followed by washing in salt solutions and, in some experiments, sodium deoxycholate
treatment (Pastor et al., 1982). They must still retain the asymmetry observed in uivo, since
glucan is protected from Zymolyase digestion in such a way that previous boiling in SDS or
pretreatment with dithiothreitol or even sodium deoxycholate facilitates the action of the
enzyme complex. Significantly, repeated SDS and Zymolyase treatments released practically all
the mannoproteins from the cell wall. On intact cells, Zymolyase is able to act only from the
external side of the wall, so it must cross the outer mannoprotein layer to gain access to glucan.
This seems to be the reason why many strains of S. cerevisiae are not transformed into
protoplasts by the action of Zymolyase or other glucan-lytic enzymes unless the cells are
pretreated with thiol reagents such as dithiothreitol or mercaptoethanol (Kidby & Davies, 1970;
Vrganskh et al., 1977; Herrero et al., 1982; Santos et al., 1982). In some strains, however, the
requirement is not so absolute and protoplasts may be obtained without the previous action of
proteases or thiol agents (our unpublished results), pointing to a heterogeneity of cell wall
morphology andfor chemistry among strains of S. cerevisiae.
Mannoproteins from S. cereuisiaewalls are rather heterogeneous as seen in the band pattern in
SDS-acrylamide gels, where about 60 bands could be detected from walls treated with urea or
SDS. This heterogeneity may be due either to differences in the peptide moieties of the several
species of molecules, or to heterogeneity in the length of polysaccharide chains or in the amount
of phosphodiester linkages in the mannan outer chain. Since SDS releases most of the
radioactivity in wall mannoproteins, the corresponding electrophoretic pattern must reflect the
composition of the wall. Although urea solubilizes less mannoprotein than SDS, the fact that
electrophoretic patterns are very similar indicates that no mannoprotein species remain
selectively insolubilized by urea. This makes the molecules released by this agent a significant
part of the population in the whole wall and, since urea may be reversibly eliminated, in most
cases its use may be a useful way to obtain mannoproteins for further studies.
The electrophoretic patterns of Zymolyase-released mannoproteins differ significantly from
those of SDS- or urea-released mannoproteins. We have previously reported (Pastor et al., 1982)
that under the conditions used for Zymolyase digestion, this enzyme complex does not decrease
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Yeast cell wall mannoproteins
1427
the amount of trichloroacetic acid-precipitable counts from labelled peptides, but this does not
exclude the possible existence of limited proteinase activity in the complex or alternatively of
mannanase or phosphodiesterase activity. In any of these cases, the action of Zymolyase would
alter the mobility of molecules in the acrylamide gels. Extensive alteration of the polysaccharide
moieties could explain the decreased capacity for precipitation with ethanol of Zymolyasereleased proteins.
Constituent mannoproteins of the yeast wall are synthesized and exported to the
extracytoplasmic space by the same pathways as in other eukaryotic cells (Novick et al., 1981;
Sentandreu et al., 1981; Sabatini et al., 1982). Once incorporated into the wall, their fate was
followed by double-labelling experiments. A drastic agent such as SDS scarcely discriminates
between recently incorporated and older molecules in their release. However, Zymolyase, when
acting both on the glucan of isolated walls and on whole cells, is more active in releasing
previously incorporated molecules, an indication that the glucan network is probably
interacting with these mannoprotein molecules. When comparing the action of Zymolyase on
pretreated and non-pretreated isolated walls, enrichment of the solubilized fraction in old
molecules is higher in the latter case, i.e. newly incorporated mannoproteins seem to be less
accessible to Zymolyase from the cytoplasmic side of the wall. Pretreatment may uncover new
molecules present at more external locations. These data are compatible with a dynamic model
of yeast wall growth in which, once the interactions between glucan polymers and
mannoproteins become saturated, new mannoprotein molecules would accumulate at external
positions, leading to wall thickening. These outer molecules might interact with each other by
both hydrogen and disulphide bonds.
This work was partially supported by grant no. 120-82 from the Comisibn Asesora de Investigacibn Cientifica y
Tecnica. F. I. J. P. received a doctoral grant from INAPE (Spanish Ministry of Education and Science).
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