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(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 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 The fungal vacuole acts as a
Ca Little is
known concerning the pathways of Ca The ubiquity of cytosolic Ca
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
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
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 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
Figure 1:
Bafilomycin inhibits V-type
H
The ATP-dependent proton
pumping, as monitored by fluorescence quenching of quinacrine, was
completely inhibited by bafilomycin A
Figure 2:
Inhibition of ATP-dependent
H
Figure 3:
Ca
Figure 4:
Effect of FCCP and bafilomycin on vacuolar
Ca
The initial rate of
H
Figure 5:
Concentration-dependence of ATP-dependent
Ca
Figure 6:
InsP
The concentration dependence of
InsP
Figure 7:
InsP
Other Ca The possibility that InsP
Figure 8:
TPMP
Figure 9:
Release of Ca
Figure 10:
Two near-saturating doses of
TPMP
The
Figure 11:
Ca
Figure 12:
Dose-response curves for La
The endomembrane channel blocker ruthenium red (Lee
and Tsien, 1983) exerted a complete blockade on Ca In contrast to its effects on
InsP One possible mode of
TPMP
Figure 13:
Sequential release by TPMP
The K
A similar explanation appears likely to account for the limited
InsP The K The
specificity of the Ca Of the
inhibitors tested, low molecular weight heparin is considered to be a
good probe for the presence of an InsP The potentiation of
InsP
Lanthanides are known to
block stretch-activated channels in Xenopus oocytes (Yang and
Sachs, 1989) as well as vacuolar voltage-sensitive Ca The inhibitory effects of EGTA suggest that Ca
Another notable
difference between the two pathways is the role of cytosolic
Ca
Downstream signaling events ensuing a projected
rise in cytosolic free Ca The wide range
of signaling events with which Ca
Volume 270,
Number 13,
Issue of March 31, 1995 pp. 7272-7280
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mobilization Pathways at the Vacuolar Membrane of Candida
albicans (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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). 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. 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). 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.
Culture Conditions and Spheroplast
Formation
C.albicans (strain C316) from the
Glaxo (Greenford, United Kingdom) culture collection was used
throughout. Liquid cultures of the yeast form were grown in 1% (w/v)
glucose-supplemented yeast nitrogen base medium (Difco, Detroit, MI) in
an orbital incubator at 37 °C and a rotation speed of 200
revolutions/min. Cells were harvested by filtration in the
mid-exponential phase of growth, washed, and resuspended in buffer (1.2 M sorbitol in 30 mM BTP adjusted to pH 7.5 with MES)
to a concentration which gave an absorbance of 6.0 at 800 nm.
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.Membrane Preparation
Vacuolar membrane vesicles
were prepared using a method modified from Uchida et
al.(1988). Briefly, each pellet was resuspended in Buffer A (10
mM Tris adjusted to pH 6.9 with MES, 0.1 mM MgCl
and 12% (w/v) Ficoll 400), and homogenized with a
loose fitting glass homogenizer in order to lyse the spheroplasts.
Unlysed spheroplasts and cell debris were removed by centrifugation at
4,500 g in a SW 27 rotor for 10 min, and the
supernatant was retained for vacuole isolation. All subsequent
manipulations were carried out at 4 °C. To isolate vacuoles, 20-ml
volumes of supernatant were transferred to SW 27 rotor tubes, and 10 ml
of Buffer A was layered on top. This was then centrifuged at 51,900
g for 30 min. The white wafer floating on top of the
Ficoll was carefully removed using a spoon-shaped spatula (which had
been prewetted with Buffer A) and resuspended in Buffer A. To further
purify this vacuolar suspension, 15-ml volumes were overlaid with 15 ml
of Buffer B (10 mM Tris-MES, pH 6.9, 0.5 mM MgCl, 8% (w/v) Ficoll 400) and recentrifuged under the
same conditions., 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. Marker Enzyme Assays
Marker enzyme assays were
employed to determine the purity of the vesicle preparation. Plasma
membranes were identified using vanadate-sensitive ATPase activity,
mitochondrial membranes using azide-sensitive ATPase activity (Widell
and Larsson, 1990), and vacuolar membranes using bafilomycin
A-sensitive ATPase activity (Bowman et al., 1988).
ATPase activity was measured by estimating total P
release
(Ames, 1966). Bafilomycin A stock solutions were prepared
in MeSO and stored at -20 °C. IDPase was used as
a marker for Golgi membranes and was measured using the method of
Green(1983). The reaction medium (final volume 1.0 ml) comprised 3
mM IDP, 1 mM MgCl, and 50 mM Tris buffered to pH 7.0 with HCl. The IDPase assay was carried out
at 25 °C and was initiated by the addition of membrane vesicles
(10-50 µg of protein). The reaction was stopped after 1 h by
the addition of ice-cold 20% (w/v) trichloroacetic acid. The
precipitated protein was removed by centrifugation, and the supernatant
was assayed for P using the method of Ames(1966).
Nonspecific phosphatase activity was measured by the method of Leigh
and Walker(1980). Membrane vesicles were incubated at 37 °C with
reaction medium (30 mM BTP-MES, pH 6.0 or 8.0, ±30
mM molybdate), and the reaction was initiated by the addition
of 3 mM disodium p-nitrophenol phosphate. The
reaction was stopped after 1 h by the addition of 1 ml of 10% (w/v)
NaCO and the absorbance read at 400 nm. The
assay was calibrated using p-nitrophenol as a standard. NADPH
cytochrome c reductase was used as a marker for endoplasmic
reticulum membranes (Schekman, 1982). The activity of cytochrome c reductase was measured as described by Hodges and Leonard(1974).
The reaction was initiated by the addition of 3 mM NADPH to
reaction medium containing membrane protein (10-50 µg), 0.45
mM cytochrome c (oxidized), 50 mM potassium
phosphate buffer, pH 7.7, and two inhibitors of respiratory chain
complexes (50 mM NaCN and 0.4 µM antimycin A).
The increase in absorbance (as a result of cytochrome c reduction) was monitored continuously at 550 nm.Fluorescence Assays
Transmembrane pH gradients
generated in response to activation of the H-ATPase
were assayed using the pH gradient-sensitive dye quinacrine
(Rottenberg, 1979). Fluorescence measurements were made in a
Perkin-Elmer LS-5 luminescence spectrometer at 22 °C. Data were
collected on-line and subsequently analyzed using the program LSR
(Jennings et al., 1988) on an IBM PC., 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 NHCl (5 mM). 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.Calcium Transport Assays
Calcium transport was
assayed using a radiometric-filtration technique. Membrane vesicles (20
µg/ml) were incubated at 22 °C in reaction medium (3.5 mM BTP-MES, pH 8.0, 0.3 M glycerol, 50 mM KCl, 4.5
mM MgSO, 2 mM NaN 0.1, mM VO, and 4.0 mM BTP-ATP) to
generate a steady transmembrane H gradient. After 5
min 
Ca (Amersham International,
Amersham, U.K.: 2.02 mCi ml
on arrival) diluted with
cold CaCl was added at the desired concentration. Samples
(50 µl) were removed from the medium at specified times, pipetted
onto prewetted filters (Whatman cellulose nitrate membrane filters,
pore diameter 0.45 µm), and washed rapidly under vacuum using a
Millipore filtration unit, with 6 ml ice-cold wash medium. Wash medium
contained 3.5 mM BTP-MES, pH 8.0, 0.3 M glycerol, and
CaCl at a concentration 20 times that used to initiate
uptake. The filtration rate was approximately 0.7 ml of wash medium
s. Radioactivity on the filters was then counted by
standard liquid scintillation techniques. Ca uptake
is expressed as nmol Ca/mg protein. Controls were
performed in the absence of ATP to account for any
``passive'' uptake and have been subtracted from the results
except when the minus ATP results are clearly depicted on the same
graph. The amount of Ca binding to the filter was
estimated by repeating the experiment without membranes and did not
exceed 0.03% of the total Ca normally retained by
membrane vesicles.Ca
After
Ca-Release Assays had been accumulated to a steady state within the
vesicles, potential agonists of Ca release
(InsP and TPMP) were added from buffered
stock solution with rapid mixing. Potential inhibitors of
Ca release were added 1 min prior to the initiation
of the H/Ca antiport. Ca release is expressed as a percentage in relation to the total
ionophore (A23187)-sensitive Ca accumulation.
Membrane Characterization
Bafilomycin was used
as a V-type ATPase inhibitor. Of the total ATPase activity in the
membrane vesicle preparation, at least 70% was bafilomycin sensitive (Fig. 1). This suggests that the dominant membrane fraction in
the preparation was vacuolar. The I
for inhibition of
ATPase activity in membrane vesicles by bafilomycin was 0.6 µM (Fig. 1), a value comparable to the I values
for other V-type ATPases (Bowman et al., 1988). The
preparation was contaminated predominantly with mitochondrial and
plasma membranes. Thus, ATPase activity was inhibited 13% by azide and
8% by vanadate, with the residual activity (about 10%) accounted for by
the action of nonspecific phosphatases. Activities of marker enzymes
for Golgi and endoplasmic reticulum were barely detectable indicating
neglible contamination by these membranes.
-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.
(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.
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.
H
Ca/Ca Antiport at the
Vacuolar Membrane uptake at the vacuolar
membrane could be driven by ATP (Fig. 3). A steady state
accumulation was achieved after 6 min and could be reversed to the
level of ATP-independent uptake by the addition of the Ca ionophore A23187. In principle, Ca uptake could
be driven either by a Ca-ATPase or by the proton
motive force set up by the primary H-ATPase. To
discriminate between these possibilities, 10 µM FCCP was
added to the reaction medium to dissipate the H gradient. Fig. 4shows that ATP-dependent Ca uptake is largely abolished by FCCP. Moreover, Ca uptake is completely eliminated by 3 µM bafilomycin
A. These results confirm that Ca accumulation is driven by a H gradient generated
by the V-type ATPase.
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 µMCaCl 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.
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
(
).
/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).
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.
Inositol 1,4,5-Trisphosphate Releases Ca
On addition
of 20 µM InsP from Vacuolar Membrane Vesicles of C. albicans to vacuolar membrane vesicles
which had been allowed to accumulate Ca to a steady
state, approximately 24% of the A23187-sensitive Ca pool was released (Fig. 6). This reproducible
Ca release was rapid, and no reuptake was observed.
The time course for InsP-induced release was not
discernible within the time resolution of the filtration assay (the
fastest sampling time is approximately 15 s). The specificity of the
InsP response was examined with respect to InsP and InsP: neither of these compounds elicited any
Ca release at concentrations up to 50
µM.
-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 µMCaCl, 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.
-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).
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.
Inhibitor Studies on InsP
Several inhibitors known to block
Ca-stimulated
Ca Release channels in animals and plants were tested for
their ability to block InsP-induced Ca release. The results are summarized in Table 1(left-hand
column). Low molecular weight heparin, a potent antagonist of
InsP-induced Ca release in animals
(Koybayashi et al., 1988) and plants (Brosnan and Sanders,
1990), inhibited Ca release, but only by 40% at 10
µM (0.05 mg ml). Other established
InsP-gated Ca release blockers,
dantrolene and TMB-8, were without effect at concentrations of 200
µM.
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.-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.Membrane Potential-driven Ca
Addition of the lipophilic cation TPMP Release to membrane vesicles generated a physiological inside-positive
membrane potential indicated by the quenching of oxonol V fluorescence (Fig. 8, inset). The quench was sustained and could be
recovered by the addition of detergent. The imposition of a
with 10 mM TPMP elicited Ca release from Ca-loaded membrane vesicles (Fig. 8). Ca release was dependent on the
concentration of TPMP applied (Fig. 9). Thus,
half-maximal release of Ca was attained at 5.9
± 0.8 mM TPMP, with maximal release
accounting for 82% of the A23187-sensitive Ca pool.
In order to test whether Ca release was complete
after a single application of TPMP, two
near-saturating doses of TPMP (20 mM) were
administered to the vesicles. Fig. 10shows that two successive
Ca release responses of equivalent magnitude were
elicited.
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.
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.
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.
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.
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.
Effect of Inhibitors on
Several Ca-sensitive
Ca Release channel antagonists were examined for their effects on
TPMP-induced Ca release (Table 1). The first group included two lanthanides
(Gd and La) and two divalent
cations (Zn and Mn). Gd and La fully blocked, and Mn (1 mM) partially (up to 80%) blocked Ca release, whereas Zn (100 µM) had
no effect on Ca release. The dose-response
relationships for inhibition of TPMP-elicited
Ca release by La and Gd are shown in Fig. 12. The derived values for half-maximal
inhibition are 0.87 ± 0.38 and 8.4 ± 3.6 µM,
respectively.
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.
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).-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.-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.InsP
When
TPMP- and -gated Ca Release Appear to be Mediated by Separate Pathways and a saturating dose of InsP are
added sequentially (in either order), Ca release to
approximately the same level is observed (Fig. 13). This
suggests that Ca release by TPMP and
InsP is independent and represents different release
pathways. The disparate pharmacological profile of these pathways
(summarized in Table 2) provides further evidence for separate
pathways.
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 H
The present results demonstrate that uptake of
Ca/Ca Antiporter at the vacuolar membrane of C. albicans can be fuelled by H/Ca antiport. The possibility of a parallel uptake pathway involving
Ca-ATPase activity, and analogous to that postulated
for the vacuolar membrane of S. cereviseae (Cunningham and
Fink, 1994), cannot be discounted as an additional in vivo mechanism, although were it to exist, it would be rendered
inoperative by the presence of VO in the
uptake media. This protocol served to select for Ca uptake by vacuolar membrane vesicles. 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.InsP
At 24%, the proportion of Ca-gated Ca Release
Pathway released
by a saturating dose of InsP from vacuolar membrane
vesicles is similar to that reported for other non-animal systems (e.g. 26% in oat roots: Schumaker and Sze, 1987; 20% in red
beet: Brosnan and Sanders, 1990). This limited release has been
quantitatively accounted for as resulting from the low native density
of InsP regulated channels in vacuoles, which results in
the formation of many vesicles which lack the channels (Brosnan, 1990). -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). 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). 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). 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.-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).Membrane Potential Sensitive Ca
The Release-dependent Ca release elicited by TPMP raises the strong
possibility that voltage-operated Ca release channels
reside in the vacuolar membrane of C. albicans. There are
clear parallels with vacuolar cation channels reported from S.
cereviseae (Bertl and Slayman, 1990) in that both pathways are
opened at cytosol-negative (&cjs0809;inside-positive)
thought to prevail in vivo as a result of the operation of the
electrogenic H-ATPase. 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. 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).Parallel Pathways of Ca
Table 2summarizes the evidence for the independence of the two
Ca Release release pathways described in this article. The
differences include: the size of the inducer-sensitive Ca pool, response to successive doses of inducer, and
pharmacological differences. These differences may provide the answer
to how the two pathways may operate in vivo. Voltage-sensitive
release appears to be controlled by desensitisation, while
InsP-induced Ca release is controlled by
the presence of InsP. Thus, successive doses of InsP to membrane vesicles loaded with Ca prompt only
one response, whereas successive changes in the membrane potential give
two Ca release responses. 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.Physiological Relevance of the Ca
If these Ca Release Pathways release
pathways are of physiological significance, there must be a signal
which triggers the Ca release. One physiological
signal for Ca release in C. albicans could
be the cytosolic alkalinization observed after induction of the
dimorphic transition (Stewart et al., 1988). This pH change
during germ tube formation could stimulate voltage-sensitive
Ca release from the vacuole either directly or
indirectly through a change in if the alkalinization were
generated through activation of the V-type H-ATPase.
It is therefore relevant that Bertl and Slayman (1992) report that the
cation channel at the vacuolar membrane in Saccharomyces opens
at alkaline pH. Activation of the InsP-gated release
pathway might arise through elevation of cAMP levels which have been
observed on germination (Chattaway et al., 1981). In Saccharomyces cAMP is known to activate PI and PIP kinases
which results in enhanced InsP production (Kato et
al., 1989). 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). 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.
),
inositol 1,4,5-trisphosphate; , membrane potential; BTP,
bis-tris propane; cAMP, cyclic adenosine 3`5`-monophosphate;
dantrolene,
(1-[(5-[p-nitrophenyl]fur-furylidene)amine]hydantoin);
FCCP, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone;
InsP, inositol 4,5-bisphosphate; InsP, inositol
1,3,4,5-tetrakisphosphate; MeSO, dimethyl sulfoxide; MES,
2-[N-morpholino]ethanesulfonic acid; oxonol V,
bis-(3-phenyl-5-oxoisooxazol-4-yl)pentamethine oxonol; PIP,
phosphatidyl inositol 4,5-bisphosphate; Quinacrine,
6-chloro-9-{[diethyl-amino)-1-methylbutyl]amino}-2-methoxy-acridine
hypochloride; TMB-8,
8-(N,N-diethylamino)-octyl-3,4,5-trimethylbenzoate;
TPMP, methyltriphenylphosphonium ion
(C
H
P).
We are grateful to Prof. K. H. Altendorf
(Osnabrück) for the gift of bafilomycin A and to Dr R. Irvine (Cambridge) for the supply of pure
InsP. We also thank Glaxo Group Research (in particular Dr.
J. Houston) for scientific assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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