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Plant Physiol. (1999) 119: 1331-1340 Changes in Phosphoinositide Metabolism with Days in Culture Affect Signal Transduction Pathways in Galdieria sulphuraria1
Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-7612 (I.H., I.Y.P., W.F.B.); and Institut für Pflanzenphysiologie und Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany (I.H., W.G.)
The metabolism of
phosphatidylinositol-4,5-bisphosphate (PIP2) changed during
the culture period of the thermoacidophilic red alga Galdieria
sulphuraria. Seven days after inoculation, the amount of
PIP2 in the cells was 910 ± 100 pmol g
The unicellular thermoacidophilic red alga Galdieria
sulphuraria occurs in hot, acidic, volcanic springs (up to 55°C
and pH < 2.0; Merola et al., 1981 In both plants and animals, the phosphoinositide pathway is involved in
the perception and transduction of external stimuli. In vitro lipid
phosphorylation with microsomal membranes from G. sulphuraria has been shown to yield primarily
polyphosphoinositides (Gross and Boss, 1993 It had been shown in several systems that the metabolism of
phosphoinositides changes with growth or senescence (Heim and Wagner,
1986 To study phosphoinositide signaling in G. sulphuraria, we
first had to characterize the phosphoinositide metabolism in the alga.
Microsomal membranes were prepared at various times during the culture
period and compared with regard to their ability to phosphorylate
lipids in vitro. These data suggested changes in the prevalence of
phosphoinositides in the cells during the culture period. In our study,
we determined that PIP kinase specific activity and
PIP2 levels of G. sulphuraria cells
were different between d 7 and 12 of the culture period. The aim of
this work was to elucidate how these differences in phosphoinositide
metabolism affected IP3 signaling after
osmostimulation of G. sulphuraria.
In addition to serving as the precursor of the second messengers
IP3 and DAG, PIP2 can
perform a variety of cellular functions in different locations of the
cell. PIP2 can function as a direct affector of
proteins (for review, see Toker, 1998 Cell Culture
In Vivo Labeling To study the incorporation of [2-3H]myo-inositol into inositol phospholipids, 0.1 to 0.3 g fresh weight of cells from d 7 and 12 was incubated overnight with 0.1 to 10 µCi of [2-3H]myo-inositol in a 2-mL culture. Cells were washed twice in deionized water before lipid extraction. To label polyphosphoinositides with 32Pi, between 0.2 and 0.5 g fresh weight of cells from d 7 and 12 was incubated with 10 µCi of carrier-free 32Pi for 10 min or 2 h in a 2-mL culture. Cells were washed twice in deionized water before extraction of lipids. Cells were lysed with 20% (v/v) TCA and washed in deionized water, and total lipids were extracted according to the method of Cho et al. (1992) .
Osmotic Stimulation Before stimulation, cells were equilibrated overnight in 50 mL of culture medium in 200-mL culture flasks at 26°C in the dark with shaking (150 rpm). Cells were stimulated by the addition of 5 mL of NaCl, KCl, or methyl-Man in conditioned culture medium to final concentrations, as indicated. Conditioned culture medium was obtained immediately before each experiment by centrifuging 100 mL of cell culture for 10 min at 2000g and decanting the medium. NaCl, KCl, and methyl-Man solutions were prepared fresh in conditioned medium for each experiment and adjusted to 26°C before use. Methyl-Man was used as an osmotically active sugar derivative, because unlike sorbitol or mannitol, it is not taken up and metabolized by G. sulphuraria (W. Gross, personal communication).Preparation of Microsomes Cells from 50-mL cultures (0.5-1 g fresh weight) were harvested by centrifugation at 2,500g for 30 s and homogenized in 20 mL of ice-cold buffer (250 mM Suc, 3 mM EDTA, 2 mM EGTA, 14 mM -mercaptoethanol, 2 mM DTT, and 50 mM Tris-HCl,
pH 7.4) with 0.1 g of polyvinylpolypyrrolidone using a blender
(VirTis Co., Gardiner, NY) and glass beads. For time-course
experiments, the times indicated denote the initiation of
homogenization. Arabidopsis vegetative tissue and whole rat liver were
ground in the same buffer using a VirTis blender and rotating blades.
Microsomal membranes were prepared by centrifuging the homogenate for
15 min at 2,500g and then centrifuging the 2,500g
supernatant for 60 min at 41,000g. The 41,000g
pellets were resuspended in 30 mM Tris, pH 6.5, containing 15 mM MgCl2.
Protein was estimated using the Bradford assay (Bio-Rad) with BSA as a
standard.
Lipid Kinase Assays PI kinase and PIP kinase activities were assayed as described previously (Cho and Boss, 1995) using 20 µg of microsomal membrane protein per assay in 50 µL of reaction mixture containing 30 mM Tris-HCl, pH 6.5, 7.5 mM
MgCl2, 1 mM sodium molybdate, 0.01%
(v/v) Triton X-100, and 0.9 mM
[ -32P]ATP (0.2 µCi/nmol). Reactions were
incubated for 10 min at room temperature with intermittent mixing. For
assays containing exogenous substrate, PI or PIP presolubilized in 1%
(v/v) Triton X-100 was added to give a final concentration of 25 µg
of lipid in 0.1% (v/v) Triton X-100 per reaction. After incubation,
inositol phospholipids were extracted using an acid-extraction method
(Cho et al., 1992). The DAG kinase assay contained 0.01% (v/v)
3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid instead of Triton X-100. To quantify DAG levels, lipids were extracted from equal amounts (500 µg of microsomal protein) of microsomes from G. sulphuraria, Arabidopsis, or rat liver.
The extracted lipids were incubated with 0.9 mM
[-32P]ATP (0.2 µCi/nmol) and 1 unit per
assay of a recombinant Escherichia coli DAG kinase
(Calbiochem) under the conditions used to measure endogenous DAG
kinase activity.
Separation of Phospholipids Lipids were separated by TLC on LK5D silica-gel plates (Whatman) using a CHCl3:methanol:NH4OH:H2O (86:76:6:16) solvent system (Cho and Boss, 1995). The
32P-labeled phospholipids were quantified using a
scanner (system 500, Bioscan, Inc., Washington, DC).
32P-labeled phosphoinositides were hydrolyzed
when the samples were incubated on the TLC plate with 0.5 M
KOH for 10 min before development, indicating that they were not
phosphosphingolipids (data not shown). To distinguish between 4- and
3-phosphorylated PIP, lipids were separated in the presence of
boric acid, as described previously by Walsh et al. (1991). Under the
conditions used for the in vitro lipid phosphorylation assays, no
PI-3-P could be detected (data not shown).
Quantification of IP3 and PIP2 Contents For IP3 measurements after stimulation, cells were harvested by centrifugation at 2000g for 10 to 30 s in preweighed test tubes. The supernatant was discarded and cells were immediately frozen in liquid N2. The times indicated denote the times the cells were placed in liquid N2. The frozen cells were weighed, and 500 µL of ice-cold 20% (v/v) PCA was added to each sample. After a 20-min incubation on ice, precipitated proteins were pelleted by centrifugation at 2000g for 15 min at 4°C. For IP3 assays, the supernatant was transferred to a clean tube and adjusted to pH 7.5 using ice-cold 1.5 M KOH, 60 mM Hepes containing universal pH indicator dye (Fisher Scientific). The neutralized samples were assayed for IP3 content using the [3H]IP3 receptor-binding assay (Amersham). Assays were carried out at 4°C according to the manufacturer's instructions using 50 µL of sample per assay.
Expression and Purification of Inositol Polyphosphate 5-Phosphatase I To rule out the possibility that inositol phosphate metabolites in G. sulphuraria samples other than IP3 affected the displacement of [3H]IP3 in the IP3 receptor-binding assay, aliquots from G. sulphuraria samples were pretreated with a recombinant human inositol polyphosphate 5-phosphatase I. The recombinant protein was induced for 3 h by the addition of isopropyl--D-thiogalactosidase (0.5 mM final concentration). Bacterial cells
expressing the His-tagged phosphatase were lysed by sonication and
resuspended in 50 mM sodium phosphate, pH 8.0, and 300 mM NaCl. The recombinant protein was
purified by metal-affinity chromatography on a nickel-nitrilotriacetic acid agarose resin (Qiagen, Dusseldorf, Germany) and eluted from the
column with the same buffer containing 50 to 500 mM imidazole. The activity of purified fractions
was tested on commercially available IP3. Samples
from G. sulphuraria osmostimulated for 90 s were
treated with active or heat-denatured phosphatase at room temperature
or at 37°C and assayed for IP3 content. The
phosphatase pretreatment eliminated the IP3 from
G. sulphuraria samples. Heat-denatured phosphatase had no
effect. The human inositol polyphosphate 5-phosphatase I cDNA
(Auethavekiat et al., 1997) was a gift from Dr. Phil Majerus (Washington University School of Medicine, St. Louis, MO).
Presentation of Data Products of in vitro phosphorylation that did not migrate on TLC (origin) were excluded from the quantification of phosphorylated phospholipids presented in Figure 1B. The data shown in Figure 4 were calculated as the percentage change of the osmostimulated samples over the conditionedmedium controls at each time point measured. Experiments presented in Figure 4 were repeated two (A + B) or four times (C + D), samples were assayed in duplicate, and values were averaged from these values before calculation of the percentage change to allow the comparison of changes between different experiments.
Northern Blots G. sulphuraria total RNA was isolated using an RNeasy (plant) kit (Qiagen). The RNA (25 µg/lane) was fractionated by electrophoresis on formaldehyde-containing agarose gels. It was then transferred to a nylon membrane (Magna Graph, Micron Separations, Inc., Westborough, MA) and cross-linked in UV light. The blots were probed with an Arabidopsis cDNA that was identical to that of the Arabidopsis PIP 5-kinase cDNA (Satterlee and Sussman, 1997) radiolabeled by random
priming with [ -32P]dCTP. Hybridizations were
carried out at 42°C in hybridization buffer containing 50% (v/v)
formamide. The blots were washed sequentially in 1× SSPE (10 mM
NaH2PO4, 1 mM EDTA, and 149 mM NaCl)
containing 0.1% (w/v) SDS, followed by a final wash in 0.1× SSPE and
0.1% (w/v) SDS at 55°C. Autoradiography was carried out using Kodak X-Omat autoradiography film.
Lipid Phosphorylation Changes with the Time in Culture To detect lipid-mediated signaling events in response to a stimulus, basal levels of key phospholipids (PA, PIP, and PIP2) had to be characterized. When [2-3H]myo-inositol or 32Pi was added to G. sulphuraria cultures at d 7 or 12 of the growth period, only trace amounts of radiolabeled phosphoinositides could be detected. Because of the low level of incorporation of the radiolabel into phospholipids and to avoid the difficulties in the identification of comigrating phospholipids after in vivo labeling, we conducted in vitro lipid phosphorylation assays to study the phosphoinositide metabolism of G. sulphuraria. Microsomal membranes were isolated from G. sulphuraria cells at 5, 7, 10, and 12 d after transfer and incubated with [-32P]ATP. The
changes in the lipid-phosphorylation profile with the culture age are
illustrated in Figure 1A. In vitro
lipid-phosphorylation products were quantified using a scanner
(Bioscan) (Fig. 1B). Between 5 to 7 d and 12 d of the 20-d
culture period, PA decreased from about 40% of the total
32P-labeled phospholipid to less than 5%. In
contrast, at the same time PIP2 increased from
about 1% at d 7 to 9% by d 12 (Fig. 1B). The most prevalent
phospholipid formed in vitro during the growth period was PIP, with
values between 62% and 83% of the total phosphorylated lipid.
PIP2 Biosynthesis and PIP2 Levels
Increase with Time in Culture
Regulation of PIP Kinase Activity
Phosphoinositide Metabolism of G. sulphuraria Changes in Response to the Addition of Solutions to the Culture Medium G. sulphuraria cells were very sensitive to the addition of solutions to the culture medium. Because the solutions used for osmostimulation were prepared with conditioned medium, before studying the effects of osmostimulation we had to characterize the effects of adding conditioned medium alone. Figure 3 illustrates changes in PIP2 formation and IP3 levels of d-12 cells that were untreated, treated with conditioned medium, or osmostimulated by the addition of 25 mM KCl in conditioned medium. The basal PIP2 formation from endogenous substrate for untreated cells was 200 ± 30 pmol min 1
mg1 protein (Fig. 3A, dashed line). When d-12
cells were treated with conditioned medium, there was a transient 50%
increase of in vitro PIP2 formation. For a
comparison, the transient increase of in vitro
PIP2 formation in response to stimulation of d-12 cells with 25 mM KCl in conditioned medium is
also shown. Although the changes of in vitro PIP2
formation in microsomes from d-12 cells resulting from
conditioned-medium treatment were small compared with those observed
after osmostimulation, they were reproducible. Therefore, controls with
conditioned medium were used for all time-course experiments to correct
for the described effects. Conditioned medium had no effect on the
specific PIP kinase activity of d-7 cells (data not shown).
Hypertonic Stimulation Results in a Transient Increase in IP3 Large differences in PIP kinase specific activity were detected between G. sulphuraria cells from 7 and 12 d after transfer (compare Fig. 2A). Because the ability of the cells to synthesize PIP2 was so different, we sought to determine whether there would be differences in their ability to produce IP3 in response to a stimulus. Cells from 7 and 12 d after transfer were stimulated by adding KCl, NaCl, or methyl-Man in conditioned culture medium (Fig. 4). For a better comparison, the data shown in Figure 4 are reported relative to the conditioned-medium control at each time point. Because methyl-Man gave identical responses as equal osmolal concentrations of KCl or NaCl, salt effects can be ruled out.
An Increase in PIP2 Biosynthesis Precedes the Increase
in IP3 in Osmostimulated d-12 Cells
There were distinct differences in lipid phosphorylation of microsomes from cells harvested throughout the growth cycle. First, at the transition from the logarithmic to the stationary phase of growth, PA formation decreased. Because microsomal DAG levels were not significantly different between growth stages of G. sulphuraria, the decrease in PA production must have resulted from a decrease in DAG kinase activity. The decreased DAG kinase activity in stationary G. sulphuraria cells may reflect a decreased demand for membrane lipid biosynthesis.
* Corresponding author; e-mail wendy_boss{at}ncsu.edu; fax 1-919-515-3436. Received August 31, 1998;
accepted December 18, 1998.
Abbreviations: DAG, diacylglycerol. IP3, inositol-1,4,5-trisphosphate. PA, phosphatidic acid. PCA, perchloric acid. PI, phosphatidylinositol. PIP, phosphatidylinositol monophosphate. PIP2, phosphatidylinositol-4,5-bisphosphate.
We thank Dr. Phil Majerus (Washington University School of Medicine) for the inositol polyphosphate 5-phosphatase I cDNA and Dr. Bjørn Drøbak (John Innes Institute, Norwich, UK) for helpful discussion.
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Abstract
Introduction
)
or methyl-Man (
) in conditioned medium. The effects of the
stimulation on in vitro PIP2 formation by microsomes from
7- and 12-d-old cells are shown in A and B, respectively. A transient
increase in PIP2 formation in vitro was observed only in
12-d-old cells. Data represent the means of 6 values from three
experiments. Vertical bars indicate the range. C and D, In vivo levels
of IP3 were measured in whole cells after the addition of
25 mM KCl in conditioned medium (
) to the cultures. The
effects of both treatments on 7- and 12-d-old cells are illustrated in
C and D, respectively. At both stages, a transient decrease in
IP3 levels could be observed, followed by a transient
increase. Data are the averages of 10 values from five experiments.
Vertical bars indicate the range.
