d-3 phosphoinositides of the ciliate tetrahymena ...d-3 phosphoinositides of the ciliate...
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Biochimica et Biophysica Act
D-3 phosphoinositides of the ciliate Tetrahymena: Characterization and
study of their regulatory role in lysosomal enzyme secretion
George Leondaritisa, Arno Tiedtkeb, Dia Galanopouloua,*
aLaboratory of Biochemistry, Department of Chemistry, University of Athens, 15771 Athens, GreecebInstitute for General Zoology and Genetics, University of Münster, D-48149 Münster, Germany
Received 28 February 2005; received in revised form 16 June 2005; accepted 20 June 2005
Available online 18 July 2005
Abstract
Phosphatidylinositol 3-phosphate, PtdIns(3)P, is a phosphoinositide which is implicated in regulating membrane trafficking in both
mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns(3,5)P2, a
phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive
lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns(3)P and PtdIns(3,5)P2 were found to comprise 16% and 30–40%
of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1–10 AM) resulted in thedepletion of PtdIns(3)P and PtdIns(3,5)P2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of
D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin
resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a
T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal
enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a
substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Tetrahymena; Phosphoinositide 3-kinase; Wortmannin; LY294002; Lysosome; Trafficking
1. Introduction
Phosphatidylinositol (PtdIns) is a minor membrane
component of all eukaryotic cells studied and it serves as
the precursor of several mono- and poly-phosphorylated
analogues (phosphoinositides, PIs) which are involved in
0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2005.06.011
Abbreviations: PtdIns, phosphatidylinositol; PI, phosphoinositide;
PtdInsP, phosphatidylinositol phosphate; PtdInsP2, phosphatidylinositol
bisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P,
phosphatidylinositol 4-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-
bisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Gro-
PInsP, glycerophosphoinositol phosphate; Vps, vacuolar protein sorting;
Fab, formation of aploid and binucleate; FYVE, Fab1-YOTB-Vac1-EEA1-
homology domain; PX, phagocyte oxidase-homology domain; EEA1, early
endosomal antigen1
* Corresponding author. Tel.: +30 210 7274471; fax: +30 210 7274476.
E-mail address: [email protected] (D. Galanopoulou).
the regulation of important cellular functions including
signalling, cell growth and differentiation, actin cytoskeletal
arrangement and intracellular vesicular trafficking [1–4].
Recently, new information has been obtained from the study
of D-3 phosphorylated PIs (D-3 PIs) and the kinases
involved in their biosynthesis. PtdIns and D-4 PIs serve as
substrates to various members of the PI 3-kinase family that
catalyze the addition of a phosphate group to the D-3
position of the inositol ring of these lipids [2]. Of the four
distinct D-3 PIs identified so far in mammalian cells,
PtdIns(3)P and PtdIns(3,5)P2 are considered to be con-
stitutively present and not in general implicated in signalling
[2].
In mammalian cells, PtdIns(3)P is found in amounts
corresponding to 2–5% of total PtdInsP; the most part of
this PtdIns(3)P pool is likely to be produced by type III PI
3-kinase (yeast Vps34p homolog) [5,6], although type II PI
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3-kinases and other pathways such as dephosphorylation of
PtdIns(3,4)P2 might contribute under certain conditions [2].
In contrast to mammalian cells, S. cerevisiae PtdIns(3)P is
found in almost equimolar amounts to PtdIns(4)P and it is
produced by the sole PI 3-kinase identified so far in the
yeast genome, Vps34p [7,8]. Studies on PtdIns(3)P local-
ization with the use of FYVE- or PX-domain probes have
shown that PtdIns(3)P is restricted to endomembranes of the
early and late endosomal network and in the vacuole of
yeast cells [9] and, also, in phagosomal membranes in
macrophages [10,11]. This localization pattern is in accord-
ance with PtdIns(3)P proposed function in regulating early/
late endosomal network trafficking [3,4]. In addition,
PtdIns(3)P serves as a substrate for type III PtdInsP kinases,
two of which have been studied and cloned so far, PIKfyve
and Fab1p. The product of these kinases is PtdIns(3,5)P2, a
phosphoinositide which has been recently identified in
mammalian, plant and yeast cells [12,13].
Several studies have implicated PtdIns(3)P in the
regulation of membrane trafficking. Most of them have
expanded early observations in S. cerevisiae , where
inactivation of the VPS34 gene causes missorting of a
subset of newly synthesized vacuolar hydrolases to the
vacuole and, as a consequence, aberrant secretion of
vacuolar proenzymes to the medium [8]. A generally
accepted model for PtdIns(3)P role in trafficking pathways
is based on the highly specific interactions of PtdIns(3)P-
enriched membrane microdomains with FYVE- or PX-
domain-containing proteins which participate in membrane
budding or fusion reactions [3,4]. Few effectors or binding
proteins of PtdIns(3,5)P2 have been described so far
[14,15]. However, in vivo studies of PIKfyve and Fab1p
in mammalian and yeast cells, respectively, have shown that
PtdIns(3,5)P2 is implicated in vacuole/late endosome
homeostasis and multivesicular body formation [16,17].
Information on PtdIns(3)P (and PtdIns(3,5)P2) and its
role in trafficking pathways has largely relied on studies in
yeast and mammalian cells. One still unanswered question is
whether these PIs are present in lower eukaryotes, like
ciliates, and if they posses similar roles in membrane
trafficking. Studies in these systems may well reveal
additional roles or modes of action or simply facilitate
analysis of PI roles in mammalian-type trafficking pathways
which are present in ciliates but not in yeast, e.g.,
phagocytosis or regulated exocytosis.
In this paper, we provide evidence that PtdIns(3)P and a
putative PtdIns(3,5)P2 are constitutive membrane lipids of
the ciliated protozoan Tetrahymena and that their synthesis
in vivo is inhibited by the mammalian PI 3-kinase inhibitor
wortmannin. Based on this inhibition, we proceed in
establishing these PIs as putative regulators of lysosome
homeostasis in Tetrahymena by showing that their depletion
is accompanied by an enhancement of constitutive lysoso-
mal enzyme secretion, a pathway which is essential for the
extracellular digestion in this organism [18]. Moreover,
using two different mutant Tetrahymena strains, deficient in
enzyme secretion and phagosome formation, respectively,
we propose that the regulation takes place at the step of
phagolysosome formation.
2. Materials and methods
2.1. Materials
Yeast extract, ferrous sulphate/chelate solution, adenine
nucleotides, p-nitrophenyl substrates, bovine brain PIs and
wortmannin were obtained from Sigma. Proteose-peptone
and silica gel H were obtained from Merck, peptone from
Serva, methylamine (33% solution in ethanol) from Fluka
and LY294002 from Calbiochem and Alexis. D-myo
[2-3H]inositol (specific activity 21.0 Ci/mmol) was pur-
chased from ICN, [3H]PtdIns(4,5)P2 (specific activity 6.5
Ci/mmol) from ARC and [3H]Ins(1,4,5)P3 was from
Amersham (TRK1000). HPLC column was purchased from
MZ Analysentechnik and scintillation fluid Floscint IV from
Packard. All other chemicals and solvents were of analytical
grade.
2.2. Cells and cell culture
Tetrahymena pyriformis (strain W) was routinely
cultured in a 2% proteose-peptone, 0.5% glucose and
0.2% yeast extract standard medium. Tetrahymena ther-
mophila CU438.1 (wild type with respect to secretion and
phagocytosis: pmr1-1/pmr1-1; pm-s, IV) and secretion
mutant (sec�) MS-1 (chx1-1/chx1-1; cy-r, sec�, II) cells
were grown in the standard medium enriched with 1%
ferrous sulphate/chelate solution. Tetrahymena thermophila
phagocytosis mutant (phg�) A2 cells (pmr1-1/pmr1-1; pm-
r, phg�, IV) (A. Tiedtke, unpublished data) were grown in
a medium of Fe2+-enriched standard medium and supple-
mented synthetic medium at a ratio of 4:1, as described for
phagocytosis mutant strains [19]. Working cultures (10–25
mL) were incubated at 26 -C under constant shaking (75–100 rpm) for approximately 72 h (late-logarithmic phase of
growth). Final cell densities ranged between 0.6–1�106cells/mL for T. pyriformis and 1–2�106 cells/mL forFe2+-enriched medium grown cells. S. cerevisiae MUCL
27831 strain was cultured in a 2% peptone, 1% glucose
and 1% yeast extract medium under constant shaking (150
rpm). Washed rabbit platelets were prepared essentially
according to Pinckard et al. [20] and suspended in a Ca2+-
free Tyrode-gelatin buffer, pH 6.5, at a final concentration
of 0.5�1010 plts/mL.
2.3. Labelling with myo-[3H]inositol, lipid extraction and
TLC analysis
Tetrahymena cells were labelled in vivo with [3H]inositol
at a concentration of 1 ACi/mL during the logarithmic phaseof growth. [3H]Inositol was added to 24 h-cultures (cell
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G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341332
density 5–10�104 cells/mL) and, after additional 48 h ofincubation, cells were first chilled on ice and then harvested
by centrifugation (1000�g for 8 min) and lipids wereextracted using the Schacht method with previously
described modifications [21]. S. cerevisiae cultures (10–
20 mL) were labelled with [3H]inositol at 1 ACi/mL for 19 h(final cell density 0.9�107 cells/mL) and the lipids wereextracted in the presence of glass beads according to
Wurmser and Emr [22]. Washed rabbit platelets resuspended
in Ca2+-free Tyrode-gelatin buffer, pH 6.5, were labelled
with [3H]inositol (15 ACi/mL) for 2 h prior to lipidextraction.
Total [3H]inositol-labelled lipids from all sources were
separated by TLC on oxalate-impregnated silica gel plates,
prepared by mixing silica gel H and 1% potassium oxalate
at a ratio of 1:2.6. A basic solvent system, chloroform/
methanol/ammonium hydroxide/water (86:76:6:16), was
used for the separation [21]. In order to detect and purify
PIs from total lipids, bovine brain standards were added to
the lipid extract prior to chromatographic separation. For
further analysis, lipids were extracted from silica gel and the
radioactivity of [3H]inositol-labelled PIs was measured by
liquid scintillation counting after resuspension in 0.5 mL
methanol and addition of a toluene-based scintillation
cocktail [21].
2.4. Methylamine deacylation and HPLC analysis of
glycerophosphoinositol phosphates
TLC-purified, [3H]inositol-labelled Tetrahymena, yeast
or rabbit platelet PtdInsP and PtdInsP2, or total Tetrahy-
mena lipids, were deacylated using methylamine as
described previously [23]. One to 2 mL of freshly
prepared methylamine reagent (methylamine in 33%
ethanol/water/n-butanol, 10:3:1) was added to dried lipids,
the suspension was briefly sonicated in a sonication bath
and, after incubation at 53 -C for 50 min, samples werecooled on ice and dried under a stream of nitrogen without
heating. The water-soluble glycero-derivatives were resus-
pended in water, washed twice with a mixture of n-
butanol/petroleum ether (bp 40–60 -C)/ethyl formate(20:4:1) and the washed aqueous phase was recovered
and freeze-dried. Glycerophosphoinositol phosphates (Gro-
PInsPs) were finally dissolved in filtered HPLC-grade
water and the pH was adjusted to 7–7.5 with HEPES/
KOH 50 mM, pH 7.5.
The separation was performed using a Hewllet Packard
HPLC on a 250�4.0 mm Partisphere 5-SAX column.Separation of GroPInsP isomers was performed with a
gradient system based on buffers A (water) and B
(NH4H2PO4 1.4 M, pH 3.8 at 25 -C with H3PO4) at aconstant flow rate of 1 mL/min as follows: 0 min, 0% B; 5
min, 0% B; 39 min, 17% B; 41 min, 37% B; 51 min, 37% B;
61 min, 100% B; 71 min, 100% B; 72 min, 0% B. Separation
of GroPInsP2 isomers was performed with the following
gradient: 0 min, 10% B; 5 min, 10% B; 45 min, 30% B; 50
min, 100% B; 55 min, 100% B; 56 min, 10% B. When
total [3H]inositol-labelled deacylated lipids were analyzed
and in order to separate all PtdInsP and PtdInsP2 isomers
in the same run, the following system was used: 0 min, 0%
B; 5 min, 0% B; 55 min, 25% B; 60 min, 100% B; 65 min,
100% B; 66 min, 0% B. In all cases, samples (250 AL)were mixed with ATP, ADP and AMP (final concentration
0.03 mM each) and the pH was adjusted to 7–7.5 with
HEPES/KOH 50 mM, pH 7.5. Samples were centrifuged in
order to precipitate particulate matter and the supernatant
was injected into a 300-AL HPLC loop. Fractions werecollected at regular intervals (0.5 min) throughout the run
and the radioactivity was determined by liquid scintillation
counting after addition of 3–5 mL of Floscint scintillation
fluid. The gradient system was evaluated using AMP, ADP,
ATP, [3H]GroPIns derived from methylamine-deacylated
purified Tetrahymena [3H]PtdIns [21], [3H]GroPIns(4)P
derived from methylamine-deacylated rabbit platelet
PtdInsP (see Results), [3H]GroPIns(4,5)P2 derived from
methylamine-deacylated standard [3H]PtdIns(4,5)P2 and
standard [3H]Ins(1,4,5)P3. Adenine nucleotide elution was
monitored by UV detection at 254 nm. In order to
accurately determine the [3H]GroPInsP and [3H]GroPInsP2content of samples, fractions were counted for 5 min and
the radioactivity of base line fractions was subtracted from
the radioactivity of peak fractions. The amount of
[3H]GroPInsP and [3H]GroPInsP2 was corrected for
recovery and quenching and was referenced to the total
radioactivity of [3H]GroPIns in the same sample. Thus, the
amount of all PIs is expressed as the % of PtdIns in each
sample.
2.5. Treatment of Tetrahymena with PI 3-kinase inhibitors in
vivo
T. pyriformis W cells were cultured and labelled in vivo
with [3H]inositol as described above. Cells were collected
by centrifugation at room temperature (650�g for 10 min),washed in an inorganic medium (Tris–HCl 10 mM, pH 7.0
or Na2HPO4 3.7 mM, KH2PO4 2.9 mM, MgSO4 1 mM, pH
7.0) and resuspended in the same medium at a final cell
density of 2–3�106 cells/mL. After further incubation for30 min, samples were treated with DMSO (0.1% final
concentration) or inhibitors (wortmannin or LY294002 at
0.1–10 AM and 50–100 AM final concentrations, respec-tively, from 1000-fold concentrated solutions in DMSO).
Samples were gently swirled during the incubation (10–20
min) after which, ice-cold perchloric acid was added (0.5 M
final concentration) and the samples were rapidly transferred
on ice. After 15–20 min on ice, samples were centrifuged at
2500�g for 5 min, the supernatant was aspirated and thepellet was suspended by addition of 3 mL of ice-cold
chloroform/methanol (1:2) and vigorous vortexing. After 20
min on ice, samples were brought to room temperature and
total lipids were extracted and deacylated as described
above.
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G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341 333
2.6. Lysosomal enzyme secretion assays
T. pyriformis or T. thermophila cells were grown to late-
logarithmic phase and then centrifuged at room temperature
as above and washed twice with Tris–HCl 10 mM, pH 7.0.
After incubation for 15 min, a 1-mL aliquot was withdrawn
for the determination of the time point 0, the rest of the
suspension was divided into flasks and DMSO (0.1% final
concentration) or inhibitor was added. Cells were incubated
at 26 -C with shaking and at regular time intervals (20–180min) 1-mL aliquots were withdrawn, incubated on ice for 5
min and centrifuged for 1 min in a microfuge. A 500-ALsample of the cell-free supernatant was recovered and the
rest of the supernatant was aspirated without damaging the
cell pellet. Cells were resuspended in 500 AL of ice-coldTris–HCl 10 mM, pH 7.0 and they were homogenized by
probe sonication on ice (3�30 s bursts with 30-s intervals).Acid phosphatase and h-hexosaminidase activities of
supernatants and cell homogenates were assayed as pre-
viously described, using the corresponding p-nitrophenyl
substrate [24]. Extracellular or cellular activity is expressed
as nmol/min/mL of initial cell suspension and the secretion
is calculated as the percentage of extracellular activity to the
total (sum of extracellular and cellular) activity. Data in
Figs. 4 and 5 were derived by subtraction of the % secretion
at time point 0; this value ranged between 1.1–4.9% for
acid phosphatase and 6.6–12% for h-hexosaminidase andwas due to incomplete removal of already secreted enzymes
during growth. In these experiments, lactate dehydrogenase
and alkaline phosphatase, which served as controls for non-
specific secretion or cell leakage, were assayed in the same
samples as described previously [25,26]. The extracellular
activity of these enzymes was negligible in both control and
wortmannin-treated samples.
3. Results
3.1. Identification of PtdIns(3)P and PtdIns(3,5)P2 in
Tetrahymena
Early studies have shown that phosphoinositides are
present in Tetrahymena [27] and subsequent experiments
from our group have partially characterized these phospho-
lipids and particularly PtdIns [21]. In addition, a recent study
provided in vitro evidence for the presence of a PI 3-kinase-
like activity in Tetrahymena [28]. However, our attempts in
demonstrating the presence of PtdIns(3)P in Tetrahymena
using a borate-based TLC solvent system yielded incon-
sistent results [21]. In order, therefore, to confirm or exclude
the presence of PtdIns(3)P in this organism, we followed the
approach of analyzing [3H]inositol-labelled deacylation
products of T. pyriformis PIs by HPLC chromatography on
an anion-exchange column. A gradient system for the
resolution of GroPInsP isomers was developed based on
earlier studies [5,7,12]. For PtdInsP isomer analysis, total
[3H]inositol-labelled Tetrahymena phospholipids were sep-
arated by TLC and the PtdInsP spot was scrapped off the
plate, extracted from the silica gel and deacylated by
methylamine. The water-soluble [3H]inositol-labelled deacy-
lation products were chromatographed on a Partisphere 5-
SAX column under the conditions depicted in Fig. 1. This
resulted in the resolution of two [3H]inositol-labelled
compounds with retention times expected for GroPIns(3)P
and GroPIns(4)P (Fig. 1A). These two compounds were
identified by comparison to parallel HPLC chromatograms
of TLC-purified, methylamine-deacylated, [3H]inositol-
labelled PtdInsP from S. cerevisiae cells and washed rabbit
platelets (Fig. 1B). As shown in Fig. 1B, deacylated yeast
PtdInsP was resolved into GroPIns(3)P and GroPIns(4)P as
expected [7], while platelet PtdInsP consisted mainly of
PtdIns(4)P. In 5 independent isolations of T. pyriformis
phospholipids, PtdIns(3)P was found to represent
16.5T2.5% of the total PtdInsP pool of the cell, a valuehigher than in any other cell type or organism examined so
far except yeast. These experiments were performed using
cells which were labelled during the logarithmic phase of
growth. Similar results concerning the relative abundance of
Tetrahymena PtdIns(3)P have been obtained by using
alternative conditions, namely labelling of cells during
starvation in inorganic buffer (Tris 10 mM, pH 7.4) or
during lag phase of growth (data not shown). Therefore, we
conclude that, under basal conditions, PtdIns(3)P is a major
PI in Tetrahymena comprising approximately 16% of the
amount of total PtdInsP. It is worth-mentioning that this is
the first report on D-3 PI presence in ciliated protozoa.
We followed the same approach in order to analyze the
PtdInsP2 isomers in Tetrahymena. The gradient system was,
therefore, optimized for the resolution of these isomers as
described in Materials and methods. HPLC analysis of
[3H]inositol-labelled, TLC-purified, methylamine-deacy-
lated PtdInsP2 from T. pyriformis cultures is shown in
Fig. 1C. Tetrahymena [3H]GroPInsP2 is resolved into two
labelled compounds one of which is co-chromatographed
with standard methylamine-deacylated [3H]PtdIns(4,5)P2.
The other compound, which constituted approximately 30%
of total GroPInsP2, was identified as a putative Gro-
PIns(3,5)P2 by comparison to parallel HPLC chromato-
grams of S. cerevisiae GroPInsP2. In all HPLC analyses,
PtdIns(3,5)P2 accounted for 30–40% of total PtdInsP2levels. Such a high relative abundance of this PI has never
been reported for any mammalian cell type or unicellular
organism studied so far.
3.2. Wortmannin but not LY294002 inhibits PtdIns(3)P and
PtdIns(3,5)P2 synthesis in Tetrahymena
Wortmannin has been widely used as a specific inhibitor
of PI 3-kinases especially in mammalian cells [2], although
at high (micromolar) concentrations it has been shown to
inhibit also PtdIns 4-kinases [29,30]. T. pyriformis cultures
were labelled with [3H]inositol and the cells were pelleted,
-
Fig. 1. Analysis of Tetrahymena PtdInsP and PtdInsP2 isomers by HPLC. (A) Tetrahymena [3H]PtdInsP and (B) [3H]PtdInsP from S. cerevisiae (open
diamonds) and rabbit platelets (filled diamonds) were isolated as described in the text, deacylated with methylamine and water-soluble products were mixed
with adenine nucleotide standards and chromatographed on a 250�4.0 mm column using a gradient system based on water and NH4H2PO4 1.4 M, pH 3.8(shown only in B). Fractions (0.5 mL) were collected and radioactivity was measured by liquid scintillation counting, while adenine nucleotide elution was
monitored by UV detection at 254 nm. Chromatograms are representative of approximately 20 individual runs. Platelet [3H]GroPIns3P is readily detected when
larger amounts of radioactivity are analyzed (not shown). (C) Tetrahymena [3H]PtdInsP2 was isolated as described in the text, deacylated with methylamine
and water-soluble products were chromatographed using a modified gradient system based on water and NH4H2PO4 1.4 M, pH 3.8. The elution positions of
[3H]GroPIns(3,5)P2 and [3H]GroPIns(4,5)P2 from S. cerevisiae and [
3H]GroPIns(4,5)P2 standard chromatographed in parallel are shown (arrows).
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341334
washed and resuspended as described in Materials and
methods. Aliquots of the cell suspension were treated with
wortmannin 1 AM in DMSO or DMSO alone (0.1% finalconcentration), total [3H]inositol-labelled lipids were
extracted and deacylated and glycerophosphoinositols were
chromatographed on the HPLC column. Representative
HPLC profiles of deacylated PIs from wortmannin-treated
and control samples are illustrated in Fig. 2. Wortmannin
caused a substantial reduction of Tetrahymena PtdIns(3)P
levels without any effect on PtdIns(4)P (Fig. 2A). This
suggests that the target of this compound in Tetrahymena is
a PtdIns 3-kinase. The reduction of PtdIns(3)P levels was
already maximal at 10 min, as further incubation with
wortmannin for 20 min did not cause any additional
lowering of PtdIns(3)P levels (data not shown). Such a
rapid wortmannin-induced depletion of PtdIns(3)P levels in
vivo has been observed in all mammalian cells studied
[6,31]. A titration curve (Fig. 2B) showed that the reduction
of PtdIns(3)P levels proceeds with an IC50 value of
approximately 200 nM. This value is substantially higher
than the values in mammalian cells, which are in the low
nanomolar range [6,12,31]. However, no in vivo data are
available from studies with unicellular organisms. More-
over, considering the uniqueness of Tetrahymena, it should
be added that the apparent differences in the efficacy of both
wortmannin and LY294002 (see below) could be due to a
reduced availability of inhibitors at specific locations/
compartments within the cell. Tetrahymena cells are
characterized by an extensive and complex pellicle which
permits direct communication of plasma membrane with
intracellular space only at specific sites in sharp contrast to
the case in most mammalian cells studied. This would
explain why highest inhibitor concentrations are required for
efficient in vivo inhibition in contrast to what is expected
from in vitro experiments.
As illustrated in Fig. 2C, wortmannin treatment reduced
also [3H]PtdIns(3,5)P2 levels to undetectable values (
-
Fig. 2. Effects of wortmannin in vivo treatment on D-3 and D-4 phosphoinositides. (A) [3H]inositol-labelled T. pyriformis cells were incubated for 10 min in
the presence of wortmannin 1 AM (filled circles) or DMSO (open circles) and, after quenching with perchloric acid, total lipids were extracted, deacylated andchromatographed on a HPLC column as shown in Fig. 1. Representative samples from more than 10 independent experiments and only the appropriate parts of
the chromatograms are shown. The recovered [3H]GroPIns of the samples was 90,000–100,000 cpm. (B) [3H]inositol-labelled T. pyriformis cells were
incubated for 10 min in the presence of different wortmannin concentrations or DMSO (control) and deacylated [3H]inositol-labelled PIs were
chromatographed as in Fig. 1. The [3H]GroPIns(3)P content was referenced to the total radioactivity of [3H]GroPIns. Results are expressed as % of the relative
abundance of PtdIns(3)P in DMSO-treated samples (0.7–0.9% of PtdIns) and they are the meanTS.D. of 3 independent experiments. (C) [3H]inositol-labelled
T. pyriformis cells were incubated for 10 min in the presence of DMSO (open circles) or wortmannin 10 AM (filled circles) and total lipids were extracted,deacylated and chromatographed using a modified gradient as described in Materials and methods, which permitted the separation of all PI isomers in the same
run. Only the appropriate parts of the chromatograms are shown. The recovered [3H]GroPIns of samples was 230,000 (wortmannin) and 260,000 cpm
(DMSO).
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341 335
[3H]PtdIns(4)P that was sensitive to LY294002 was 25–
30%. Although we cannot exclude that this reduction might
be due to a non-specific effect because of the high
concentrations used, the above results indicate that the
metabolism of PIs in Tetrahymena bears rather unique
specificities to two mammalian PI 3-kinase inhibitors:
wortmannin inhibits a PtdIns 3-kinase, while LY294002
apparently inhibits a PtdIns 4-kinase (see also Discussion).
It should be added that all experiments described so far have
been reproduced in another Tetrahymena species, T.
thermophila CU438.1 with similar results (D. Deli, G.
Leondaritis and D. Galanopoulou, unpublished results).
3.3. Wortmannin enhances the secretion of lysosomal
enzymes
Having established the occurrence of D-3 PIs in
Tetrahymena, we proceeded in investigating their possible
implication in the regulation of lysosomal vesicular
trafficking. Tetrahymena research, however, lacks a
clean-cut tool for the dissecting Golgi to lysosome traffic
in contrast to S. cerevisiae and mammalian cells where
pulse-chase experiments of newly-synthesized carboxypep-
tidase Y and cathepsin D, respectively, have provided the
first evidence for a function of PtdIns(3)P in this process
[8,33]. We decided, therefore, to study the effect of
wortmannin on the constitutive lysosomal enzyme secre-
tion pathway that has been well characterized in this cell
[18].
T. pyriformis cells were grown and treated as described
in previous sections and the activities of two lysosomal acid
hydrolases with different secretion rates, acid phosphatase
and h-hexosaminidase, were assayed in both supernatantsand cell homogenates. Acid phosphatase secretion in T.
pyriformis cells occurred at a rate of 3.9–4.8% of total
activity/h (Fig. 4A), while h-hexosaminidase secretionoccurred at a rate of 14–20% (Fig. 4B), both consistent
with previously published data [25]. When T. pyriformis
cells were treated with 10 AM wortmannin, an enhancementof the secretion of both acid phosphatase and h-hexosami-nidase was observed (Fig. 4A–B). This increased secretion
was evident at 20 min (the earliest time point examined, not
shown), peaked at 1 h after addition of wortmannin and
accounted for 65–88% and 67–71% increases over control
values for acid phosphatase and h-hexosaminidase, respec-tively. The increased secretion of acid phosphatase and h-
-
Fig. 3. Effects of LY294002 in vivo treatment on D-3 and D-4 phosphoinositides. (A) [3H]inositol-labelled T. pyriformis cells were incubated for 10 min in the
presence of LY294002 50 AM (filled circles) or DMSO (open circles) and deacylated [3H]inositol-labelled PIs were chromatographed as in Fig. 1. Only theappropriate parts of the chromatograms are shown. The recovered [3H]GroPIns of the samples was 90,000–97,000 cpm. (B) Samples were treated with 50 or
100 AM LY294002 or DMSO and the [3H]GroPIns(3)P (open bars) and [3H]GroPIns(4)P (filled bars) content was referenced to the total radioactivity of[3H]GroPIns. Results are expressed as % of the relative abundances of the lipids in DMSO-treated samples and they are the meanTS.D. of 3 independent
experiments. (C) [3H]inositol-labelled T. pyriformis cells were treated as in (A) and deacylated [3H]inositol-labelled PIs were chromatographed as in Fig. 2C.
Only the appropriate parts of the chromatograms are shown. The recovered [3H]GroPIns of samples was 230,000 (LY294002) and 260,000 cpm (DMSO).
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341336
hexosaminidase was progressively eliminated and, after a 3-
h incubation with wortmannin, secretion returned to control
rates (Fig. 4A–B). As shown in Fig. 4C, total activity of
acid phosphatase in control and wortmannin-treated cells
was very similar during the time course of these experi-
ments, a similar behavior was observed for h-hexosamini-dase as well.
The comparison of the titration curves of wortmannin-
induced PtdIns(3)P depletion (Fig. 2B) and acid phospha-
tase secretion (Fig. 4D) illustrates that both effects occur at
approximately the same EC50 of wortmannin. Moreover, as
expected from the ineffectiveness of LY294002 in the
inhibition of PtdIns(3)P synthesis, LY294002 did not
increase the initial rate of acid phosphatase or h-hexosami-nidase secretion in parallel experiments (data not shown).
3.4. Wortmannin effect in secretion is different in two T.
thermophila strains defective in distinct steps of the
phagolysosomal trafficking pathway
Lysosomal enzyme trafficking pathway in Tetrahymena
consists of two branches. Mature lysosomes or vesicles
containing mature lysosomal enzymes are either targeted to
plasma membrane for secretion or they fuse to early
acidified phagosomes to produce phagolysosomes [18,24].
In fact, a substantial fraction of intracellular acid phospha-
tase and h-hexosaminidase in growing Tetrahymena cells isfound associated with phagosomes [24].
We found it intriguing to examine wortmannin-induced
secretion of lysosomal hydrolases in two T. thermophila
mutant strains that exhibit distinct defects in the phagolyso-
somal trafficking pathway, namely the secretion mutant T.
thermophila MS-1 and the phagocytosis mutant T. thermo-
phila A2. In T. thermophila MS-1 cells, biosynthesis and
processing of proenzymes is unaltered compared to wild type
cells, but the secretion of mature enzymes is blocked,
probably at late steps of the secretory pathway [18,34–36];
importantly, T. thermophila MS-1 cells display a normal
phagocytic pathway [24,34] and are apparently wild type
concerning other Tetrahymena secretory pathways like
regulated exocytosis of dense-core granules [34]. In T.
thermophila A2 cells, phagocytosis is blocked and therefore
no phagosomes are formed when cells are incubated with
Indian Ink or iron dextran particles under different growth
conditions (our unpublished results). Previous studies of
several independent phagocytosis mutant strains have shown
that the basal secretion of lysosomal enzymes does not rely on
the presence of a functional phagocytic pathway [18].
Wortmannin treatment of wild type T. thermophila
CU438.1 cells induced depletion of PtdIns(3)P and
PtdIns(3,5)P2 levels similar to this of T. pyriformis and
enhancement of lysosomal enzyme secretion, although at
-
Fig. 4. Effects of wortmannin in vivo treatment on lysosomal enzyme secretion in Tetrahymena. T. pyriformis cells were treated with DMSO (open bars) or
wortmannin 10 AM (filled bars) as described in Materials and methods and, at regular time intervals, aliquots were withdrawn and centrifuged and acidphosphatase (A) or h-hexosaminidase (B) activity was assayed in supernatants and cell homogenates. Secreted enzyme activity is expressed as % of the totalactivity of each sample. Results are meansTS.D. of duplicate samples from 2 independent experiments. (C) Cells were treated with DMSO (open bars) or
wortmannin 10 AM (filled bars) and total acid phosphatase activity (sum of supernatant and homogenate activity) is expressed as % of the initial value (448nmol/min/106 cells). Results are meansTS.D. of duplicate samples from 2 independent experiments. (D) T. pyriformis cells were treated with different
concentrations of wortmannin or DMSO for 1 h and the secreted acid phosphatase activity was calculated as in (A) and was referenced to the value obtained
with 10 AM wortmannin which was taken as 100%. Results are the meansTS.D. of 3–4 experiments.
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341 337
lower (10-fold) wortmannin concentrations. Interestingly,
treatment of T. thermophila CU438.1 with 1 AM wortman-nin caused hypersecretion of both acid phosphatase and h-hexosaminidase (Fig. 5B for acid phosphatase), which
persisted even after 3 h; this permitted us to use 1 AMwortmannin which would allow for even a slight increase in
secretion, particularly in the case of T. thermophila MS-1
cells, to be detected and also after prolonged incubation.
As shown in Fig. 5A, T. thermophila MS-1 cells exhibit a
defect in the secretion of acid phosphatase, as was originally
described by Hünseler et al. [34], while T. thermophila A2
cells exhibit a very similar basal secretion of acid phosphatase
when compared to T. thermophila CU438.1 cells. One
noticeable difference, however, was that the total activity of
acid phosphatase of this mutant was lower compared to that
of wild type cells (approximately 248 and 481 nmol/min/106
cells, respectively). The result of wortmannin treatment on
the secretion of all strains is shown in Fig. 5B and Table 1.
The secretion of acid phosphatase induced by wortmannin in
T. thermophila MS-1 cells was negligible even when higher
concentrations (10 AM) were used (data not shown). At thesame time, in T. thermophila A2 cells, wortmannin induced
an increase in secretion which, after 1 h of treatment,
accounted for 50–60% of the increase observed in T.
thermophila CU438.1 cells. The difference was more
obvious after prolonged treatment since, after 3 h, wortman-
nin-induced secretion in T. thermophila A2 was 15–20%
compared to that of T. thermophila CU438.1 cells (Fig. 5B
and Table 1).
4. Discussion
Despite the establishment of D-3 PIs as important
regulators of vesicular trafficking in mammalian cells, a
similar role in unicellular organisms has been studied in
detail only in the yeast S. cerevisiae. We decided to proceed
in the characterization of putative D-3 PI isomers and
investigation of their involvement in Tetrahymena traffick-
ing pathways since the ciliated protozoan Tetrahymena
provides an excellent and rare opportunity for identifying
potential sites of action of PIs in trafficking: it possesses
three well-characterized pathways, regulated exocytosis of
dense-core granules, secretion of lysosomal enzymes and
phagocytosis, all in a genetically tractable and amenable
cellular context [37].
PtdIns(3)P and PtdIns(3,5)P2 were identified in T.
pyriformis cells by means of in vivo [3H]inositol labelling,
methylamine deacylation and HPLC analysis of the corre-
sponding glycero-derivatives. PtdIns(3)P was found to
represent approximately a 16% fraction of total PtdInsP
pool, while PtdIns(3,5)P2 was found to represent 30–40% of
-
Table 1
Wortmannin-induced acid phosphatase secretion is different in T. thermo-
phila mutant strains
T. thermophila strain Wortmannin-induced acid phosphatase
secretion (% of total)
60 min 180 min
CU438.1 (wild type) 2.8T0.2 12.0T2.4MS-1 (sec�) 0.4T0.2 1.0T0.4
A2 (phg�) 1.5T1.1 2.2T0.5
T. thermophila strains were treated as in Fig. 5B and net secretion induced
by wortmannin was calculated by subtraction of the values of DMSO-
induced secretion. Thus, values in the table represent the fraction of total
acid phosphatase activity that is mobilized for secretion by wortmannin at
each time point and they are meansTS.D. of duplicate samples from 3
independent experiments.
Fig. 5. Effect of wortmannin on lysosomal enzyme secretion in T.
thermophila MS-1 and A2 cells. (A) T. thermophila CU438.1 (diamonds),
MS-1 (triangles) and A2 (circles) cells were resuspended in inorganic
medium and, at regular time intervals, aliquots were withdrawn and acid
phosphatase activity was assayed. Secreted acid phosphatase activity is
expressed as in Fig. 4. Results are meansTS.D. of duplicate samples from 2
to 3 independent experiments. Total acid phosphatase activity was 481
nmol/min/106 cells (CU438.1), 470 nmol/min/106 cells (MS-1) and 248
nmol/min/106 (A2 cells). (B) T. thermophila CU438.1, MS-1 and A2 cells
were treated with DMSO (open bars) or wortmannin 1 AM (filled bars) for60 or 180 min and the secreted acid phosphatase activity was calculated as
described above. Results are meansTS.D. of duplicate samples from 3
independent experiments.
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341338
the PtdInsP2 pool. Concerning PtdIns(3,5)P2, Tetrahymena
seems to represent an interesting case: this lipid has been
found in minor amounts ranging from 0.2–0.5% of the
PtdInsP2 pool in mammalian cells [12] to 5% in S.
cerevisiae [13]. In any case, the calculated PtdIns(3)P/
PtdIns(3,5)P2 ratio in Tetrahymena (approximately 18:1)
has a value close to that of S. cerevisiae (20–30:1, in the
absence of hyperosmotic shock) [13]. However,
PtdIns(3,5)P2 concentration in S. cerevisiae increases
dramatically upon hyperosmotic stress reaching levels
similar to those of PtdIns(4,5)P2 [13]. We have been
unable so far to characterize an osmodependent activation
of PtdIns(3,5)P2 synthesis in Tetrahymena cells (D. Deli,
G. Leondaritis, and D. Galanopoulou, unpublished
results). Nevertheless, it is still an open question whether
this PtdIns(3,5)P2-based signalling pathway represents a
more widespread osmosensing pathway in eukaryotic
cells; apart from yeasts, only Chlamydomonas and some
plant cells have been reported to exhibit robust hyper-
osmotic activation of PtdIns(3,5)P2 synthesis [38].
In order to connect the presence of PtdIns(3)P and
PtdIns(3,5)P2 to functions in Tetrahymena, we decided to
use wortmannin and LY294002, two compounds that have
served as specific PI 3-kinase inhibitors in vivo and in vitro
in mammalian cell studies [2,32]; in fact, much of the work
that connected PtdIns(3)P to membrane trafficking has been
obtained using these compounds [4,33].
The data presented in Figs. 2 and 3 illuminate several
aspects of D-3 and D-4 PI metabolism in Tetrahymena.
Wortmannin causes a rapid, concentration-dependent,
reduction of PtdIns(3)P levels, quite similar to that in
mammalian cells, except the higher IC50 wortmannin
concentration which characterizes the effect in Tetrahy-
mena. Moreover, wortmannin treatment reduced the levels
of PtdIns(3,5)P2 as well. The above results predict the
presence of a PtdIns 3-kinase which is wortmannin-sensitive
and provide evidence for the synthesis of PtdIns(3,5)P2 via
a PtdIns(3)P 5-kinase activity in Tetrahymena. Whether
these enzymes are the Tetrahymena homologs of class III PI
3-kinases and type III PtdInsP kinases awaits future studies.
Interestingly, putative genes that show extended homology
to Vps34-PI3K III and Fab1/PIKfyve-type III PtdInsP
kinases have already been identified in the ongoing
Tetrahymena sequencing project (Suppl. Fig. 1).
Wortmannin has been shown to inhibit PtdIns 4-kinases
as well. In mammalian cells, the PtdIns 4-kinase that
confers wortmannin-sensitivity is the type III PtdIns 4-
kinase [29]. Additionally, in S. cerevisiae cells, the main
target of wortmannin in vivo and in vitro is a PtdIns 4-
kinase, the product of STT4 gene and not the Vps34p
PtdIns 3-kinase [39]. We believe that, apart from an
apparent absence of a wortmannin-sensitive PtdIns 4-
kinase in Tetrahymena, our results provide a strong basis
for the use of wortmannin in experiments aimed at
identifying specific roles of PtdIns(3)P in Tetrahymena
cellular functions. At the same time, LY294002 was
ineffective in reducing PtdIns(3)P and PtdIns(3,5)P2 levels
in Tetrahymena; instead PtdIns(4)P levels were slightly but
reproducibly reduced. Therefore, our results raised the
-
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341 339
question whether the preferred target of LY294002 in
Tetrahymena is a wortmannin-insensitive PtdIns 4-kinase.
This idea might not be excluded since these two
compounds use slightly different mechanisms for inhibition
of class I PI 3-kinases: although they both interfere with
the ATP-binding site, wortmannin is covalently bound and
effects a distortion in the catalytic domain while LY294002
makes extensive contacts with both the adenine and ribose
binding sites of the site [40]. These observations correlate
well with a recent study which showed that wortmannin
and LY294002 have different efficiencies in inhibiting
Tetrahymena PI kinase activity in vitro and in vivo [28].
We postulate, however, that the main target of LY294002
in vivo in Tetrahymena might not be a PtdIns 4-kinase but
possibly another protein or lipid kinase (see below).
Tetrahymena PtdIns 3-kinase seems to be rather different
from mammalian-type enzymes concerning sensitivity to
wortmannin and LY294002, at least in vivo. Therefore, we
sought to establish whether this discrepancy could be
explained at the molecular level. Towards this end, we
screened the ongoing Tetrahymena sequencing project for
genes homologues to mammalian-type PI 3-kinases using as
a bait the sequence of PI3Kg for which the crystal structure ofenzyme–inhibitor complexes has been resolved [40]. Inter-
estingly, four putative genes were recovered from this screen
and apart from a putative Vps34-type PI3K III (see above),
three genes showed extended similarity to class I PI3Ks
(Suppl. Fig. 1). A detailed comparative analysis of residues
implicated in wortmannin and LY294002 binding to human
PI3Kg showed that small differences do exist (Suppl. Fig. 2);however, these cannot clearly explain the differential
sensitivity to these two inhibitors and future studies will
hopefully clarify this issue. Different PI3Ks (with different
sensitivity to inhibitors) may as well account for the differ-
ential and concentration-dependent effects of wortmannin
and LY294002 on T. vorax differentiation [41] and Tetrahy-
mena phagocytosis (our unpublished results and [28]).
In mammalian and yeast cells, PtdIns(3)P serves as a
signal for membrane recruitment of FYVE- and PX-domain
containing proteins that regulate vesicular trafficking steps
in various aspects of endosomal trafficking [3,4]. Few
studies, however, have been focused on the lysosomes per
se: incubation of mammalian cells with wortmannin results
in a slight shift of lysosomal markers (including lysosomal
enzymes) towards less dense fractions in fractionation
gradients [42,43]. It has been proposed that, in this case,
wortmannin inhibits dense lysosome reformation after the
late endosome–lysosome fusion into a proteolytically active
hybrid organelle [43,44]. Several recent studies have also
highlighted the involvement of PtdIns(3)P in the maturation
of early phagosomes to phagolysosomes [11,45]. Incubation
of macrophages with wortmannin results in the inhibition of
PtdIns(3)P production on phagosomal membranes [10,11]
and in a block in the acquisition of early and late endosomal
markers (i.e., EEA1 and lyso-bisphosphatidic acid, respec-
tively) by nascent phagosomes [11,45].
Interestingly, Tetrahymena lysosomes serve at least two
functions: first, they fuse to – as yet unidentified – sites of
the plasma membrane and release their contents to the
extracellular space and second, they fuse to maturing
phagosomes which results in the formation of phagolyso-
somes [18,24,37]. The phagosomal maturation pathway in
ciliates is similar to that of mammalian macrophages in
several aspects: newly-formed phagosomes are rapidly
acidified by fusion with endosomal type organelles and
subsequently they become competent for fusion with
lysosomes or lysosomal vesicles containing active hydro-
lases [46]. Not surprisingly, pathogens, which escape
degradation by reprogramming the phagosomal maturation
pathway in mammalian cells [45], seem to do the same in
Tetrahymena as well [47].
The results of wortmannin-induced secretion of the two
T. thermophila mutant strains clearly implicate phagosomes/
phagolysosomes as putative target compartments of
PtdIns(3)P (and/or PtdIns(3,5)P2) function in Tetrahymena.
If the wortmannin-induced secretion involved a mechanism
of action that bypasses the normal secretory pathway, this
compound would induce the secretion of a fraction of total
lysosomal hydrolases in T. thermophila MS-1 cells com-
parable to that of wild type cells. As shown in Fig. 5B, this
is not the case. On the other hand, wortmannin treatment of
a strain devoid of phagosomes shows a decrease in
lysosomal enzyme secretion as high as 85% compared to
the secretion of wild type cells (Fig. 5B and Table 1).
Therefore, the presence of functional phagosomes/phagoly-
sosomes is necessary for the wortmannin-induced secretion
as, a significant part of it, is dependent on their presence. A
possible interpretation would be that PtdIns(3)P (and/or
PtdIns(3,5)P2) regulate the proper secretion at the level of
phagosome–lysosome fusion such that, in the absence of
PtdIns(3)P, lysosomal enzymes destined for delivery to
phagosomes could be diverted instead to the secretory
pathway thus resulting in a hypersecretion effect. This idea
is further suggested by the fact that wortmannin at the same
time causes an arrest of phagosomal maturation in early
stages (our unpublished results). The notion that blocking of
lysosome–phagosome fusion can result in a hypersecretion
effect of mature lysosomal enzymes has been suggested in
studies with chloroquine- and bafilomycin A1-treated J774
macrophages [48]. To our best knowledge, a similar
question has not been addressed in studies of wortmannin-
inhibition of phagosomal maturation in macrophages.
In conclusion, we have demonstrated the presence of D-3
PIs in the lower eukaryote Tetrahymena. Our data indicate a
role for these lipids in lysosomal trafficking and provide the
basis for future studies on their exact targets, which
probably lie in the phagolysosomal pathway of the cell.
Furthermore, the different effects of wortmannin and
LY294002 on Tetrahymena PtdInsP synthesis show that
this organism could be used for dissecting wortmannin and
LY294002 cellular targets which, until now, are considered
as identical in mammalian cells studies.
-
G. Leondaritis et al. / Biochimica et Biophysica Acta 1745 (2005) 330–341340
Acknowledgements
We wish to thank Dr. M. Typas (Department of Biology,
University of Athens, Greece) for the gift of the S.
cerevisiae MUCL 27831 strain and Dr A. Lykidis (Institute
for Biomedical Research of the Academy of Athens,
Greece) for critically reading of the manuscript. This work
was partially supported by a University of Athens grant (KA
70/4/2507).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bbamcr.
2005.06.011.
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D-3 phosphoinositides of the ciliate Tetrahymena: Characterization and study of their regulatory role in lysosomal enzyme secretionIntroductionMaterials and methodsMaterialsCells and cell cultureLabelling with myo-[3H]inositol, lipid extraction and TLC analysisMethylamine deacylation and HPLC analysis of glycerophosphoinositol phosphatesTreatment of Tetrahymena with PI 3-kinase inhibitors in vivoLysosomal enzyme secretion assays
ResultsIdentification of PtdIns(3)P and PtdIns(3,5)P2 in TetrahymenaWortmannin but not LY294002 inhibits PtdIns(3)P and PtdIns(3,5)P2 synthesis in TetrahymenaWortmannin enhances the secretion of lysosomal enzymesWortmannin effect in secretion is different in two T. thermophila strains defective in distinct steps of the phagolysosomal trafficking pathway
DiscussionAcknowledgementsSupplementary dataReferences