(Received for publication, December 7, 1994)
From the
Vacuolar membrane vesicles were isolated from Candida
albicans protoplasts, and marker enzyme assays were employed to
identify the membranes as vacuolar in origin. The mechanisms of
Ca uptake and Ca
release at the
vacuolar membrane were investigated. Ca
accumulation
by vacuolar membrane vesicles can be generated via
H
/Ca
antiport. The inside-acid pH is
in turn generated by a vacuolar-type H
-ATPase, as
demonstrated by the sensitivity of Ca
uptake to
ionophores and the vacuolar H
-ATPase inhibitor
bafilomycin A
. Vacuolar membrane vesicles exhibit two
Ca
release pathways: one induced by inositol
1,4,5-trisphosphate (InsP
) and the other by inside-positive
voltage. These two pathways are distinct with respect to the amount of
Ca
released, the nature of response to successive
stimuli, and their respective pharmacological profiles. The
InsP
-gated pathway exhibits a K
for
InsP
of 2.4 µM but is not activated by
inositol 4,5-bisphosphate or inositol 1,3,4,5-tetrakisphosphate at
concentrations up to 50 µM. Ca
release
by InsP
is blocked partially by low molecular weight
heparin. Ca
released by the voltage-sensitive pathway
occurs at membrane potentials estimated to be over a physiological
range from 0 to 80 mV. The voltage-sensitive Ca
release pathway can be blocked by lanthanide ions and organic
channel blockers such as ruthenium red and verapamil. Furthermore, the
voltage-sensitive Ca
release pathway exhibits
Ca
-induced Ca
release. These
findings are discussed in relation to the mechanism of
Ca
-mediated cellular signaling in C. albicans and other fungi.
Candida albicans is a dimorphic yeast, which grows either as an ellipsoidal bud (often referred to as the blastophore or the yeast form) or in a filamentous fashion producing pseudohyphae or true septate hyphae (Odds, 1988). C. albicans is an opportunistic pathogen which generally produces mild superficial infections. Although, inspection of infected tissues reveals a mixture of budding, mycelial and pseudomycelial C. albicans cells (Odds, 1988), the pathogenicity of C. albicans is often linked to structural dimorphism. Thus the ability to grow filamentously may be advantageous during tissue invasion, and hyphal formation may be an escape mechanism from a phagocytosing host cell.
The transition from
yeast to hyphal growth in C. albicans can be initiated by
several factors (reviewed by Shepherd et al., 1985; Odds,
1988). Significantly, Ca is one of the factors that
is able to regulate the dimorphic potential in C. albicans.
Roy and Datta(1987) demonstrated inhibition of germ tube formation by
Ca
ionophores and calmodulin inhibitors. Exogenous
Ca
can also induce the dimorphic transition (Sabie
and Gadd, 1989), and germ tube forming cells have more active
calmodulin (Paranjape et al., 1990). The importance of
Ca
in dimorphism has parallels in other filamentous
fungi (Muthukumar and Nickerson, 1984; Gadd and Brunton, 1992).
Furthermore, cytosolic free Ca
is thought to play a
crucial regulatory role in hyphal tip growth in a diverse range of
fungi (Jackson and Heath, 1993).
The fungal vacuole acts as a
Ca buffering system, maintaining low cytosolic
Ca
concentrations. Halachmi and Eilam(1989) estimated
the cytosolic free Ca
in Saccharomyces cerevisiae with the Ca
-sensitive fluorescent dye indo-1 as
346 nM while the vacuolar concentration was calculated as 1.3
mM. A lower estimate of 116 ± 90 nM for
cytoplasmic Ca
concentration was obtained by Iida et al.(1990), and in Neurospora crassa cytosolic free
Ca
measured with ion-selective microelectrodes is 92
± 15 nM (Miller et al., 1990). One mechanism
for vacuolar accumulation of Ca
is an exchange of
Ca
for nH
via a
H
/Ca
antiporter (Ohsumi and Anraku
1983; Okorokov et al. 1985), although there is also evidence
for a Ca
-ATPase at the same membrane in yeast
(Cunningham and Fink, 1994). Conclusive evidence for the Ca
homeostatic function of the vacuole in S. cerevisiae arises from work on the mutant
vma4, which is deficient in
functional vacuolar-type H
-ATPase (Ohya et
al., 1991) and hence in Ca
uptake by
Ca
/H
exchange: this mutant is unable
to control cytosolic Ca
concentration.
Little is
known concerning the pathways of Ca release from
fungal vacuoles, and, hence of the likely mechanisms of intracellular
Ca
mobilization during cell signaling. Patch clamp
studies on yeast vacuoles have revealed the presence of
voltage-sensitive ion channels which conduct Ca
(Bertl and Slayman, 1990). In addition, InsP
, (
)a second messenger widely involved in intracellular
Ca
mobilization in animal cells (Berridge and Irvine,
1989), elicits Ca
release from vacuoles of N.
crassa (Cornelius et al., 1989) and S. cerevisiae (Belde et al., 1993). The physiological relevance of
InsP
-elicited Ca
release is supported by
findings that the elements of a phosphoinositide cycle are present in
both yeast and some filamentous fungi (Kaibuchi et al., 1986;
Kato et al., 1989; Robson et al., 1991). It is not
yet known whether both voltage- and InsP
-sensitive pathways
for Ca
release from fungal vacuoles occur in the same
species, as is the case in plants (Johannes et al., 1992).
The ubiquity of cytosolic Ca as a second messenger
in eukaryotic cells, coupled with the likelihood that Ca
plays a specific role in C. albicans dimorphism and
clear indications that the vacuole contains the major mobilizable
intracellular Ca
store in fungi, suggested the need
to characterize the Ca
transport pathways at the
vacuolar membrane of C. albicans. We demonstrate here
the presence of two such pathways which appear to be discrete. The
first is gated open by InsP
, whereas the second is gated
open by cytosol-negative transmembrane voltages in the physiological
range.
The
enzymes (all supplied by Sigma-Poole, U.K.) used for spheroplast
formation were lyticase from Arthrobacter luteus, chitinase
from Serratia marcescens, and glucuronidase type H2 from Helix pomatia. Cell suspension (200 ml) was preincubated for
15 min with 2 mM dithiothreitol and then incubated with 50 ml
of lyticase (100 units/ml), 16 units of chitinase, and 200,000 units of
glucuronidase for 3 h at 30 °C with shaking (150 revolutions/min).
Spheroplasts were harvested by centrifugation for 15 min at 2200
g using a Beckman Ti-35 rotor, washed in spheroplast
buffer (1 M sorbitol, 30 mM BTP adjusted to pH 8.0
with MES) and reharvested.
The vacuoles recovered from the top of Buffer B
were then converted into vesicles by diluting them first with an equal
volume of Buffer C (10 mM Tris-MES, pH 6.9, 0.5 mM MgCl, 25 mM KCl), and then two volumes of
Buffer D (20 mM Tris-MES, pH 6.9, 1.0 mM MgCl
, 50 mM KCl). The vesicles were pelleted
at 37,000
g for 20 min. The pellet was resuspended in
35 mM BTP-MES, pH 8.0, 0.3 M glycerol, 2 mM dithiothreitol, and 1% (w/v) bovine serum albumin (fraction V,
protease-free). Protein concentration of the membrane vesicle
preparation was determined using a Bio-Rad (Hemel Hempstead, U.K.)
assay kit with bovine serum albumin (fraction V, essentially fatty
acid-free) as the standard. Typically the protein concentration of the
lysed protoplasts was 360 mg which yielded 7.5 mg of membrane vesicles.
Membrane vesicles
were incubated with 5 µM quinacrine in 1 ml of reaction
medium comprising 4.0 mM ATP, 50 mM KCl, 3.5 mM BTP-MES, pH 8.0, 2 mM NaN, 0.1 mM VO
and 0.3 M glycerol. All
solutions were stirred constantly. The transport-related percent quench
in fluorescence on addition of 10 mM MgCl
was
estimated from the percent quench recovered on the addition of an
uncoupler such as NH
Cl (5 mM).
The membrane
potential of the vacuolar membrane vesicles was monitored by loading
membrane vesicles with 5 µM oxonol V (Molecular Probes,
Junction City, OR). On attainment of a steady fluorescence reading, 10
mM TPMP was added to generate an inside
positive-membrane potential.
was estimated using the Nernst
equation, after preloading the membrane vesicles for 5 min in reaction
medium containing 1 mM TPMP
.
Figure 1:
Bafilomycin inhibits V-type
H-ATPase activity in C. albicans. Vacuolar
membrane vesicles (10 µg) were assayed for
H
-ATPase activity in reaction medium comprising 4.0
mM ATP, 2.0 mM MgSO
, 50 mM KCl,
and 50 mM BTP-MES, pH 8.5, ± bafilomycin A
.
Results are the mean of three separate experiments and are fitted to a
rectangular hyperbola (solid line) using nonlinear
least-squares.
The ATP-dependent proton
pumping, as monitored by fluorescence quenching of quinacrine, was
completely inhibited by bafilomycin A (Fig. 2). This
result is significant as it demonstrates that intravesicular
acidification is generated by the V-type H
-ATPase
alone. In total, these marker enzyme results suggest that this membrane
vesicle preparation is suitable for the study of ion transport at the
vacuolar membrane.
Figure 2:
Inhibition of ATP-dependent
H pumping by bafilomycin A
. H
pumping was assayed as quenching of quinacrine fluorescence, as
described under ``Experimental Procedures.'' A, 100
µg of membrane protein was preincubated for 5 min in the presence
of 3 µM bafilomycin. B, relaxation of steady
state pH gradient by addition of 3 µM bafilomycin.
Figure 3:
Ca uptake by vacuolar
membrane vesicles. 50 µg of membrane protein was preincubated for 5
min with reaction medium as detailed under ``Experimental
Procedures,'' with (
) or without (
) 4.0 mM ATP. 10 µM
CaCl
was then
added at time = 0 and mixed rapidly. The time scale refers to
the time when aliquots of reaction medium were filtered. 10 µM A23187 was added at the time shown to release the Ca
accumulated. Each point represents the mean of at least three
independent experiments ± S.E.
Figure 4:
Effect of FCCP and bafilomycin on vacuolar
Ca accumulation. 40 µg of membrane protein was
preincubated in reaction medium in the absence (
) or presence of
either 10 µM FCCP (
) or 3 µM bafilomycin
(
).
The initial rate of
H/Ca
antiport (assayed after 15 s)
displayed saturation kinetics with respect to Ca
concentration, and possesses a K
of 7.3
± 1.5 µM (Fig. 5).
Figure 5:
Concentration-dependence of ATP-dependent
Ca uptake into vacuolar vesicles. 20 µg of
membrane protein were preincubated in 0.5 ml of reaction medium with or
without 4.0 mM ATP. The initial influx was calculated as
ATP-dependent Ca
uptake after 15 s. Each point is the
mean of three determinations ± S.E. The solid line is a
nonlinear least-squares fit (Marquardt, 1963) to the Michaelis-Menten
equation.
Figure 6:
InsP-induced Ca
release from vacuolar membrane vesicles. 40 µg of membrane
protein were preincubated for 5 min with Ca
uptake
reaction medium, as detailed under ``Experimental
Procedures.'' Ca
uptake was initiated at t = 0 by addition of 10 µM
CaCl
, followed by rapid mixing. 10
µM InsP
and 10 µM A23187 were
added to the reaction medium at the times shown and mixed rapidly. Each
point represents the mean of at least three independent experiments.
The concentration dependence of
InsP-elicited Ca
release is shown in Fig. 7. These data exhibit monophasic saturation kinetics and
yield a K
for InsP
-induced
Ca
release of 2.4 ± 0.2 µM, with
maximal release at saturating InsP
concentrations amounting
to 24%. After an initial dose of 20 µM InsP
,
no further release of Ca
was observed on subsequent
application of InsP
(data not shown).
Figure 7:
InsP release of Ca
is dependent on InsP
concentration. The amount of Ca
released from the
A23187-sensitive Ca
pool was measured in varying
concentrations of InsP
. The InsP
stock solution
was diluted appropriately so that the same volume of InsP
was added each time. Other experimental details are described in
the legend to Fig. 3. Data (the mean ± S.E. of three
separate experiments) were fitted by nonlinear least-squares to the
Michaelis-Menten equation.
Other Ca channel blockers were
examined as potential inhibitors. The lanthanides Gd
and La
both blocked Ca
release when applied at a concentration of 100 µM. 1
mM Mn
had no effect on Ca
release. The endomembrane calcium channel blocker ruthenium red
completely inhibited Ca
release at 100
µM. Verapamil (100 µM) did not exert any
inhibitory effects on InsP
-induced Ca
release.
The possibility that InsP-elicited
Ca
release is regulated by cytosolic free
Ca
was investigated by addition of 200 µM EGTA 2 min prior to the addition of InsP
. Although
EGTA itself had no effect on preaccumulated Ca
, this
chelation of Ca
in the medium resulted in a 60%
enhancement of InsP
-elicited Ca
release.
Figure 8:
TPMP releases
Ca
from vacuolar membrane vesicles. Vacuolar membrane
vesicles were preloaded with Ca
, as detailed in the
legend to Fig. 6. 10 mM TPMP
was added
with rapid mixing at the time shown. Each point represents the mean of
four separate experiments. Inset,
TPMP
-induced quenching of oxonol V fluorescence. 20
µg of membrane protein was incubated in 0.5 ml of Ca
uptake reaction medium containing 5 µM oxonol V. 10
mM TPMP
was added as indicated to impose an
inside-positive membrane potential (and hence induce the fluorescence
quench), and then 0.02% (v/v) Triton X-100 was added to disrupt the
vesicles. The trace shown is representative of two
experiments.
Figure 9:
Release of Ca is
dependent on TPMP
concentration. Vacuolar membrane
vesicles were allowed to accumulate Ca
as described
in the legend to Fig. 6. After 6 min TPMP
was
added with rapid mixing. Aliquots were then removed from the reaction
medium 1 min after TPMP
addition and assayed for
Ca
retained by the vesicles. The data are fitted to
the Michaelis-Menten equation by nonlinear least-squares. Each data
point represents the mean of three
experiments.
Figure 10:
Two near-saturating doses of
TPMP elicit two responses of equal size. After
Ca
loading and accumulation to a steady state (as in Fig. 6), 20 mM TPMP
was added to the
reaction medium. Two aliquots were removed, and the amount of
Ca
retained was determined. A further dose of
TPMP
was then added, and the amount of Ca
retained was measured. The results are the mean ± S.E. of
three experiments.
The dependence of Ca
release
was quantified by preincubating vesicles in the presence of 1 mM TPMP
. The
resulting on subsequent
addition of various concentrations of TPMP
was then
calculated by application of the Nernst equation. The results are shown
in Fig. 11, and demonstrate measurable Ca
release over a range of intravesicular
between 0 and
80 mV. Since the membrane potential of intact yeast vacuoles is also
thought to reside around positive potentials, when referenced to lumen
cytosol (Bertl et al., 1992), the results in Fig. 11are in accord with activation of Ca
release over a physiological range of membrane potentials.
Figure 11:
Ca release increases
with membrane potential. 40 µg of membrane protein were
preincubated with Ca
uptake reaction medium
containing 1 mM TPMP
. After subsequent uptake
of Ca
for 6 min, TPMP
was added at
the desired concentration, and the amount of Ca
released after 1 min was measured. The results are expressed as
Ca
release as a percent of the amount of
Ca
accumulated in the steady state. Results shown are
the data from three independent determinations ±
S.E.
Figure 12:
Dose-response curves for La and Gd
inhibition. Various concentrations of
La
(A) or Gd
(B)
were added to the Ca
uptake reaction medium. Vesicles
were then loaded with
CaCl
for 6 min prior to
the addition 10 mM TPMP
. The data (mean
± S.E. of three independent experiments) are fitted to the
Michaelis-Menten equation using nonlinear
least-squares.
The endomembrane channel blocker ruthenium red (Lee
and Tsien, 1983) exerted a complete blockade on Ca release at 100 µM. Verapamil (an inhibitor of animal
plasma membrane Ca
channels: Biden et al.,
1984) also significantly reduced the amount of Ca
released (7% released).
In contrast to its effects on
InsP-elicited Ca
release, EGTA (200
µM) considerably reduced TPMP
-generated
Ca
release to only 5% of the A23187-sensitive pool.
Thus, it appears likely that voltage-sensitive Ca
release requires the presence of cytosolic free Ca
for full activity.
One possible mode of
TPMP-induced Ca
release might be
that the shift in membrane potential induced by TPMP
reverses the antiport mechanism. However, this is very unlikely
since we detected no effect of La
on Ca
uptake via H
/Ca
antiport.
Figure 13:
Sequential release by TPMP and InsP
. Vacuolar membrane vesicles were preloaded
with Ca
as described in the legend to Fig. 6.
When Ca
uptake was at a steady state, 10 mM TPMP
or 10 µM InsP
was
added, and two aliquots were removed from the reaction medium to
estimate the amount of Ca
retained. Following this a
subsequent dose of InsP
or TPMP
was added
(the reduction in reaction volume was accounted for), and the amount of
Ca
retained was estimated. Results are the means of
two independent experiments ± standard
deviation.
The K of 7 µM for H
-coupled
Ca
transport into vacuolar vesicles of C.
albicans is in close agreement with the K
for
Ca
uptake in some other vacuolar vesicles. In plant
cells, Schumaker and Sze(1986) reported a K
of 10
µM Ca
for
H
/Ca
exchange in oat root vacuoles,
and Bush and Sze(1986) obtained a K
of 21
µM for Ca
uptake in tonoplast vesicles
from cultured carrot cells. Previous estimates of the K
for vacuolar Ca
uptake in yeasts are, however,
somewhat higher, and range from 60 µM in Saccharomyces
carlsbergensis to 100 µM in S. cerevisiae (Okorokov et al., 1985; Ohsumi and Anraku,
1983). In all cases the K
for Ca
transport was calculated on the basis of Ca
added to the reaction medium, so the actual K
for free calcium may be lower than these values indicate if there
is significant Ca
chelation. Nevertheless, since
cytosolic free Ca
in fungi resides normally at
submicromolar levels (see Introduction), it seems likely that one major
function of vacuolar H
/Ca
antiport
would be to clear cytosolic Ca
when it is abnormally
high. Such conditions might apply locally, and especially in the
vicinity of the vacuolar membrane, during stimulus-evoked
Ca
mobilization.
A similar explanation appears likely to account for the limited
InsP-gated Ca
release from C.albicans vesicles. Furthermore, it is possible that not
all of the small vacuoles present in the blastospore of C. albicans possess an InsP
receptor. This could provide a
mechanism for short bursts of localized Ca
elevation
in the cytosol, possibly advantageous for actin localization at the
apex of the developing hyphae in C. albicans (Lasker and
Riggsby, 1992). The loss of responsiveness of membranes to a second
saturating dose of InsP
is characteristic and can be
attributed to saturation of the InsP
receptor (Prentki et al., 1984).
The K of 2.4 µM for InsP
-induced Ca
release in C. albicans also compares favorably with values
reported for other systems. In Neurospora vacuoles, the K
is 5.2 µM (Cornelius et
al., 1989), and in Saccharomyces the K
is 0.4 µM (Belde et al., 1993). In plants
the K
for InsP
mobilization of
Ca
varies from 8 µM in corn coleoptile
microsomes (Reddy and Poovaiah, 1987) to as little as 0.2 µM in Acer vacuoles (Ranjeva et al., 1988) and 0.5
µM in red beet microsomes (Brosnan and Sanders, 1990). In
pancreatic acinar cells, Ca
is released from
non-mitochondrial stores by InsP
with a K
of 1.1 µM (Streb et al., 1983).
The
specificity of the Ca release for InsP
suggests that the response is mediated by a defined receptor in
the vacuolar membrane vesicles of C. albicans. Such
specificity has previously been demonstrated in plants (Ranjeva et
al., 1988; Schumaker and Sze, 1987) and Saccharomyces (Belde et al., 1993) but was not observed in Neurospora, as several inositol phosphates also elicited
Ca
release (Schultz et al., 1990).
Of the
inhibitors tested, low molecular weight heparin is considered to be a
good probe for the presence of an InsP receptor (Ghosh et al., 1988) and is thought to interact directly with the
InsP
receptor as it is able to displace bound InsP
(Cullen et al., 1988; Brosnan and Sanders, 1993).
Heparin is not a very effective inhibitor in C. albicans (present work) or Neurospora (Cornelius et al.,
1989), and this may reflect differences in receptor structure between
fungi and other eukaryotes.
The potentiation of
InsP-dependent Ca
release by EGTA
suggests that cytosolic Ca
exerts an effect on the
InsP
response. This result is in agreement with previous
work done on animal systems where extravesicular Ca
has been demonstrated to inhibit Ca
release by
optimal doses of InsP
(Jean and Klee, 1986; Chueh and Gill,
1986).
Lanthanides are known to
block stretch-activated channels in Xenopus oocytes (Yang and
Sachs, 1989) as well as vacuolar voltage-sensitive Ca release channels in plants (Allen and Sanders, 1994).
Gd
is also an inhibitor of the mechanosensitive
plasma membrane calcium channel in the fungus Uromyces
appendiculatus (Zhou et al., 1991). The inhibitory
effects on voltage-gated Ca
release of the ions
tested in the present study might be explained in terms of a physical
blockade (Stein, 1990). The unhydrated ionic radius of Ca
is 0.099 nm, and La
and Gd
are close to this with radii of 0.106 and 0.094 nm, respectively.
The unhydrated ionic radius of Mn
is 0.080 nm, which
is considerably smaller than that of Ca
, and smaller
still is Zn
at 0.074 nm. As Mn
partially inhibits the TPMP
-induced
Ca
release, and Zn
has no effect,
this may reflect a critical size of radius required for inhibition.
The inhibitory effects of EGTA suggest that Ca release is controlled by external Ca
. Elevation
of cytosolic Ca
to micromolar levels is known to
enhance the activity of the voltage-dependent slowly activating
vacuolar (SV) channel at the plant vacuolar membrane (Hedrich and
Neher, 1987) which has recently also been demonstrated to operate as a
Ca
release channel (Ward and Schroeder, 1994). Cation
channels at the vacuolar membrane of Saccharomyces are also
activated by Ca
, albeit at high levels (1
mM) (Wada et al., 1987). Later work on yeast vacuolar
cation channels which can conduct Ca
reports that
this unphysiologically high Ca
requirement can be
lowered to 1 µM in the presence of a reducing agent (1
mM dithiothreitol or 10 mM 2-mercaptoethanol; Bertl
and Slayman, 1990).
Another notable
difference between the two pathways is the role of cytosolic
Ca concentration. Voltage-sensitive Ca
release requires the presence of Ca
: if free
Ca
is substantially lowered by EGTA, then no
Ca
release is observed. For InsP
-induced
Ca
release, endogenous Ca
may also
be involved, as Ca
removal by EGTA partially
stimulates the release of Ca
. These results hint at
an interesting phenomenon; Ca
-regulated
Ca
release pathways at the vacuolar membrane of C. albicans. This difference in the two pathways
might mean that, functionally, the pathway for Ca
release from the vacuole would depend on the prevailing
cytoplasmic Ca
concentration. The different responses
of the two pathways to cytoplasmic Ca
concentration
may suggest a mechanism whereby limited Ca
release
elicited by InsP
could serve to trigger more substantial
Ca
-induced Ca
release. The
requirement for cytoplasmic Ca
in voltage-sensitive
release can be viewed as a positive feedback mechanism, as observed in Saccharomyces (Bertl and Slayman, 1992). This would ensure
fast and effective release of Ca
from the vacuole.
Downstream signaling events ensuing a projected
rise in cytosolic free Ca could follow a well
established pattern. Intracellular mobilization of free Ca
will result in activation of calmodulin, which is known to be
present in C. albicans (Muthukumar and Nickerson, 1987) and
which has been implicated in the dimorphic transition of C.
albicans (Sabie and Gadd, 1989; Paranjape et al., 1990).
Calmodulin could then activate various phosphodiesterases and protein
kinases (Miyakawa et al., 1989), and it is therefore
noteworthy that an increase in protein phosphorylation has been
observed in germinating cells (Roy and Datta, 1987).
The wide range
of signaling events with which Ca has been associated
in C. albicans also includes regulation of chitin synthase
activity (Datta, 1992) and clustering of actin granules at the tip of
the germ tube (Schmid and Harold, 1988; Soll, 1986). The presence of
discrete pathways for intracellular Ca
mobilization
potentially endows cells with the capacity for modulation in the
spatial or temporal patterns of Ca
release. Thus,
despite the wide range of signaling events with which cytosolic free
Ca
is likely to be associated in C.
albicans, elements of specificity in stimulus-response coupling
have the potential to be attained.