Laboratoire de Bioénergétique et Microbiologie, Université de Genève, 3 Place de l'Université, CH-1211 Genève 4, Switzerland
* Author for correspondence (e-mail: Mukti.Ojha{at}bota.unige.ch)
Accepted 5 December 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Allomyces arbuscula, Calcium-dependent neutral protease, Immunogold labeling, Intracellular localization, Hyphal tips, -tubulin a specific target
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The filamentous growth form, evolved and perfected by fungi, represents an
extreme example of polarized growth because of its unrestricted nature in
suitable environments in contrast to the restricted growth pattern of pollen
tubes and nerve cells. In these polarized cell types, a small and restricted
region, about the size of an average cell (approximately 10-20 µm
diameter), the tip, is believed to be the center for growth-related
activities. Numerous studies have emphasized the role of Ca2+ in
polarized tip growth. Tip-high gradients of total and free cytoplasmic
Ca2+ have been measured in the pollen tube and growing fungal
hyphae (Brownlee and Wood,
1986; Nobiling and Reiss,
1987
; Schmid and Harold,
1988
; Jackson and Heath,
1993
; Pierson et al.,
1994
; Torralba and Heath,
2001
). In actively growing hyphae of Saprolegnia ferax, a
steep tip-high gradient extends from the tip for about 5 µm, in contrast to
Neurospora crassa where the gradient peaks about 3 µm from the tip
(Levina et al., 1995
;
Hyde and Heath, 1997
;
Silverman-Gavrila and Lew,
2001
). The precise role of the tipbased Ca2+ gradient
is not clear but one possibility is that it may be involved in the regulation
of secretion vesicles, which are abundant in this region, and the activities
of Ca2+-activated proteins.
We identified a Ca2+-activated protease (CDP II) in the actively
growing vegetative cultures of Allomyces arbuscula and found that it
disappeared when the hyphae were induced to differentiate reproductive
structures by transfer to dilute salts solution, thus interrupting the hyphal
growth (Ojha and Turian,
1985). The enzyme was purified and shown to be a doublet of
Mr 43-40 kDa in SDS-PAGE, irrespective of the method and
speed of purification (Ojha and Wallace,
1988
). Both peptides were active and contained serine residues
that can be phosphorylated (Ojha and
Favre, 1991
; Ojha et al.,
1994
). The in vitro catalytic activity of the enzyme had an
absolute requirement for Ca2+ and reduced cysteine SH group(s).
Ca2+ seemed to induce conformational changes in the protein with
two major consequences, at µM concentrations the enzyme bound to the plasma
membranes, but at mM concentration of Ca2+ the catalytic activity
was activated (Ojha, 1989
). A
second Ca2+-dependent enzyme, CDP I, eluting at lower ionic
concentrations than CDP II, has also been identified
(Ojha, 1996b
) and shown to be
developmentally regulated (Ojha,
1996a
).
Indirect immunofluorescence microscopic studies of the spatial distribution
of CDP II showed it was predominantly localized in the growing hyphal tips of
A. arbuscula and in fungi as divergent as ascomycete, N.
crassa and basidiomycete, Uromyces appendiculatus
(Huber and Ojha, 1994;
Barja et al., 1999
).
Cytoskeletal proteins, particularly the actins and tubulins, have also been
shown to be abundantly present in the growing hyphal tips
(Raudaskoski et al., 1991
;
Barja et al., 1991
;
Huber and Ojha, 1994
). The
ultrastructural studies of hyphal tips had already shown an abundance of
microtubules and microfilament in the tip region of the hyphae
(Roos and Turian, 1977
;
Howard, 1981
;
Vargas et al., 1993
),
suggesting that these elements are probably involved in the maintenance of
form and growth of the tips. Our finding that CDP II localized to the same
region led us to assume that this colocalization must have some meaning. In
this report we show the spatial and intracellular localization of CDP II at a
higher resolution using immunogold labeling in the two growing cell types of
A. arbuscula, namely hyphal and rhizoidal tips. The evidence obtained
demonstrates the existence of an apico-basal gradient of the enzyme, which is
mainly cytosolic, although a small amount is also associated with nuclei and
the plasma membrane. We also show that
-tubulin is a selective target
of CDP II in response to a specific Ca2+ signal,
proteolysis of
-tubulin can modify microtubule arrangements in the
elongating hyphae.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strain, cultural conditions and light microscopy
Allomyces arbuscula strain Burma LD, our experimental strain, was
maintained on solid YPSs medium (Emerson,
1941). For light microscopy, A. arbuscula were grown in
liquid culture for 18 hours. Actively growing hyphal apex was prepared from a
culture grown for 48 hours on solid YPSs medium. A thin plug of mycelium, a
few mm from the colony front of the culture, was cut with a Pasteur pipette
and mounted in 100 µl YPSs medium and grown for 3-4 hours in a sterile
humidified chamber with further addition of the medium when necessary. Hyphal
apices were observed with Nomarski differential interference contrast optics
in a Zeiss axioplan microscope, the images were taken with Hamamatsu color
chilled 3CCD camera and developed by Raster Ops video captor and treated using
the Adobe PhotoShop 6 program.
Mycelia for the preparation of subcellular fractions or for electron
microscopy and immunogold labeling of CDP II were obtained by inoculation of
zoospores in liquid GCY medium (Turian,
1963). After 18-20 hours of growth at 30°C with forced
aeration (Ojha and Turian,
1981
), a small amount of the culture was saved for immunogold
labeling and the rest was harvested by filtration. The mycelia were washed
with de-ionized water, squeeze-dried and used for the subcellular fractions as
described below.
Purification of CDP II, preparation of antibodies and
immunoblotting
Details of the methods for enzyme purification, raising of anti-CDP II
antibodies and their affinity purification have been described elsewhere
(Ojha and Wallace, 1988;
Huber and Ojha, 1994
). Western
blot experiments were done essentially according to the protocol of Towbin et
al. (Towbin et al., 1979
) with
minor modifications. Proteins of cellular fractions were separated by SDS-PAGE
(11%) and electroblotted onto nitrocellulose membrane, blocked overnight at
4°C with 5% BSA in TBS-Tween, incubated for 4 hours with 1:2000 diluted
CDP II antibodies, washed and re-incubated for 2 hours with anti-rabbit IGg
antibodies coupled to horseradish peroxidase, re-washed and developed with
DAB-H2O2 as described earlier
(Huber and Ojha, 1994
).
Electron microscopy and immunogold labeling
The mycelia from an 18 hour culture were fixed for 2 hours at room
temperature. In order to maintain the integrity of the culture and to keep any
alteration owing to fixation at minimum, a concentrated solution of the
fixative was added directly to the medium to obtain the final concentration of
0.5% glutaraldehyde, 4% paraformaldehyde and 100 mM phosphate buffer
(NaH2/K2HPO4, pH 7.4). The mycelia were
collected by centrifugation and, pre-embedded in 1.5% agar at 45°C,
dehydrated respectively in 70% and 100% ethanol (for 30 minutes each). The
samples were then infiltrated sequentially in 2:1 (v/v) ethanol: LR White
resin (Polysciences), 1:1 (v/v) ethanol: LR White for 30 minutes each, 1:2
(v/v) ethanol: LR White for 1 hour and finally 100% LR White for 24 hours at
50°C for polymerization.
Ultrathin sections were taken on nickel grids, incubated at room temperature (RT) for 2 hours in 50 mM phosphate buffer (K2/KH2PO4, pH 7.0) containing 2% BSA, and then for another 2 hours (at RT) with anti-CDP II (polyclonal antibodies) diluted to 1:30 in phosphate buffer containing 2% BSA + 0.05% Tween-20. The sections were rinsed with the same buffer and incubated for 1 hour at RT with goat anti-rabbit antibodies conjugated to 20 nm gold particles diluted to 1:30 in the same buffer. After incubation, sections were washed with phosphate buffer, rinsed in distilled water, stained for 10 minutes in 2% uranyl acetate, 5 minutes in Reynold's lead citrate and examined at 60 kV in Philips M400 transmission electron microscope. The controls consisted of the use of pre-immune serum or CDP II antibody pre-incubated with purified enzyme at ratios of 1:0.8 and 1:1 for 30 minutes at room temperature and diluted to 1:30, as primary antibody.
Subcellular fractionation
Subcellular particles were prepared by a modification of the procedure
described by Hahn and Covault (Hahn and
Covault, 1990) for the isolation of the nuclei. Squeeze-dried
mycelia from an 18 hour culture were extensively minced by a sharp onion
cutter with frequent addition of small amounts of buffer A (0.3 M sucrose, 60
mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 5 mM spermidine and 15 mM
MOPS, pH 7.5). The pulp was then ground without abrasive to a very fine
homogenate in pre-cooled mortar. Buffer A was added to complete the final
ratio of 10 ml buffer per g of squeeze-dried mycelia. The homogenate was
filtered through a four-layer cheese cloth and the filtrate was centrifuged
for 15 minutes at 300 g. The supernatant was recovered and
re-centrifuged for 20 minutes at 2000 g in a Beckman JA20
rotor. The pellet contained the nuclei, and the supernatant the mitochondria
and plasma membrane. The nuclear pellet was suspended in 3 ml buffer B (which
is the same as buffer A except for the addition of EDTA and EGTA, both at 0.1
mM, and BSA, at 10 mg/ml). Percoll was then added to a final concentration of
27% and the mixture centrifuged for 20 minutes at 27,000 g.
The fluffy nuclear layer at the bottom of the tube was removed with a
siliconized Pasteur pipette, diluted 10 times with buffer A, deposited
carefully on a pad of 1 ml storage buffer (50% glycerol, 75 mM NaCl, 5 mM
Mg-acetate, 0.85 mM DTT, 0.125 mM PMSF, 20 mM Tris-HCl, pH 7.9) in a
siliconized corex tube and centrifuged at 2000 g for 20
minutes. The nuclear pellet thus obtained was suspended in storage buffer and
kept until use. The post-nuclear supernatant was centrifuged at 18,000
g for 20 minutes and the pellet containing mitochondria and
contaminating nuclei was suspended in 3 ml buffer B, made up to 27% percoll
and re-centrifuged at 27,000 g for 20 minutes. The fluffy
mitochondrial layer from the top was recovered, diluted to 10 volumes with
buffer A and re-centrifuged at 18,000 g for 20 minutes to
pellet mitochondria, which were suspended in storage buffer.
Plasma membranes were sedimented from the post-mitochondrial supernatant by
centrifugation at 41,300 g for 30 minutes and the pellet was
washed by centrifugation and suspended in 200 µl buffer A. The supernatant
from this centrifugation was subjected to ultracentrifugation at 100,000
g for 1 hour using a centricon TFT rotor. The pellet
containing ribosomes, fragmented membranes and microbodies was suspended in 50
µl buffer A, and the clear supernatant was recovered and considered as
soluble fraction. The enzyme activity in different fractions was measured
using Bz-Arg-pNA as a substrate according to the procedure described earlier
(Ojha et al., 1999).
In vitro proteolysis of tubulins in cell-free extract
The idea of using cell-free extract for testing the substrate specificity
was based on the assumption that in crude extract there would be thousands of
proteins, and given a choice the enzyme would cleave proteins only
selectively, and these can be traced using specific antibodies. We chose
tubulins because of their colocalization with CDP II in the tip region.
Actively growing 18-hour culture was harvested, mycelia washed with cold
distilled water, squeeze-dried, ground with quartz sand in a pre-cooled mortar
and suspended in MOPS buffer containing protease inhibitors
(Ojha et al., 1999). The
homogenate was centrifuged in an Eppendorf microcentrifuge at 13,000
g, the supernatant collected and concentration of proteins
determined with Coomassie according to Bradford
(Bradford, 1976
). A pool of six
reactions containing 75 µg of soluble protein was mixed with 3 µg CDP
II, made up to a total volume of 120 µl with the enzyme reaction buffer
containing 5 mM Ca2+ and 10 mM ß-mercaptoethanol and incubated
at 37°C. 20 µl samples (12.5 µg protein) were withdrawn at
intervals, supplemented with 5 µl of 5x concentrated SDS-PAGE sample
buffer, boiled for 3 minutes and processed on SDS-PAGE. As the control, two
samples (containing 12.5 µg soluble proteins each) were incubated in the
reaction mixture without CDP II or Ca2+. The digested proteins in
the samples were separated by SDS-PAGE and processed for western blotting as
described elsewhere (Ojha et al.,
1999
; Barja et al.,
1999
).
In situ proteolysis and immunofluorescent localization of
tubulins
Actively growing hyphae from the 18-hour culture were gently removed, fixed
in 3% paraformaldehyde in phosphate buffer (unless stated otherwise, the
phosphate buffer was 50 mM, pH 7.0), washed three times in the same buffer,
the last wash being with the phosphate buffer, pH 6.5. The hyphae were then
incubated at room temperature in a solution of Novozyme (5 mg/ml in phosphate
buffer, pH 6.5) for 10 minutes, washed three times, for 5 minutes each, with
phosphate buffer, pH 7.0. The plasmamembrane was permeabilised by treatment
with 0.1% Triton-X100 for 10 minutes at room temperature, re-washed three
times with phosphate buffer and then incubated for 1 hour at 37°C with CDP
II (0.22 µg/µl, specific activity 1.176 mmol, pNA released
L1 min1) in the enzyme reaction buffer (20
mM Tris-HCl, pH 7.5, 3 mM EDTA, 4 mM MgCl2, 5 mM Ca2+
and 10 mM ß-mercaptoethanol). The hyphae were then washed five times, for
5 minutes each, with phosphate buffer and incubated for 2 hours at 37°C in
phosphate buffer containing 3% BSA and further incubated with - or
ß-tubulin-specific antibodies (Amersham N-356) at 1:10 dilution for 1
hour at the same temperature, washed five times, for 5 minutes each, with
phosphate buffer and then incubated for another 2 hours with secondary
antibody (goat anti-mouse IgG conjugated to FITC) diluted to 1:100 in
phosphate buffer containing 3% BSA + 2% skimmed milk. The hyphae were then
washed five times, for 5 minutes each, in phosphate buffer. The preparation
was mounted on a slide in phenylenediamine and observed on a Leitz Orthoplan
epi-illumination microscope equipped with fluotar optics with selective filter
combination for viewing FITC conjugated antibodies. Photographs were taken on
HP5 Ilford black and white films. The controls consisted of hyphae without CDP
II treatment and
- or ß-tubulin-specific antibody,
respectively.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Spatial distribution CDP II
Immunogold labeling of CDP II in ultrathin near median longitudinal
sections of hyphal apices covering a length of approximately 32 µm from the
tip showed an apico-basal gradient of the enzyme. Labeled grains were more
abundant in the proximal than distal regions
(Fig. 2A). This was further
confirmed by actual counting of the labeled grains
(Table 1) in three different
regions of the section representing the apical exclusion zone
(Fig. 2B), the proximal region
rich in mitochondria but devoid of nuclei
(Fig. 2C) and the zone further
down where nuclei first appear (Fig.
2D). The 0-4.2 µm tip region (exclusion zone) contained
approximately 1.8 times more enzyme than the sub-apical region, which was
devoid of nuclei but rich in mitochondria, and about 2.5 times more than the
region where nuclei start to appear, in the region about 26-32 µm further
down (4 grains/µm2 in the exclusion zone against 1.6
grains/µm2 in the region between 26-32 µm from the tip).
However, we think that this is an underestimate because the large number of
grains counted in the mitochondria may not represent the reality, since in
purified mitochondria very little labeling was observed (see
Fig. 5A,B).
|
|
|
|
This spatial distribution was more spectacular in the growing rhizoidal apex where a clear apico-basal gradient was visible. The labeled grains in the tip gave the image of projected spray from the tip and the number of grains in the 1 µm tip was twice that of the sub-apical region (Table 2; Fig. 3). The distribution of grains in the ultimate tip (0-1 µm) was cytoplasmic, but in the sub-apical regions, they were mainly localized along the plasma membrane and sometimes associated with the microtubules (Fig. 3B). The long filamentous mitochondrium present in the sub-apical region was devoid of labeled grains, which indicates the absence of the enzyme.
|
Controls using an incubation mixture containing a solution from which the CDP-II-specific antibody was stripped off by incubation with purified enzyme at ratios of 1:0.8 and 1:1 (w/w) showed only a few grains in the cytoplasm or organelles with the former ratio and none at all with a ratio of 1:1 (Fig. 4A,B). Similarly, very few labeled grains were observed when the hyphae were incubated with the pre-immune serum (Fig. 4C). These results indicated that the labeling reactions presented in Figs 2 and 3 were specific.
|
Intracellular distribution of CDP II
In the hyphal tip, labeled grains were predominantly located in the
cytoplasm, but some were also present in the nuclei and attached to the plasma
membrane (Fig. 2 and
Fig. 5C-G). Occasionally, the
enzyme was seen entrapped in the nuclear pore as if it were in the process of
translocation from the cytosol to the nuclei (data not shown). The labeling in
mitochondria was sparse, and grains were only seen in a few; by contrast, the
enzyme was totally absent in the vacuoles
(Fig. 2C,D;
Fig. 5A). In the cytosol where
the majority of the labeling was observed, single grains were mostly isolated,
although sometimes aggregates of two to three grains were seen. As mentioned
in the preceding section, in the rhizoid, grains were mainly cytoplasmic in
the exclusion zone but predominantly associated with plasma membrane in the
distal region (Fig. 3;
Fig. 5G). Some aggregates of
the enzyme were also found along the microtubules in the tip region
(Fig. 3B). A comparison of the
distribution of labeled grains in different zones showed that the major
proportion of the enzyme is cytoplasmic. In the tip region, which was devoid
of cell organelles, the labels were entirely cytoplasmic
(Fig. 2A). Some labeling was
found to be associated with mitochondria and nuclei in the sub-apical zones,
but, the majority was cytoplasmic. The label counts in mitochondria could be
an overestimate, as there was a total absence of labeling in the purified
mitochondria and in the single large mitochondrium of the rhizoid
(Fig. 3,
Fig. 5A,B). Further, the
purified mitochondrial fraction had very little enzyme activity
(Table 3) and lacked enzyme
antigens, as observed with western blot experiments
(Fig. 6).
|
|
Immunogold labeling of CDP II in purified nuclei, mitochondria and
plasma membranes
Localization of the enzyme in organelles was further studied by immunogold
staining in purified organelles, and its distribution was compared to its in
situ localization. As shown in Fig. 5B and
D, the enzyme distribution in the isolated organelles corresponded
well to that obtained using in situ labeling except for in mitochondria, which
contained much fewer labeled grains in purified preparations than those in
situ.
Cellular distribution of enzyme activity
Cellular distribution of the enzyme was further quantified by differential
centrifugation of cell homogenate (prepared by mild cell disruption and the
use of a buffer system known to protect the integrity of cell organelles) and
measurement of the activity in different fractions. The results showed that
90% of the activity was associated with the soluble fraction and the remaining
activity was distributed between the nuclei (0.2%), mitochondria (0.5%),
plasma membrane (0.7%) and microsomal fractions (4%)
(Table 3). The presence or
absence of EGTA in homogenizing buffer did not affect the amount of activity
recovered in the soluble fraction, indicating that the high activity in this
fraction was not due to solubilization of the membrane bound enzyme by EGTA or
EDTA present in the buffer (data not shown).
Cellular distribution of the enzyme was further verified by western blot experiments with purified CDP II antibodies. As shown in Fig. 6, besides a small amount of reacting antigens present in the nuclear and plasma membrane (Fig. 6, lanes 2 and 4), the soluble fraction contained most of the antigens reacting with CDP II antibodies (Fig. 6, lane 5).
-tubulin is a specific target of CDP II
A search for the integrity of tubulins in the cell-free extract after
treatment with CDPII in the presence of Ca2+ by western blotting
using specific antibodies showed a rapid proteolysis of -tubulin.
ß-tubulin in the same extract was not affected, even after prolonged
incubation. (Fig. 7C,D). An
examination of the relative intensity of ponceau-stained bands after transfer
from SDS-PAGE to nitrocellulose membrane did not show any massive general
proteolysis (Fig. 7A,B).
Similar analysis for the integrity of actins did not show any modification
although purified protein was degraded rapidly (data not shown).
|
Incubation of fixed and permeabilised hyphae with CDP II in the enzyme
reaction mixture and processing for immunofluorescence using - or
ß-tubulin-specific antibodies showed a complete digestion of
-tubulin, with little effect on ß-tubulin
(Fig. 8A-D). These results
confirmed those obtained with the crude extracts.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunogold labeling of the enzyme in ultrathin sections of the hyphal tip
clearly demonstrated an apico-basal gradient, which confirmed our previous
results obtained by indirect immunofluorescence microscopy
(Huber and Ojha, 1994). The
0-4.2 µm tip region had 2.5 times more CDP II than the distal region
(Table 1). An even more
pronounced apico-basal gradient was observed in the rhizoidal tip
(Fig. 3,
Table 2) where labeling was
absent in the filamentous mitochondria. The predominance of CDP II in the
rhizoidal apex is interesting from a fundamental point of view. It raises the
questions how is sustained growth maintained at long distances in the
absence of nuclei and nuclear gene related transcription and how is CDP II,
coded by a nuclear gene, constantly transported to the tip?
In growing hyphal tips many investigators have documented the abundance of
cytoskeletal proteins (Jackson and Heath,
1990; Kaminskyj and Heath,
1995
; Barja et al.,
1993
; Huber and Ojha,
1994
; Srinivasan et al.,
1996
), Ca2+
(Jackson and Heath, 1993
;
Hyde and Heath, 1997
) and the
presence of a pH gradient (Robson et al.,
1996
). The intracellular pH gradient (a pH of 8.2 in the tip and
6.8 in the distal region) in the growing hyphae of N. crassa showed a
steep correlation between the magnitude and length of the pH gradient and the
rate of hyphal extension (Robson et al.,
1996
). A decrease in the intracellular pH by weak acids resulted
in the inhibition of growth in S. ferax
(Bachewich and Heath, 1997
).
The optimal pH for the catalytic activity of the Allomyces
Ca2+-dependent proteases is in the range 7.4-8.0; thus the hyphal
tip region provides an ideal environment for its catalytic activity. Although
recent experiments by Parton et al. discount the role of pH in the regulation
of tip growth (Parton et al.,
1997
), the tip is rich in various proteins with different pH
requirements for their activity, and it is difficult to conceive that proteins
that are optimally active at a given pH would function normally in a
sub-optimal atmosphere. The pH, Ca2+ concentration and presence of
colocalized proteins in the tip lead us to suggest a role for CDP II in the
selective modification of cytoskeletal protein interactions between themselves
and with the plasma membrane; thus, plasticity is maintained in this dynamic
region. The experiments presented here have clearly demonstrated the
predominant cytosolic localization and apico-basal gradient of the enzyme. A
small amount of CDP II is also present in the nuclei and associated with the
plasma membrane and microtubules. The localization of the enzyme along the
plasma membrane is interesting as Hyde and Heath have shown an apico-basal
Ca2+ gradient in the peripheral region along the plasma membrane in
the growing hyphal tip of S. ferax
(Hyde and Heath, 1997
). If a
similar gradient exists in the hyphal tip of Allomyces, the localized
increase in Ca2+ along the plasma membrane and the translocation of
the enzyme from the cytosol to the plasma membrane in response to a rise in
the free intracellular Ca2+ could activate its catalytic activity
(Ojha, 1989
).
The nuclear location could have a functional role in the regulation of the
activity of transcriptional factors or other nuclear proteins; likewise, the
enzyme aggregates along microtubules may be involved in the turnover of
tubulin. Our results clearly identified -tubulin as a specific target
of the enzyme, although there might be many more proteins as well. The
experimental conditions used to demonstrate this specificity are not
necessarily physiological; more experiments are needed to understand the in
vivo mechanism of CDP II activation, which must involve interactions with
other proteins. The catalytic property of the enzyme has been studied using in
vitro reactions, where its activation requires mM concentrations of
Ca2+, a concentration that is not available physiologically; this
mystery of in vivo activation of the enzyme is unresolved. The free
intracellular concentration of Ca2+ is considered to be around
106 to 107 M; however, a localized
increase in Ca2+, as along the plasma membrane
(Hyde and Heath, 1997
), can
not be ruled out, although it can never attain concentrations in the mM range.
Therefore, some other activator must exist that could lower the
Ca2+ requirement of the enzyme activity. In this context it is
interesting to note that the extensively investigated
Ca2+-dependent proteases (calpains) in the mammalian cells, the in
vitro activation reactions, also require a non-physiological concentration of
Ca2+. But it has been shown recently that in vitro activation
reactions can be activated by an endogenous protein activator
(Melloni et al., 2000
) at
physiological Ca2+ concentration.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bachewich, C. L. and Heath, I. B. (1997). The cytoplasmic pH influences hyphal tip growth and cytoskeleton-related organization. Fung. Genet. Biol. 21, 76-91.[CrossRef]
Barja, F., Nguyen Thi, B.-N. and Turian, G. (1991). Localization of actin and characterization of its isoforms in the hyphae of Neurospora crassa. FEMS Microbiol. Lett. 77,19 -24.
Barja, F., Chappuis, M.-L. and Turian, G. (1993). Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa. FEMS Microbiol. Lett. 107,261 -266.[Medline]
Barja, F., Jaquet, Y., Ortega Perez, R., Hoch, H. C. and Ojha, M. (1999). Identification and localization of calcium-dependent protease II in Neurospora crassa and Uromyces appendiculatus. Protoplasma 210, 85-91.
Berridge, M. J. (1997). Elementary and global aspects of calcium signaling. J. Physiol. 499,291 -306.[Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Brownlee, C. and Wood, J. W. (1986). A gradient of cytoplasmic free Ca2+ in growing rhizoid cells of Fucus serratus. Nature 320,624 -626.
Carafoli, E. (1987). Intracellular calcium. Annu. Rev. Biochem. 56,395 -433.[CrossRef][Medline]
Clapham, D. E. (1995). Calcium signaling. Cell 80,259 -268.[Medline]
Emerson, R. (1941). An experimental study of the life cycles and taxonomy of Allomyces. Lloydia 4, 77-144.
Gow, N. A. R. (1994). Growth and guidance of the fungal hypha. Microbiology 140,3193 -3205.[Medline]
Grove, S. N. and Bracker, C. E. (1970). Protoplasmic organization of hyphal tips among fungi vesicles, Spizenkörper. J. Bacteriol. 104,989 -1009.[Medline]
Grove, S. N., Bracker, C. E. and Morré, D. J. (1970). An ultrastructural basis for hyphal tip growth in Pythium ultimum. Am. J. Bot. 57,245 -266.
Hahn, C.-G. and Covault, J. (1990). Isolation of transcriptionally active nuclei from striated muscle using percoll density gradients. Anal. Biochem. 190,193 -197.[Medline]
Howard, R. J. (1981). Ultrastructural analysis of hyphal tip cell growth in fungi: Spizenkörper, cytoskeleton and endomembranes after freezesubstitution. J. Cell Sci. 48,89 -103.[Abstract]
Huber, D. and Ojha, M. (1994). Immunocytochemical localization of Ca2+-dependent protease in Allomyces arbuscula. FEBS Lett. 341,268 -272.[CrossRef][Medline]
Hyde, G. J. and Heath, I. B. (1997). Ca2+ gradients in hyphae and branches of Saprolegnia ferax.Fung. Genet. Biol. 21,238 -251.[CrossRef]
Jackson, S. L. and Heath, I. B. (1990). Evidence that actin reinforces the extensible hyphal apex of the oomycete Saprolegnia ferax. Protoplasma 157,144 -153.
Jackson, S. L. and Heath, I. B. (1993). Roles of calcium ions in hyphal tip growth. Microbiol. Rev. 57,367 -382.[Abstract]
Kaminskyj, S. G. W. and Heath, I. B. (1995).
Integrin and spectrin homologues and cytoplasm-wall adhesion in tip growth.
J. Cell Sci. 108,849
-856.
Levina, N. N., Lew, R. R., Hyde, G. J. and Heath, I. B.
(1995). The roles of Ca2+ and plasma membrane ion
channels in hyphal tip growth of Neurospora crassa. J. Cell
Sci. 108,3405
-3417.
Melloni, E., Minafra, R., Salamino, F. and Pontremoli, S. (2000). Properties and intracellular localization of calpain activator protein. Biochem. Biophys. Res. Commun. 272,472 -476.[CrossRef][Medline]
Nobiling, R. and Reiss, H.-D. (1987). Quantitative analysis of calcium gradients and activity in growing pollen tubes of Lilium longiflorum. Protoplasma 139, 20-24.
Ojha, M. (1989). Allomyces Ca2+-activated neutral protease: Interaction with phospholipids and plasma membranes. Plant. Sci. 59,151 -158.[CrossRef]
Ojha, M. (1996a). Purification, properties and developmental regulation of a Ca2+-independent serine-cysteine protease from Allomyces arbuscula. Int. J. Biochem. Cell Biol. 28,345 -352.[CrossRef][Medline]
Ojha, M. (1996b). Ca2+-dependent protease I from Allomyces arbuscula. Biochem. Biophys. Res. Commun. 218,22 -29.[CrossRef][Medline]
Ojha, M. and Turian, G. (1981). DNA synthesis during zoosporangial differentiation in Allomyces arbuscula. J. Gen. Microbiol. 122,263 -269.
Ojha, M. and Turian, G. (1985). Developmentally regulated proteases in Allomyces arbuscula. Plant Sci. 39,151 -155.[CrossRef]
Ojha, M. and Wallace, C. J. A. (1988). Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula. J. Bacteriol 170,1254 -1260.[Medline]
Ojha, M. and Favre, B. (1991). In vitro and in vivo phosphorylation of calpain-like protease of Allomyces arbuscula.Plant Sci. 74,35 -44.[CrossRef]
Ojha, M., Cattaneo, A. and Norberg, W. (1994). Structure and properties of casein kinase IIs from Allomyces arbuscula phosphorylating serine residues. Exp. Mycol. 18,349 -362.
Ojha, M., Cattaneo, A. and Schwendimann, B. (1999). Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.Biochimie 81,765 -770.[CrossRef][Medline]
Parton, R. M., Fischer, S., Malho, R., Rapasouliotis, O.,
Jelitto, T. C., Leonard, T. and Read, N. D. (1997).
Pronounced cytoplasmic pH gradients are not required for tip growth in plant
and fungal cells. J. Cell Sci.
110,1187
-1198.
Pierson, E. S., Miller, D. D., Callaham, D. A., Shipley, A. M.,
Rivers, B. A., Cresti, M. and Hepler, P. K. (1994). Pollen
tube growth is coupled to the extracellular calcium ion flux and the
intracellular calcium gradient: Effect of BAPTA-type buffers and hypertonic
media. Plant Cell 6,1815
-1828.
Raudaskoski, M., Rupes, I. and Timonen, S. (1991). Immunofluorescence microscopy of the cytoskeleton in filamentous fungi after quick-freezing and low-temperature fixation. Exp. Mycol. 15,167 -173.
Robson, G. D., Prebble, E., Rickers, A., Hosking, S., Denning, D. W., Trinci. A. P. J. and Robertson, W. R. (1996). Polarized cell growth in fungal hyphae is defined by an alkaline pH gradient. Fung. Genet. Biol. 20,289 -298.[CrossRef]
Roos, U. P. and Turian, G. (1977). Hyphal tip organization in Allomyces arbuscula. Protoplasma 93,231 -247.
Schmid, J. and Harold, F. M. (1988). Dual roles for calcium ions in apical growth of Neurospora crassa. J. Gen. Microbiol. 134,2623 -2631.[Medline]
Silverman-Gavrila, L. B. and Lew, R. R. (2001). Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa. Eur. J. Cell Biol. 80,379 -390.[Medline]
Srinivasan, S., Vargas, M. M. and Roberson, R. W. (1996). Functional, organizational, and biochemical analysis of actin in the hyphal tip cells of Allomyces macrogynus.Mycologia 88,57 -70.
Torralba, S. and Heath, I. B. (2001). Cytoskeletal and Ca++ regulation of hyphal tip growth and initiation. Curr. Top. Dev. Biol. 51,135 -187.[Medline]
Towbin, H., Staehlin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl Acad. Sci. USA 76,4350 -4354.[Abstract]
Trump, B. F. and Berezesky, I. K. (1995).
Calcium mediated cell injury and cell death. FASEB J.
9, 219-228.
Turian, G. (1963). Synthèse différentielle d'acide ribonucléiques et différenciation sexuelle chez l'Allomyces. Dev. Biol. 6, 61-72.[Medline]
Turian, G. (1981). Low pH in fungal bud initials. Experientia 37,1278 -1279.
Turian, G. (1983). Polarized acidification at germ tube outgrowth from fungal spores (Morchella ascospores, Neurospora conidia). Bot. Helv. 93, 27-32.
Vargas, M. M., Aronson, J. M. and Roberson, R. W. (1993). The cytoplasmic organization of hyphal tip cells in the fungus Allomyces macrogynus. Protoplasma 176, 43-52.