May 27, 1992 - The PIP and PIP2 phospholipase C in wheat root plasma membranes was .... pase C activity and not phospholipase D or some other enzyme ...
Received for publication May 27, 1992 Accepted June 26, 1992
Plant Physiol. (1992) 100, 1296-1303 0032-0889/92/100/1296/08/$01 .00/0
Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.4' Orientation of Active Site and Activation by Ca2" and Mg2" Christophe Pical, Anna Stina Sandelius, Per-Martin Melin, and Marianne Sommarin* Department of Plant Biochemistry, University of Lund, P.O. Box 7007, S-220 07 Lund, Sweden (C.P., M.S.); Department of Plant Physiology, Botanical Institute, University of Goteborg, Carl Skottsbergs Gata 22, S-413 19 Goteborg, Sweden (A.S.S.); and Department of Biochemistry, Chemical Centre, University of Lund, P.O. Box 124, S-220 01 Lund, Sweden (P.-M. M.). ABSTRACT
animal cells, phospholipase C-catalyzed production of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol from PIP22 is a key event in the transduction of agonist-dependent signals over the plasma membrane (1, 3, 15). PIP2 is formed by a two-step phosphorylation of PI by PI kinase and PIP kinase, enzymes that are also present in plant plasma membranes (18, 24, 29). Because phospholipase C, preferentially active on PIP and PIP2, has also been identified in plasma membranes from plants (7, 17, 30), there is an enzymic basis for signal transduction through hydrolysis of polyphosphoinositides. However, very little is known about the mechanism operating in the plant plasma membrane, in part due to the scant knowledge about phospholipase C. In this communication, we present evidence for the localization of polyphosphoinositide phospholipase C activity in wheat (Triticum aestivum L. cv Drabant) plasma membranes to the cytoplasmic surface of the membrane. We also present a further characterization of the in vitro properties of the enzyme activity.
Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (7080% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/ v) sodium deoxycholate, whereas in fractions enriched in insideout (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP2 phospholipase C was dependent on Ca21 with maximum activity at 10 to 100 Mm free Ca2 and half-maximal activation at 0.1 to 1 Mm free Ca2. In the presence of 10 gM Ca21, 1 to 2 mM MgCI2 or MgSO4 further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba2", Co2+, Cu2+, Mn2+, Ni2+, and Zn2+) inhibited the enzyme activity. The stimulatory effect by Mg2+ was observed also when 35 mm NaCI was included. Thus, the PIP and PIP2 phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP2 as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca2 , with maximum activity at 1 mm CaC12, and could not be further stimulated by Mg2+.
MATERIALS AND METHODS
Materials
[2-3H]PI, [2-3H]PIP, and [2-3H]PIP2 were from Amersham. Unlabeled phosphoinositides were purified from a phosphoinositide-rich brain extract (Sigma type I) by chromatography on immobilized neomycin (26). All other chemicals used were of analytical grade. Plant Material Wheat seedlings (Triticum aestivum L. cv Drabant) were grown hydroponically in the dark for 8 d (28).
Plant phosphoinositide metabolism shows many similarities to the metabolism involved in mammalian polyphosphoinositide transmembrane signaling systems (8, 12, 25). In
Preparation of Plasma Membranes Highly purified plasma membranes were isolated from the microsomal fraction (10,000-30,000g pellet) of wheat shoots
'This work was supported by grants from the Swedish Council for Forestry and Agricultural Research (M.S.), the Swedish Natural Science Research Council (A.S.S.), and the Carl Tesdorpf Foundation (M.S.).
2 Abbreviations: PIP2, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate. 1296
POLYPHOSPHOINOSITIDE PHOSPHOLIPASE C IN PLASMA MEMBRANES
and roots by partitioning in aqueous polymer two-phase systems as described earlier (28) with minor modifications (22). The homogenization medium was 50 mm Mops-1,3bis[tris (hydroxymethyl)methylamino]propane (pH 7.5), 0.25 M sucrose, 5 mM EDTA, 0.2% (w/w) BSA (protease free), 0.2% (w/v) casein (Sigma type I, enzymic hydrolysate that was boiled for 10 min before addition to the medium), 2 mm DTE, and 0.5 mm PMSF. The final upper phases consisting of 85 to 95% tightly sealed, right-side-out (exposing the original apoplastic side out) shoot and root plasma membrane vesicles were diluted severalfold in 0.25 M sucrose, 10 mM Hepes-KOH (pH 7.5), and 1 mm DTE. They were pelleted at 100,000g for 1 h, resuspended in the same medium to 15 to 20 mg of protein m-1', and used within 4 h of isolation or immediately frozen and stored in aliquots in liquid nitrogen until use. Alternatively, when inside-out plasma membrane vesicles from wheat roots were to be produced and isolated, the final upper phases were washed and resuspended in 0.25 M sucrose, 5 mm potassium phosphate (pH 7.8), 50 mi KCl, and 1 mM DTE. Formation and Isolation of Inside-Out Plasma Membrane Vesicles
The highly purified right-side-out plasma membrane vesicles were frozen in liquid nitrogen and thawed in water at 200C a total of four times to produce a mixture of inside-out and right-side-out vesicles (21). The freeze/thawed plasma membranes (8-10 mg of membrane protein in 0.8 mL) were added to a 7.2-g phase mixture to give an 8.0-g phase system with a final composition of 7.0% (w/w) dextran T500, 7.0% (w/w) PEG 3350, 0.25 M sucrose, 5 mm potassium phosphate (pH 7.8), 7.5 mm KCl, and 1 mm DTE (at 40C). The membranes were further subfractionated by countercurrent distribution, with three transfers of the upper phase to fresh lower phases, to produce one fraction enriched in sealed, insideout vesicles and another fraction enriched in sealed, rightside-out plasma membrane vesicles (11, 19, 21). As when isolating the original plasma membrane fraction, the rightside-out vesicles partitioned to the upper phase. In contrast, the inside-out plasma membrane vesicles have surface properties similar to other intracellular membranes and, therefore, partitioned to the lower phase plus interface. This difference in surface properties made the separation of inside-out and right-side-out vesicles possible. The fractions enriched in sealed, inside-out vesicles and in sealed, right-side-out vesicles were diluted about 10-fold with 0.25 M sucrose, 10 mM Hepes-KOH (pH 7.5), and 1 mirm DTE before pelleting the plasma membranes at 100,000g for 1 h. The pellets were resuspended in the same medium and immediately used for phospholipase C assays and control of sidedness by ATPase latency and proton-pumping capacity. Proton uptake into the vesicles was measured as the decrease in absorbance at 495 nm of the ApH probe acridine orange (22). ATPase activity was measured in the presence and absence of 0.05% (w/v) Brij 58 to estimate latency, using an ATP-generating system in which ADP produced was coupled to oxidation of NADH, which was followed at 340 nm (23).
1 297
Phospholipase C Assay The standard incubation mixture contained 50 mm Trismaleate (pH 6.0), 10 AM free Ca2" (Ca2+/EGTA mixture [20]), 0.2 mm 3H-labeled phosphoinositide (1000 dpm/nmol) in micellar solution, and an appropriate amount of enzyme protein in a final volume of 50 ,tL (17). The phosphoinositide solutions were prepared by evaporating the lipids in solvent to dryness under a stream of nitrogen, followed by sonication in a water bath for 10 min in the incubation buffer. The reaction was started by addition of phosphoinositide or enzyme and performed at 250C, usually for 5 min. It was stopped by addition of 1 mL of chloroform:methanol (2:1, v/ v), followed by 250 ,uL of 1 M HCI. After the mixture was vortexed and centrifuged for 30 s in a Beckman microfuge, 400 ,tL of the upper phase was transferred to a scintillation vial and supplemented with 2.5 mL of Beckman Ready Safe, before the radioactivity was measured in a liquid scintillation counter. Values presented have been recalculated to correspond to the total upper phase. The analyses were performed in duplicate when the reaction rate was proportional to incubation time and amount of protein. Reaction products formed were analyzed by ion-exchange chromatography on Dowex AG1-X8 columns (9). 3H-Labeled phosphoinositides remaining after incubation were also checked by TLC on silica gel H (Merck) impregnated with potassium oxalate (10, 14).
Protein Determination Protein was determined according to the method of Bearden (2) with BSA as the standard. RESULTS
Effects of Sodium Deoxycholate Plant plasma membranes isolated from microsomal fractions by aqueous polymer two-phase partitioning are obtained as sealed vesicles and oriented with the apoplastic side out, i.e. they are right-side-out vesicles. When using these sealed membrane vesicles to study an enzyme with its active site located on the cytoplasmic surface, the structural barrier needs to be made permeable for the necessary enzyme substrates and/or ligands to obtain an accurate measure of the total activity of the enzyme being investigated. Usually a detergent is used as membrane perturbant. We previously showed that plasma membrane vesicles isolated from wheat shoots and roots possess polyphosphoinositide phospholipase C activity (17) as determined in the absence of detergent. To estimate the total polyphosphoinositide phospholipase C activity present in the membrane, we tested the effect of sodium deoxycholate (a detergent often used when assaying animal phospholipases C) over a concentration range (Fig. 1). The enzyme activity was increased severalfold, using either PIP (Fig. 1A) or PIP2 (Fig. 1B) as substrate, in both shoot and root plasma membrane vesicles when 0.020 to 0.025% (w/v) sodium deoxycholate was included. Higher concentrations were inhibitory, and virtually no activity was detected at 0.15% detergent.
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PICAL ET AL.
to a number of freeze/thaw cycles, the proportion of insideout (cytoplasmic side-out) vesicles increases (11, 21). A freeze/thawed mixture of vesicles obtained from wheat root
'i Io*X
plasma membranes was further subfractionated in an aqueous polymer two-phase system to obtain one fraction highly enriched in sealed, inside-out and one fraction enriched in sealed, right-side-out vesicles (see ref. 19 for details). PIP and PIP2 phospholipase C activities, assayed without detergent (nonlatent activity), were 2.6 and 2.5 times higher, respectively, in the inside-out fraction than corresponding activities in the right-side-out fraction (Table I, experiment I). The corresponding factors were 3.9 and 2.4 for proton pumping and nonlatent ATPase activity, respectively. In another experiment, proton pumping, nonlatent ATPase, and PIP phospholipase C activities were 2.9, 3.2, and 2.9 times higher, respectively, in the inside-out fraction relative to the rightside-out fraction (Table I, experiment II). Sodium deoxycholate increased the PIP phospholipase C activity to a higher degree in the right-side-out (3-fold) than in the inside-out (1.4-fold) fraction (Fig. 2). The results presented in Table I and Figure 2 lead us to conclude that the active site of polyphosphoinositide phospholipase C indeed is located on the inner surface of the plant plasma membrane. Using PIP phospholipase C as a marker for the inner surface of the plasma membrane, we calculated a figure of 71% inside-out vesicles for the inside-out fraction (Fig. 2). This is in agreement with the figure of 69 to 77% inside-out vesicles obtained by assaying ATPase latency (Table I).
T~~~~~~~~~~~ 200
E
E
100
0
0.05
0.10 0.15
0.10 0.15
0.05
0
[Sodium deoxycholatel, % (w/v)
Figure 1. Effect of sodium deoxycholate on polyphosphoinositide phospholipase C activity. Plasma membranes were isolated from shoots (A) and roots (0) of dark-grown wheat. Freshly isolated plasma membranes were incubated with 0.2 mm PIP (A) or PIP2 (B) in the presence of 10 LM free Ca2+. The error range was between 1 and 4% from each average value presented in figure.
Localization of the Active Site
The orientation of plant plasma membrane vesicles is usually determined by assaying H+-ATPase activity (a marker for the cytoplasmic surface) in the absence and presence of the detergent Triton X-100 or Brij 58 (23, 34). The percentage of right-side-out vesicles is calculated from the ratio of latent activity (difference in activity measured with and without detergent) to total activity (activity measured with detergent). When the isolated plasma membrane vesicles are subjected
Effects of Mg2" The PIP and PIP2 phospholipase C in wheat root plasma membranes was activated by Mg2+ (16). However, the stim-
Table I. Proton Pumping, ATPase, and Polyphosphoinositide Phospholipase C Activities in Plasma Membrane Fractions Isolated from Roots of Dark-Grown Wheat A frozen/thawed plasma membrane fraction consisting of a mixture of inside-out and right-sideout vesicles was further subfractionated into two fractions by countercurrent distribution. One fraction was enriched in inside-out vesicles (10), and the other was enriched in right-side-out vesicles (RO). Plasma membrane fractions from two independent experiments (I, II) were assayed. ATPase activities were assayed in the absence or presence of 0.05% (w/v) of the detergent Brij 58, and the percentage of 10 was calculated as described in the text. PIP and PIP2 phospholipase C activities were assayed without detergent in the presence of 100,M free Ca2+. Proton
ATPase
Pumping
Phospholipase C PIP
PIP2
II I
Brij 58 +
AA495
mg
nmol mg-'
Frozen/thawed vesicles
1.07
0.72
469
10
2.12
1.23
701 (69% 287 (29% 2.4
RO
0.54
0.42
IO/RO
3.9
2.9
+
min-'
nmol mg-' min-'
990
309
892
104
198
160
1011 10) 1000 10)
629
821
200
422
276
76
146
110
(77% 10)
195 910 (21% 10) 3.2
2.6
2.9
2.5
POLYPHOSPHOINOSITIDE PHOSPHOLIPASE C IN PLASMA MEMBRANES
1299
Ir
0
Table IL. Effects of Divalent Cations on Polyphosphoinositide Phospholipase C Activities Plasma membrane fractions were isolated from shoots of darkgrown wheat. The activities were measured without (control) or with 1.5 mm divalent salt with 0.2 mm lipid substrate and 10 AM free Ca21 in the absence or presence of 0.01% (w/v) sodium deoxycholate (Na-doc). PIP2 phospholipase C was also determined in the presence of 35 mM NaCI with the above additions. The control activities are set to 100% with the actual values expressed in nanomoles per milligram of protein and minutes in parentheses.
300 F
I.) L._ 0
200 F
100 F
(.41
PIP
0
0.02
0.01
0
0.03
0.04
0.05
Figure 2. Effect of sodium deoxycholate on PIP phospholipase C activity in plasma membrane fractions of defined vesicular orientation, isolated from roots of dark-grown wheat. Plasma membrane vesicles, obtained by aqueous polymer two-phase partitioning, were subjected to four freeze/thaw cycles. The resulting membrane vesicles were subfractionated (see "Materials and Methods") into one fraction of predominantly right-side-out vesicles (0) and one fraction of predominantly inside-out vesicles (0), as determined by ATPase latency. The PIP phospholipase C activities in the absence of detergent were 136 and 378 nmol mg-1 min-', respectively, for the right-side-out and inside-out plasma membranes. They were determined with 10 jiM free Ca2' and 0.2 mm lipid substrate. The error range was between 1 and 4% from each average value presented in figure.
ulation by Mg2" could be observed only if Ca2" also was present. The maximum stimulation was 1.8- and 2.3-fold, respectively, for the PIP2- and the PIP-specific activities of the root enzyme at 2 mM MgCl2 in the absence of detergent (Fig. 3A). In the presence of detergent, the stimulation was 1.5-fold for both activities. A similar stimulation by MgC12
600AB
E 400
E
200
o
2
4
6
8
[Mg2lJ,
10 15
0
2
+35 mm NaCI
-Na-doc +Na-doc -Na-doc +Na-doc
[Sodium deoxycholatel, % (w/v)
E
PIP2
Salt
4
mM
Figure 3. Effect of Mg2+ on polyphosphoinositide phospholipase C activity in freshly isolated plasma membranes from dark-grown wheat. The incubations contained 10 jiM free Ca2', and the activities were determined in the absence (open symbols) or presence (closed symbols) of 0.025% (w/v) sodium deoxycholate. A, Root plasma membranes incubated with 0.2 mM PIP2 (0, *) or PIP (O, *). B, Shoot plasma membranes incubated with 0.2 mM PIP2 (A, A). The error range was between 1 and 4% from each average value presented in figure.
Control
100
(248) 143 152
100 (337) 126 119 111
MgCI2 MgSO4 MnCI2 70 30 78 CoCI2 -a BaCI2 NiC12 CuCI2 ZnCI2 203 184 CaCI2 a , Not determined.
-Na-doc +Na-doc
100
100
100
(282) 151 151
(389) 137 151 36 33 28
(228) 125 128 26 20 29
89 8 23 74
45
124 61 14 56 67
8 75
103
21 28 18 87 35 20 65
100 (285) 114
was also seen for the shoot enzyme using PIP2 (Fig. 3B, Table II) and PIP (Table II) as substrates. The degree of stimulation varied between plasma membrane preparations, but maximum stimulation was always obtained at 1 to 2 mM MgCl2. MgCl2 concentrations greater than 4 mm were inhibitory to membrane-bound phospholipase C. This inhibition could be due to interaction of Mg2+ with the polyphosphoinositides, perhaps by replacement of bound Ca2 . Strong interactions between PIP2 and Ca2' and Mg2' have been reported (5). The effects of several divalent salts on the shoot polyphosphoinositide phospholipase C activity were tested (data not shown). With PIP2 as substrate, the stimulation seemed cation specific for Mg2", because MgCl2 and MgSO4 stimulated to the same degree, whereas other divalent cations were more or less inhibitory. Monovalent salts (2 mm NaF, KC1, KI, and NaCl) did not significantly affect the enzyme activity (data not shown). Also, in the presence of 35 mm NaCl, the PIP2 phospholipase C activity was stimulated by Mg2' but not by other divalent cations (Table II). However, in the stimulation of PIP phospholipase C, 1.5 mM Mg2+ could be replaced by equimolar Ca2+; Mn2+ was slightly stimulatory (+11%) in the presence of detergent but inhibitory in its absence. No stimulation by Mg2+ of the PI-specific phospholipase C activity in shoot or root plasma membranes could be detected under the experimental conditions tested, i.e. in the presence of 10 or 303 jiM Ca2' and in the presence or absence of sodium deoxycholate (data not shown). Analyses of the water-soluble reaction products by ionexchange chromatography revealed that more than 85% of the radioactivity eluted in the positions of inositol bisphosphate and inositol trisphosphate when PIP and PIP2, respec-
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1300
100 7~~~~~~~~~~~~
cm
0 LI-
E -
400
B
E
300
200
100
0
1
0.1
10
100
1000
[Ca2", PiM Figure 4. Effect of free Ca2+ on (A) PIP and (B) PIP2 phospholipase C activity. Freshly isolated plasma membranes from shoots (A, A) or roots (0, 0) of dark-grown wheat were used as enzyme source. The activities were determined with 0.2 mm lipid substrate in the absence (open symbols) or presence (closed symbols) of 0.025% (w/v) sodium deoxycholate. The error range was between 1 and 4% from each average value presented in figure.
tively, were the substrates. The product pattern was the same whether Mg2" was present in the reaction or not. This control verified that the reaction investigated was due to phospholipase C activity and not phospholipase D or some other enzyme activity. No polyphosphoinositide phosphatase activities were detected under our assay conditions because no dephosphorylation of either PIP or PIP2 was found by TLC of the acid chloroform-methanol-soluble material remaining after incubation.
4A). A similar result was obtained for shoot PIP2 phospholipase C (Fig. 4B). The root PIP2 phospholipase C activity continued to increase above 10 Mm Ca2", with a 35% increase between 10 and 100 AM. At 1 mm Ca2 , the activity of shoot and root PIP phospholipase C was markedly increased, regardless of whether detergent was present or not (Fig. 4A), whereas a marked decrease in activity was found for the PIP2 phospholipase C (Fig. 4B). However, the activities obtained with the polyphosphoinositides at 1 mm Ca2+ should be interpreted with caution because precipitates are easily formed in the assay medium, causing the results to vary considerably between experiments, although the trend was always the same. We also observed that the interaction between Ca2' and the enzyme seemed to be impaired at very low concentrations of the divalent ion if sodium deoxycholate was present during assay. In the presence of detergent, for instance, only approximately 15% of the activity found at 10 $M Ca2+ was detected at 0.1 $M Ca2+ for shoot PIP phospholipase C, as compared with 40% in its absence. For shoot PIP2 phospholipase C, corresponding figures were 22 and 46%. Similar results were obtained for the root enzyme. As shown for shoot plasma membranes, addition of 1.5 mm MgCl2 affected the sensitivity of the polyphosphoinositide phospholipase C activity to Ca2+ in much the same way as sodium deoxycholate (Fig. 5). However, half-maximal activation of the polyphosphoinositide phospholipases C occurred at 0.1 to 1 ,UM Ca2+ under any assay condition tested in both shoot and root plasma membranes. The PI phospholipase C activity in wheat plasma membranes was considerably lower than the activities observed for the PIP and PIP2 phospholipase C (cf. Figs. 6 and 7 with Figs. 1 and 4). As with polyphosphoinositide phospholipase C, addition of 0.025% sodium deoxycholate increased the PI phospholipase C activity at 10 Mm Ca2" but also at higher concentrations of Ca2" (300 and 1000 AM) (Fig. 6). The root enzyme was more sensitive to inhibition by higher concentrations of sodium deoxycholate than the shoot enzyme. The
500
400 c E 300
Effects of Ca21
E
As shown before, the wheat polyphosphoinositide phospholipase C is dependent on Ca2+ for activity (16, 17). Here, we analyzed the Ca2' dependency in more detail in the absence and presence of sodium deoxycholate in both shoot and root plasma membranes. In the absence of detergent, the shapes of the Ca2' dependency curves are similar for polyphosphoinositide phospholipase C in shoot and root plasma membranes up to 100 gM free Ca2+ (Fig. 4). A rapid increase in activity was obtained from zero (in the presence of 1 mm EGTA) to 1 AM free Ca2 followed by a slower increase up to 100 Mm using either PIP or PIP2 as substrate. In the presence of sodium deoxycholate, the activity of shoot and root PIP phospholipase C reached maximum at 10 ,
Mm
free Ca2+ with no further increase up to 100 AM Ca2+ (Fig.
7-
200
E c
100 0
* 0
0.1
1
10
100
[Ca2", PM Figure 5. Effect of free Ca2", in the presence of 1.5 mM Mg2+, on polyphosphoinositide phospholipase C activity in freshly isolated plasma membranes from shoots of dark-grown wheat. Activity was determined with 0.2 mM PIP2 (A, A) or PIP (O, *) in the absence (open symbols) or presence (closed symbols) of 0.025% (w/v) sodium deoxycholate. The error range was between 1 and 4% from each average value presented in figure.
POLYPHOSPHOINOSITIDE PHOSPHOLIPASE C IN PLASMA MEMBRANES .Or-
1301
I -
II
I
Table I1. Polyphosphoinositide Phospholipase C Activities in Plasma Membrane Fractions Isolated from Various Plant Sources by Aqueous Polymer Two-Phase Partitioning The plasma membrane fractions were stored in liquid nitrogen until used. The activities were determined with 0.2 mM lipid substrate and 10 uM free Ca24 in the absence or presence of 0.01% (w/ v) sodium deoxycholate (Na-doc).
8
E
6
E
4
PIP
E 2
nI tI v
0
-Na-doc +Na-doc -Na-doc +Na-doc nmol mg-1 min-' nmol mg-' min-
* .......,0 ............. .
//..* ... ~~~~ I,,.
.
~ _,
0.05
[Sodium deoxycholatel,
PIP2
Plasma Membrane Source
"I@
,-'
0.10 0.15 X
(w/v)
Figure 6. Effect of sodium deoxycholate on Pi phospholipase C activity. Plasma membranes were isolated from shoots (A) or roots (O) of dark-grown wheat by two-phase partitioning. The freshly isolated plasma membranes were incubated with 0.2 mm Pi in the presence of 1 mm (solid lines), 300 gM (dashed lines), or 10 Mm (dotted line) Ca2". The error range was between 1 and 4% from each average value presented in figure.
Ca2" dependency of the PI phospholipase C exhibited a biphasic profile (Fig. 7). A linear increase in activity was obtained between 10 and 300 uM Ca2" whether detergent was present or not. Between 300 and 1000 uM Ca2", the activity increased considerably with no appreciable further increase above 1000 MM.
Wheat roots Oat roots Barley roots Maize mesocotyls Spruce roots Cauliflower inflorescences Sugar beet roots Sugar beet leaves Spinach leaves a , Not determined.
68 55 a
136 114
43 131 136 104
49 214 162 198
131 80 48 122 40 49 270 227 166
199 196 88 116 36 67 344 257 281
the better substrate of the two. Plasma membranes isolated from sugarbeet leaves or roots and spinach leaves exhibited the highest specific activities, and spruce roots and cauliflower inflorescences had the lowest ones. In this experiment, we used plasma membrane vesicles that had been frozen and thawed several times, thus explaining the low latency obtained for some fractions.
Occurrence of Polyphosphoinositide Phospholipase C Plasma membrane fractions isolated from several different plant sources were assayed for polyphosphoinositide phospholipase C activity (Table III). Such activity was present in all fractions tested. The enzyme was active toward both polyphosphoinositides but, when compared, PIP2 was always 10
E 6
0, E
4
E 2 0
[Ca2], PM Figure 7. Effect of free Ca2" on Pi phospholipase C activity in freshly isolated plasma membranes from shoots (A, A) or roots (0, *) of dark-grown wheat. The activities were determined with 0.2 mM lipid substrate in the absence (open symbols) or presence (closed symbols) of 0.025% (w/v) sodium deoxycholate. The error range was between 1 and 4% from each average value presented in figure.
DISCUSSION
Polyphosphoinositide-specific phospholipase C activity is present in membrane fractions isolated from several plant sources. The highest activity was obtained in plasma membrane fractions (Table III; refs. 17 and 30). Because of the presence of phosphomonoesterases and/or phospholipase D in crude wheat membrane fractions and in the cytosol, we were not able to determine whether the only location of the polyphosphoinositide phospholipase C was in the plasma membrane or whether it is also present in other subcellular compartments. However, the enzyme activity was 10 times higher in plasma membranes than in a microsomal fraction (i.e. the bottom phase membranes) depleted of right-side-out plasma membrane vesicles (but not of inside-out ones). This correlated well with the 10-fold enrichment of glucan synthase II activity (a marker for plant plasma membranes) in the plasma membrane over the microsomal fraction. Therefore, at least a major part of the polyphosphoinositide phospholipase C activity was located in the plant plasma membrane. Similar results were reported for the alga Dunaliella salina (7). The ionic detergent sodium deoxycholate increased the polyphosphoinositide phospholipase C activity 4- to 5-fold at a concentration of 0.020 to 0.025% (w/v) in freshly isolated plasma membrane vesicles (Fig. 1). Triton X-100, a nonionic detergent, increased the enzyme activity in a similar way, whereas other detergents tested were without effects or were inhibitory (16). The increase in enzyme activity obtained in
1 302
PICAL ET AL.
the presence of sodium deoxycholate can be explained in several ways: (a) the enzyme is stimulated by the detergent; (b) the lipid substrate-detergent complex is a better substrate for the enzyme than the lipid substrate alone; or (c) the detergent opens up a structural barrier and exposes latent active sites. The first two explanations do not seem valid because higher concentrations of detergent severely inhibited the enzyme, and the detergent also seemed to affect the interaction between Ca2" and enzyme at low Ca2" levels. The third explanation appears reasonable because our plasma membrane fractions, isolated from a microsomal fraction by aqueous polymer two-phase partitioning, consists mainly of tightly sealed, right-side-out vesicles. Thus, inclusion of a suitable detergent in the enzyme assay would allow access by impermeable enzyme substrates to an enzyme with its substrate-binding site located on the inner surface of the vesicle. The localization of polyphosphoinositide phospholipase C activity on the cytosolic surface of the plasma membrane has always been logically assumed but not yet demonstrated for animal or plant plasma membranes. To examine further whether polyphosphoinositide phospholipase C is localized in the cytoplasmic leaflet of the plasma membrane, we used the recently developed method to produce and subsequently separate inside-out and rightside-out plasma membrane vesicles (11, 19, 21). The two vesicle populations were assayed for proton pumping and nonlatent ATPase activities, which are both considered to be markers for the cytoplasmic surface of the plasma membrane (27, 34) and for polyphosphoinositide phospholipase C activity. The close correlation between the enrichment of all three activities in the inside-out as compared with the right-sideout vesicle fraction (Table I), together with the low increase in PIP2 phospholipase C activity in the inside-out fraction by inclusion of detergent in the assay (Fig. 2), clearly showed that the active site of polyphosphoinositide phospholipase C, indeed, is located on the inner cytoplasmic surface of the plant plasma membrane. The PIP and PIP2 but not the PI phospholipase C activity was significantly enhanced by 1 to 2 mM Mg2+. This stimulation was observed only if Ca2+ was also present. A similar enhancement of PIP2 phospholipase C activity by Mg2+ was observed in rat cerebral-cortical membranes (13) and rat liver plasma membranes (31). The effect of Mg2+ was to potentiate the stimulation by nonhydrolyzable GTP analogs on the PIP2 phospholipase C activity because the Mg2+ ions are essential for the binding of GTP and its analogs to G proteins (31). However, we were not able to detect any reproducible effects of GTP-y-S on the polyphosphoinositide phospholipase C activity in wheat plasma membranes in the presence or absence of Mg2+. Likewise, no effect of GTP-'y-S was detected in oat plasma membranes (30). In plasma membranes from the unicellular alga D. salina, 100 Mm GTP-'y-S about doubled the PIP2 phospholipase C activity over a wide range of Ca21 concentrations (7), but unlike the rat liver plasma membrane enzyme, this stimulation was also observed at zero (EGTA only) Ca2+ levels. GTP-binding proteins are present in plant plasma membranes (4), but very little is presently known about their physiological function. Recently, a blue-lightactivated GTP-binding protein was detected in etiolated pea plasma membranes (33).
Plant Physiol. Vol. 100, 1992
The plant plasma membrane polyphosphoinositide phospholipase C was dependent on Ca2" for activity and was very sensitive to changes in Ca2` concentrations in the micromolar range. When the Ca2" dependency was analyzed between zero (EGTA) and 100 FM free Ca2" at low ionic strength, the half-maximal activation of polyphosphoinositide hydrolysis ranged between 0.1 and 1 AM Ca2" whether Mg2` and/or sodium deoxycholate was present or not. At low ionic strength, an apparent activation constant of 2 MM Ca2+ was reported for oat plasma membranes in the absence of Mg2+ and detergent (30), and in D. salina plasma membranes halfmaximal activation below 1 AM Ca2+ was obtained in the presence of sodium cholate (7). In prelabeled rat liver plasma membranes, half-maximal activities were at 0.1 AM Ca2+ in the presence or absence of 2.5 mm Mg2", whereas 10 mM Mg2+ shifted the half-maximal activation up to 10 AM Ca2+ (32). In prelabeled human erythrocyte membranes, the halfmaximal activation was between 1 and 3 yM Ca2+ at low ionic strength (6), but at intracellular ionic strength and in the presence of MgATP, no phospholipase C activity was detected below 100 MM Ca2+ in these membranes. However, when exogenously added lipid was used as a substrate, a half-maximal activation at approximately 1 MM Ca2+ at high ionic strength and 2.5 mM Mg2+ and sodium cholate was observed in rat liver plasma membranes (31). The purified wheat enzyme was quite active at 10 MM Ca2+ at high ionic strength and in the presence of 4 mM Mg2+, but the affinity toward Ca2+ was clearly lower than in the absence of Mg2+ (16). Thus, the plant enzyme, like the rat liver enzyme, may well be active in unstimulated cells (low Ca2+ levels) in vivo, and we cannot exclude the possibility that the enzyme can be further activated as a result of cell stimulation (higher Ca2+ levels). However, care must be taken when extrapolating results obtained from isolated plasma membranes to the situation in the intact cell. Other regulatory factors, such as substrate supply and factors affecting the coupling of effectors to putative receptors/G proteins and polyphosphoinositide phospholipase C are likely also to play a role. The role of such other factors are unknown at present for the plant polyphosphoinositide phospholipase C. ACKNOWLEDGMENTS We are very grateful to Mrs. Hildegun Lundberg and Mrs. Inger Rohdin for their excellent technical assistance and to Dr. Bengt Jergil for discussions and critical reading of the manuscript.
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