Journal of General Microbiologj* (1993). 139, 2185-2195.
Priiiied in Great Britain
21 85
Ultrastructure of proteinase-secreting cells of Candida albicans studied by
alkaline bismuth staining and immunocytochemistry
TOMOHIRO
AKASHI,* MICHIOHOMMA,
TOSHIO
KANBEand KENJITANAKA
Lahorutory of Medical Mycology, Research hstiiute ,for Disease Mechanism and Control, N u p y a Unhersity Schuoi
Show-ku, Nagoya 466, Japan
of Medicirze,
(Received 22 January 1993; reziised 7 March 1993; accepted 17 March 1993)
The ultrastructure of Cundida ulbicans cells induced to secrete extracellular proteinase (EPR) has been studied.
Electron microscopy employing alkaline bismuth staining, a method which stains polysaccharides, clearly revealed
Golgi-like bodies and secretory vesicles in C. albicans cells. After EPR induction, there was no apparent increase
in the number of these structures. Instead, many flocculent granules appeared at the periphery of induced cells. The
granules were similar to secretory vesicles in size, but were more irregular in shape. Similar granules were observed
in non-induced cells, though less frequently than in induced cells. Brefeldin A, a specific inhibitor of membrane
transport in the secretory pathway, caused the accumulation of EPR and Golgi-like bodies in EPR-induced cells,
but did not affect the accumulation of the granules, These results suggest that the granules are unrelated to EPR
secretion. Electron microscope immunocytochemistry with affinity-purified anti-EPR antibodies showed that the
granules in EPR-induced cells were recognized by the antibodies. This recognition was completely inhibited by the
presence of glycogen, suggesting that antibodies cross-react with glycogen-like polysaccharides in the granules.
Although the location of EPR within the cells remains unclear, the results suggest that EPR might be secreted via
the constitutive secretory pathway, and that EYR is glycosylated to give a structure with some similarity to
glycogen.
Introduction
Candidu albicclns is an opportunistic pathogen and one of
the medically important yeasts. During tissue invasion it
secretes an acid proteinase (extracellular proteinase ;
EPR), and there have been many reports discussing the
relationship between EPR and the virulence of this
fungus (Cutler, 1991; Odds, 1488). Although the role of
EPR in pathogenesis is controversial, several lines of
evidence have indicated that EPR is one of the factors
which contributes to the pathogenicity of C. albicans
(Shimizu et a/.,1987).
EPR is a 43 kDa aspartyl acid proteinase (Hattori
et al., 1984; Remold et ul., 1968; Ruchel, 1981 ; Shimizu
e t al., 1987), and is induced in uitro when C. albicans is
cultured in media containing protein as sole nitrogen
source. Many reports have shown that EPR induction ill
vitro is affected by various physiological conditions
*Author for correspondence.
Abbreciurions: EM, electron microscopy ; EPR, extracellular proteinase; ER, endoplasmic reticulum.
(Odds, 1988; Homma et d.,
1993). However, few studies
have investigated the intracellular events and
modifications following EPR induction or the mechanism of EPR induction in uitrn.
Homma tlt al. (1992) showed, using cell fractionation
experiments, that intracellular EPR co-sediments with a
membrane fraction, suggesting that EPR might be
enveloped by some membrane within cells and secreted
via the membrane traffic system through the endoplasmic
reticulum (ER), Golgi bodies. and secretory vesicles. To
clarify intracellular aspects of EPR secretion, the
ultrastructural relationship between EPR secretion and
organelles of the membrane traffic system needs to be
determined. Hornma et ul, (1992) reported that the
induction of EPR caused an intracellular accumulation
of granules detected by electron microscopy (EM). In
this paper, we have tried to clarify, using cytochemical
and immunochemical EM, whether the granules are
involved in EPR secretion. We report that the granules
appearing in EPR-secreting cells are ones containing
glycogen-like polysaccharides, which are independent of
EPR secretion. Furthermore, we show that induction of
0001-8107 0 1993 SGM
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21 86
T. Akashi and others
EPR does not appear to change the structure of the
Golgi bodies or secretory vesicles.
Methods
Media. YPD medium contained 10g yeast extract (Wako Pure
Chemicals), I0 g polypeptone (Wako Pure Chemicals) and 20 g glucose
per litre of distilled water. YNB medium contained 1.7 g yeast nitrogen
base without amino acids and ammonium sulphate (Difco) per litre of
distilled water. EPR induction medium was made by adding 2 (wjv)
glucose and 0-2% (wjv) bovine serum albumin to YNB medium. EPR
suppression medium contained 0.1 M-ammonium tartrate in the EPR
induction medium.
Sfrain. The strain of Crmdida albicans used in this study was C9
(Kwon-Chung ef al., 1985). Cells were cultured in YPD medium with
shaking overnight at 30 "C before experiments.
Induction qf E P R secretion. Induction of EPR secretion was
performed according to the method of Hornma et a/. (1992). Cells were
grown to exponential phase (OD,,, of about 1.5) in YPD broth
containing 1.1 YO(w/v) casamino acids (Difco) with reciprocal shaking
(100 strokes min-lj at 37 'C, collected by centrifugation (lOOOg,
5min), resuspended in EPR induction medium or EPR suppression
medium, and incubated with reciprocal shaking (100 strokes min-') at
37 "C. At various times the cells were sedimentcd by centrifugation
(lOOOg, 5 min), and prepared for EM, SDS-PAGE and immunoblotting.
Prepparation of samples for E M . The freeze substitution method for
EM (Tanaka & Kanbe, 1986) was used with minor modifications. Cells
were frozen rapidly in liquid propane cooled with liquid nitrogen in a
Reichert KFXO apparatus. The frozen cells were transferred to absolute
acetone or 2 YO(w/v) OsO, in absolute acetone, whch was pre-cooled
to - 80 "C,and substituted for 2 d at - 80 "C.After warming gradually
to room temperature, the cells were embedded in Epon 812. Thin
sections were cut using a Reichert OmU4 ultramicrotome and a
diamond knife.
Sections were stained with uranyI acetate and lead citrate, or with
bismuth nitrate according to the method of Shinji et al. (1975) and
viewed with a JEOL lOOSX transmission electron microscope at an
accelerating voltage of SO kV.
Electron rnicroscupe immunocytuchemisfr~.
For immunocytochemistry we used samples substituted in acetone only, as described above.
Sections on nickel grids were treated with 1 YO(v/v) H,O, for I5 min.
After washing with distilled water, the grids were treated in phosphatebuffered saline (PBS ; 8 m M potassium/sodium phosphate bufl'er,
150 mM-sodium chloride, pH 6.8) containing 1 % (w/v> bovine serum
albumin (BSA) for 30 min at 37 "C, and then in PBS-BSA with the
primary antibodies for 2 h at 37 *C. The primary antibodies were
affinity-purified rabbit polyclonal anti-EPR antibodies, preimrnune
serum, or commercially available rabbit IgG (Sigma). We used affinitypurified anti-EPR antibodies diluted 50 or 100 times with PBS-BSA,
which is comparable to the origmal serum diluted 200 or 400 times with
PBS-BSA in the reaction with EPR on nitrocellulose membranes.
Preirnmune serum was diluted 5WOO times with PBS-BSA. Rabbit
IgG was used at the concentration between 5 pg ml-' and 5 mg rn-'.
After washing with PBS-BSA, the grids were treated with 5 or 10 nm
gold particle-labelled goat anti-rabbit IgG (Amersham), diluted 20
times with PBS-BSA, for 1 h at 37 "C. In each step, the grids were
floated on drops of reagent. Immunostained sections were further
stained with alkaline bismuth in some cases.
Preparation of rabbit anti-EPR serum has been described by Hornma
et al, (1992). Affinity purification of the anti-EPR antibodies was
performed with purified secreted EPR (Homma et al., 1992) coupled
to CNBr-activated Sepharose 4B (Phannacia). Eluate of affinity
chromatography was concentrated with the aid of a microdialyser
(Ultrafree-PF; Millipore).
Treatrnmt with brefeldin A . Brefeldin A (Wako Pure Chemicals) was
dissolved in dimethyl sulphoxide (DMSOj at 2 rng ml-'. After cells had
been cultured in EPR induction medium for 1 h, brefeldin A was added
to the medium to a final concentration of 10 pg ml-', and the cells were
cultured for a further 0.5 or 1 h. In the control culture, DMSO alone
was added at the same time to a final concentration of 0.5% (v/v).
Cells were cultured in EPR suppression medium containing brefeldin A
at LO pg ml-' for 0.5 and 1 h to check the effect of brefeldin A on EPRsupressed cells.
Electrophoresis and immunablatting. SDS-PAGE analysis and immunoblotting analysis were performed as described previously (Hornma
et d.,1992). Briefly, samples were prepared by mixing 0.1 ml cell
suspension with 0.02 ml sample buffer [0.4 M-Tris/HCl, 6 O h (w/v) SDS,
38 % (v/v) glycerol, 0.006 9'0 (w/v) bromophenol blue, pH 6.81 and
0-01 ml 2-mercaptoethanol, and the sample solutions were heated at
100°C for 5min. SDS-PAGE was performed by the method of
Laemmli (1970). Proteins in the gel were stained with 0.1 YO (w/v)
Coomassie brilliant blue R250. Components separated by SDS-PAGE
were transferred onto nitrocellulose sheets (Schleicher & Schuell) by the
method of Howe & Hershey (1985). Non-specific sites on the blotted
sheets were blocked by immersing for 30 min at room temperature in
3 % Iw/v) skimmed milk in TBS (20 mM-Tris/HCl, 0.5 M-NaCl,
pH 7.8). The sheets were incubated in TBS containing anti-EPR
antibodies and 1 % (w/v> skimmed milk. After washing with TBS
containing 1a?' (w/v) skimmed milk, the sheets were incubated with an
affinity-purified anti-rabbit IgG antibody conjugated with alkaline
phosphatase (AP; Promega). For detection of AP, the sheets were
incubated in a mixture of 5 ml AP buffer (100 rn~-Tris/HCI,100 mMNaCI, 5 ~ M - M ~ C IpH
, , 9.5) containing 0433 ml NBT solution [50 mg
nitro-blue tetrazolium ml-l in 70 ' ! o (v/v) dimethylformamide] and
0-0165 rnl BClP solution (50 mg 5-bromo-4-chloro-3-indolyl phosphate
m1-I in dimethylformamide).
The average content of EPR in an EPR-induced cell was estimated
by measuring the colour intensity of bands on nitrocellulose sheets
detected with anti-EPR antibodies. The colour intensity of the EPR
band in each lane was quantitated with a video image analysing system
DVS-3000 (Hamamatsu Photonics). Purified EPR was used as a
standard for calibration which was carried out three times.
Alkaline bismuth staining
The ultrastructural changes that occur during the
induction of EPR secretion in C. albicans were
investigated by conventional staining with uranyl acetate
and lead citrate, as well as polysaccharide staining with
alkaline bismuth. Several different polysaccharide-staining procedures have been used to reveal Golgi apparatus,
secretory vesicles and plasma membranes (Dargent el a/.?
1982; Garrison & Mirikitani, 1981 ; Roland et al., 1972).
Preliminary work with several of these methods showed
that alkaline bismuth staining was the most effective and
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Proteinuse secretion in C . albicans
2187
Fig. 1. Electron micrographs of sections of C. ulbicuns C9 cells prior to EPR induction stained by the alkaIine bismuth method. Cells
were freeze-substituted in acetone. ( s c ) Colgi-like bodies (G)
were stained with alkaline bismuth. In (c), small granules {arrows)were
observed beneath the plasma membrane. Bars, 0-2 prn. ( d ) Vesicles at the budding tip. Bar, 0.2 pm.( e ) Vesicles a t the septum-forming
area. Bar, 0.2 prn.
easiest way to stain Golgi-like bodies (Fig. 1 a, b, c) and
secretory vesicles (Fig. 1 d, e) in C. albicans. At the site of
bud (Fig. 1 d ) and septum formation (Fig. 1e> there were
many alkaline-bismuth-positive granular structures,
which are presumed to be secretory vesicles destined to
participate in new cell wall synthesis. Such granular
structures were rarely found in other regions of the cell.
Alkaline bismuth also stained the cell wall, particularly
at inner and outer boundaries. Plasma membrane
invaginations were stained intensely (see arrow in Fig.
3b), indicating that the intensely stained material at the
inner boundary of the cell wall was probably plasma
membrane. Alkaline bismuth also stained vacuolar
membranes although less intensely (see Fig. 3h), but did
not stain membranes of the nucleus, ER and mitochondria. The matrices of nucleus and mitochondria,
and ribosomes were also stained slightly in cells freezesubstituted with OsO, in acetone (data not shown).
Structural changes accompanying EPR indue [ion
A section of a C9 cell before EPR induction, stained with
uranyl acetate and lead citrate with normal ultrastructure
is shown in Fig. 2(a). When stained with alkaline
bismuth, few intracellular structures were stained (Fig.
2h), although Golgi-like bodies and vesicles were
observed occasionally.
Many granules were observed in cells induced to
secrete EPR, mainly near the plasma membrane (Fig. 2c,
d ) . The granules which appeared in EPR-induced cells
stained intensely with alkaline bismuth (Fig. 2 4 .
Although the g r a d e s were also stained with uranyl
acetate and lead citrate, it was difficult to observe the
granules because of their low contrast relative to the
surroundings (Fig. 2 c). They resembled the vesicles
observed at the site of bud and septum formation in size,
but differed from them in shape; the outline of the
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T. Akashi and athers
Fig. 2. Electron micrographs of sections of exponential phase cells of C9 cultured in YPD medium (a,b) and C9 cells cultured in EPR
induction medium ( r , d ) for 1 h. (a, c) Cell freeze-substituted in Os0,-acetone, stained with uranyl acetate and lead citrate. (b,b) Cell
freeze-substituted in acetone, stained with alkaline bismuth. Bars, 1 pm.The insert in (c) shows a higher magnification (bar, 0.1 pm)
and the arrows indicate the position of granules.
vesicles was smooth and clear, whilst that of the granules
was irregular and obscure (compare Fig. 1 d with Figs 2 d
and 3b), so we called them flocculent granules. These
flocculent granules began to appear 30min after induction, increased in number with time, and occupied the
whofe peripheral cytoplasm 2 h after induction in most
cells (Fig. 3a,b).
In EPR-suppressed cells, even after culture for 2 h,
granules such as those seen in EPR-induced cells were
not seen using uranyl acetate and lead citrate staining
(data not shown). However, when stained with alkaline
bismuth, small flocculent granules were observed near
the plasma membrane in these cells (Fig. 3 c , d ) . The
granules in EPR-suppressed cells were smaller and less
frequent than in the EPR-induced cells after 2 h culture,
but they enlarged and accumulated with time after a
further 4 h (data not shown). Even in some pre-induced
cells, similar granules were identified by alkaline bismuth
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Proteinase secretion in C. albicans
21 89
Fig. 3. Electron micrographs of C9 cells cultured for 2 h in EPR induction medium (a, h) and EPR suppression medium (c, d ) . Cells
were freeze-substituted in acetone. Arrow in ( b ) indicates plasma membrane invaginatian. (a, c) Bars, 1 pm;(b, d ) bars, 0.1 pm.
staining, although they were small and infrequent (Fig.
l c , arrows).
Golgi-like bodies and vesicles were observed at the site
of bud and septum formation in EPR-induced cells (Fig.
4cr-4. There were no apparent differences in their
appearance and their frequency of appearance among
pre-induced ceIls, EPR-induced cells and EPRsuppressed cells (data not shown). Flocculent granules
were seen much more in mother cells than in daughter
celIs (Fig. 4 a , b ) . EPR-induced cells did not show any
apparent changes in any other cell organelles apart from
vacuoles, which increased in size.
Eflects ufbrefddin A on the ultrustructure of EPRinduced cells
In many sections of cells treated with brefeldin A, a
specific inhibitor of protein secretion, we observed
stacked cisternae, similar to Golgi bodies of higher
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T. Akushi and others
eukaryotes (Fig. 5 a, b). The stacked cisternae were
alkaline- bismuth-posi tive. These changes were also
observed in the EPR-suppressed cells (Fig. Sb),
indicating that they were independent of EPR secretion.
In contrast to the remarkable changes in the structure of
Golgi-like bodies, the occurrence of flocculent granules
induced by EPR induction was not affected by brefeldin
A treatment.
Cross-reactim
anribadies
of
flocculent graplules with anti-EPR
To determine the location of EPR within cells, electron
microscope immunocytochemistry
using
rabbit
polyclonal anti-EPR antibodies was attempted.
In cells induced to secrete EPR, most of the gold
particles were located in an area adjacent to the plasma
membrane, virtually corresponding with the location of
the flocculent granules (Fig. 6 4 , which were observed as
electron-lucent areas in unstained sections (Fig. 6a,6).
The gold particles seemed to be associated more with the
periphery than with the contents of granules (Fig. 6b).
These reactions were not observed when preimmune
serum or commercially available rabbit IgG was used as
primary antibody, although the serum and IgG crossreacted non-specifically and intensely with the cell wall,
and randomly throughout the cytoplasm (data not
shown). The reaction of the anti-EPR antibodies to the
flocculent granules was also observed in EPR-suppressed
cells (Fig. 6c), and rarely in the pre-induced cells (data
not shown). The frequency of gold particles in each cell
appeared to correlate with the number of the flocculent
granules.
These results raised the possibility that the reaction of
anti-EPR antibodies to the granules was not due to the
specific reaction of anti-EPR antibodies to EPR. As the
granules might be glycogen granules, we examined
whether or not glycogen inhibited the reaction of the
anti-EPR antibodies.
Glycogen at concentrations at or above 0.1 mg ml-'
inhibited the reaction of anti-EPR antibodies with the
granules (Fig. 6 e ) . Glucose and maltose at 10 mg ml-'
also inhibited the reaction, whilst mannose, N-acetyl-Dglucosamine, xylose, lactose and trehalose did not. These
results indicate that the observed reaction of anti-EPR
antibodies might be due to reaction with some sugar
residues similar in structure to glycogen.
Fig. 4. Electron micrographs of C9 cells cuitured in EPR induction
medium for 1 h and freeze-substituted in acetone. (a, h) Cells with buds,
( c , d ) septum-forming cell. G, Golgi-like bodies; V, vesicles; gr,
granules beneath the plasma membrane of mother cells. (u-C> Bars,
J pm; ( d ) bar, 0.2 pm.
E s t i ~ a f i o *f
n intrflcelhlar EPR by immunoblolting
Irnmunob1otting analysis showed that the affinitypurified anti-EPR antibodies did not cross-react with
any blotted components of pre-induced and EPRsuppressed cell extracts (Fig. 7a,b, lanes 1 and 2,
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Pruteimse secretion in C. ulbicuns
2191
Fig. 5. Electron micrographs ofceIIs cultured in (a> EPR induction medium for 1 h and for an additional 1 h in the presence of brefeldin
A (10 pg ml-’) or (6) in EPR suppression medium in the presence of brefeldin A (10 pg ml-’) for 1 h. *, Golgi-like body of stacked
cisternae present in cytoplasm. In (a> part of the cell wall was broken during preparation. Bars, I pm.
respectively), and that they cross-reacted specifically with
43 and 45 kDa bands of EPR-induced cell extracts on the
blotted membrane (Fig. 7a, 6 , lanes 3). The 43 kDa band
was EPR and the 45 kDa band was a precursor form
(Homma et al., 1993). Quantitative analysis of the
blotted EPR in extracts of EPR-induced cells showed
that the cells after 2 h culture in EPR-induction medium
had 1000-2000 molecules of 43 kDa EPR per cell on
average. When EPR-induced cells were treated with
brefeldin A, much more EPR and its precursor form
were detected on the nitrocellulose membrane (Fig. 7 a ,
b, lanes 4). There were no differences in the reaction of
the anti-EPR antibodies to EPR blotted on nitrocellulose
membrane in the absence and in the presence of glycogen
(Fig. 7c).
Discussion
A Ekuline h ismu th st aining
Alkaline bismuth staining, a method used to demonstrate
glycogen (Ainsworth ef al., 1972; Shinji ef aE., 1974,
1975). intensely stained Golgi-like bodies, secretory
vesicles and plasma membranes, and lightly stained
vacuolar membranes in C. ulbicuns. It is supposed that
membrane glycolipids and/or glycoproteins were stained
by this method, because alkaline bismuth was reported
to react with 1,2-glycols(Ainsworth et al. 1972; Shinji el
al., 1975). It should be noted that a rapid freezing and
freeze-substitution method, which is known to preserve
fungal cells better than conventional fixation procedures
(Howard & Aist, 19’791, was used in the present study.
This may explain why previous papers, using conventional preparation procedures, did not describe the
advantage of alkaline bismuth for polysaccharide staining.
It is difficult to detect yeast Golgi bodies and secretory
vesicles by conventional uranyl acetate and lead citrate
staining, because of their low contrast. Alkaline bismuth
stained the structures in high contrast to a similar extent
as other staining procedures for staining polysaccharides
(Dargent et a/., 1982; Garrison & Mirikitani, 1981;
Roland et al,, 1972). Additionally, the procedures of
alkaline bismuth staining are simple, so this method will
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2 192
T. Akasbi and others
Fig. 6. Electron micrographs of sections of C9 cells prepared by freeze-substitution in acetone, immunostained with rabbit pdyclonal
anti-EPR antibodies and with gold-labelled anti-rabbit IgG antibodies. Cells were cultured in EPR induction medium for 1 h @>, 2 h
(h,d, e> or in EPR suppression medium for 2 h (c). ( d ) Cell stained with alkaline bismuth, (e) cell immunostained in the presence of
glycogen (0.1 mg rn1-l). (a,e>Bars, 1 pm; ( k d ) bars, 0.1 pm.
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Proleinme secretiori in C . nlbicans
Fig. 7. SDS-polyacrylamide gel stained with CBB-RZSO after
electrophoresis of cell lysates (a> and immunoblotting with affinitypurified anti-EPR antibodies and AP-conjugated anti-rabbit IgG
antibodies (b,c). (a, b) Lanes: I, cells prior to EPR induction; 2, cells
cultured in EPR suppression medium for 2 h; 3, cells cultured in EPR
induction medium for 2 h ; 4, cells cultured in EPR induction medium
for I h and for an additional 1 h in the same medium to which brefeldin
A was added at a final concentration of I0 pg rn1-l. (c) Effect of
glycogen on the reaction of anti-EPR antibodies to EPR blotted onto
a nitrocellulose membrane. CeIIs cultured in EPR induction medium
for 2 h were analysed by immunoblotting. The blotted sheet was cut
into strips, and each strip was treated with a solution of anti-EPR
antibodies in the absence (-G) or presence ( + G I of I mg glycogen
ml-'.
prove useful in further study of these structures in
C. albicans.
Glvcogen granules
In cells induced to secrete EPR, many flocculent granules
appeared in the cytoplasm adjacent to the plasma
membrane. The size of these granules was similar to that
of secretory vesicles seen in the region of bud and septum
formation. As these granules stained intensely with
alkaline bismuth, it is possible that they might be EPR
secretory vesicles. However, similar granules were
observed in EPR-suppressed cells, and increased in size
and number gradually in prolonged cultures. This
suggests that granule formation is independent of EPR
secretion. Further evidence for this is provided by the
results of the experiment with brefeldin A, which is
known to be an inhibitor of transport from ER to Golgi
bodies (Misumi et d.,1986; Oda e t al., 1987; Takatsuki
& Tamura, 1985) and to cause the disappearance of
Golgi bodies (Fujiwara et ul., 1988; Lippincott-Schwartz
d.,1989). Brefeldin A was reported to block the
secretion of acid phosphatase in C. albicans, (Arioka
2 193
e l nl., 1991) and we found that brefeldin A caused the
accumulation of EPR in EPR-induced C9 cells. If the
flocculent granules were secretory vesicles, brefeldin A
would have inhibited formation of the granules. Brefeldin
A radically affected the structure of Golgi-like bodies,
but did not affect the formation of granules, indicating
that the granules have no direct relation to the secretory
pathway.
The granules observed close to the plasma membrane
were similar to glycogen granules often observed in C.
ulbicuns (Rajasingham & Cawson, 1980), CV~PLOCOCCUS
yeast (Schultz & Ankel, 1970) and fungi (Garrison &
Mirikitani, 1981). In Succkaromyces cereuisiae, it has
been reported that glycogen accumulates in cells cultured
under conditions of nitrogen limitation (Lillie & Pringle,
1980).Thus, it can be postulated that the cells cultured in
YNB medium in the present experiment accumulated
glycogen granules because they were in low nutrient
conditions. The difference in the amount of granules
between EPR-induced and EPR-suppressed cells may
result from the difference in each nutrient condition. To
confirm that the granules are glycogen granules, further
biochemical and structural analyses are needed.
Electron microscope immunocytochemislry
This technique was used to determine the exact
localization of EPR within a cell, but the data obtained
were inconclusive. Results revealed that anti-EPR antibodies labelled the granules located near the plasma
membrane, indicating the possibility that EPR existed in
these granules. Other results, however, suggest that this
interpretation may be incorrect. The same labelling
pattern was seen in the EPR-suppressed cells, and
labelling was inhibited by glycogen. Thus, it appears that
the antibodies cross-react with glycogen itself or with
some polysaccharides which have structural similarity to
glycogen. The latter might be explained if EPR is a
glycoprotein and some antibodies recognize epitopes
which resemble glycogen. Indeed, there are some reports
indicating that EPR does contain polysaccharide residues
(MacDonald & Odds, 1980; Ruchel, 1981). It is now
necessary to clarify whether EPR has polysaccharide
residues and determine their identity.
Immunobl otting with affinity-purified antibodies
showed that the antibodies reacted with EPR even in the
presence of glycogen. In contrast, electron microscope
immunocytochemistry showed that glycogen completely
inhibited labelling of the cells by the antibodies. These
results suggest that proteinaceous epitopes of EPR were
detected in immunoblotting, but not detected by
immunocytochemistry . According to our estimation
from immunoblotting, the number of intracellular EPR
molecules per cell might be 2000 at most. Considering the
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T.Akushi and others
accessibility of antibodies to antigen and the limitation
of the preservation of antigenicity in the resin-embedded
samples, only a fraction of the antigen may react with the
antibodies. For example, van Tuinen & Riezman (1987)
reported that detectable antigen on a section of resinembedded yeast cells is only 1-2 % of the total amount of
antigen existing in the section. Taking these considerations into account, too many gold particles reacted with
the granules in C. albicam to represent the actual level of
EPR molecules in a cell, and most of the labelling might
be due to binding of the antibodies to materials other
than EPR.
C. albicans EPR secretion
From the quantification data, the amount of EPR
retained in cells was very low in comparison to the
amount of EPR secreted into the medium, suggesting
that the transport from inside to outside of the cells is
very fast. No apparent increase in the number of vesicles
within cells induced to secrete EPR was found, suggesting
that the proteinase is secreted via the constitutive
secretory pathway and not via some special pathway.
Field & Schekman (1980) and Linnemans et al. (1977)
have shown that the acid phosphatase of S. cerevisiae is
found in the wall precursor vesicles, which are
concentrated at the site of budding and septum
formation, and where new cell wall is actively
synthesized. S. cerevisiae invertase has also been
proposed to be located in wall vesicles (Tkacz &I Lampen,
1973; Walworth & Novick, 1987). These results suggest
that these vesicles are utilized not only for cell wall
synthesis, but also for the transport of inducible,
secretory proteins. For further analysis of EPR intracellular location, it will be necessary to examine the
relationship between the wall vesicles and EPR.
This work was supported in part by Grants-in-Aid (No. 02404005
and No. 02304007) for Scientific Research from the Japanese Ministry
of Education, Science and Culture.
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