Phosphorylation-dephosphorylation of membrane proteins controls the microsomal H+-ATPase activity of corn roots

Plant Science, 40 (1985) 153--159

153

Elsevier Scientific Publishers Ireland Ltd.

PHOSPHORYLATION-DEPHOSPHORYLATION OF MEMBRANE T H E M I C R O S O M A L H÷-ATPase A C T I V I T Y O F C O R N R O O T S

PROTEINS

CONTROLS

GRAZIANO ZOCCHI Istituto di Chimica Agraria, Universita degli Studi, Via Celoria, 2, 20133 Milano (Italy)

(Received April 2nd, 1985) (Revision received June 7th, 1985) (Accepted June 7th, 1985} The Mg2*-dependent H÷-ATPase activity of a sealed microsomal vesicle fraction isolated from corn (Zea mays L.) roots appears to be controlled by a phosphorylation-dephosphorylation cycle. Phosphorylation of the microsomal fraction is carried out by a Ca2*/calmodulin (CaM)-stimulated process. The H÷-ATPase activity decreases with increasing phosphorylation of the membranes and becomes only slightly uncoupled by ionophores and less inhibited by dieyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), NO 3- and vanadate. The inhibitory effect of phosphorylation is greater on the NO 3 -sensitive H+-ATPase activity than on the vanadate-sensitive activity. Restoration of H÷-ATPase activity is achieved by allowing the phosphorylated membranes to dephosphorylate either in the absence or presence of exogenous alkaline phosphatase. Moreover, the presence of fluphenazine during the Ca2÷/CaM-stimulated treatment inhibits membrane phosphorylation and protects the H÷-ATPase activity from inhibition. Key words: calcium; calmodulin; H*-ATPase; phosphorylation/dephosphorylation; Zea mays

Introduction T h e r e is n o w a g o o d deal o f e v i d e n c e t h a t t h e m e c h a n i s m o f p r o t o n e x t r u s i o n can be ascribed to m e m b r a n e - b o u n d , Mg2+-depen d e n t H+-ATPases [ 1 - - 5 ] . T h e use o f sealed m i c r o s o m a l vesicles o b t a i n e d f r o m several p l a n t tissues has provided more direct evidence for the existence o f such H+-ATPase activities [ 6 - - 1 2 ] . In particular two types of Mg2%dependent H*-ATPases h a v e b e e n s h o w n : a v a n a d a t e sensitive, NO3--insensitive H÷-ATPase p r o p e r of the plasma membrane, and a vanadate-

insensitive, NO3--sensitive o n e localized o n the tonoplast [6--13]. R e c e n t l y it has b e e n s h o w n t h a t t h e p r e i n c u b a t i o n o f sealed m i c r o s o m a l vesicles f r om corn roots under conditions which p r o d u c e p r o t e i n p h o s p h o r y l a t i o n inhibits t o t a l a n d i o n o p h o r e - s t i m u l a t e d A T P a s e activity [ 1 4 ] . This p r e v i o u s w o r k has b e e n ext e n d e d b y d e m o n s t r a t i n g t h a t t h e degree o f m e m b r a n e p r o t e i n s p h o s p h o r y l a t i o n is accompanied by ATPase inhibition and characterizing the phosphorylation-inhibited activity. Materials a n d m e t h o d s Plant material

Abbreviations: BSA, bovine serum albumin; CCCP, carbonylcyanide m-chlorophenylhydrazone; CaM, calmodulin; DCCD, dicyclohexylcarbodiimide; DES, diethylstilbestrol; EGTA, ethylene glycol bis(~aminoethyl ether)-N,N,N',N'-tetraacetic acid; MES, 2 N-morpholinoethanesulfonic acid; TCA, trichloroacetic acid.

Primary roots of 3-day-old etiolated c o r n seedlings ( Z e a m a y s L., cv. De K a l b X L 85) w e r e used. Seeds w e r e g e r m i n a t e d at 26°C in t h e d a r k o n f o u r layers o f filter p a p e r m o i s t e n e d w i t h 0.1 m M CaC12 a c c o r d ing t o L e o n a r d a n d H a n s o n [ 1 5 ] .

0168-9452/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

154

Preparation of sealed vesicles from the microsomal fraction The microsomal fraction was prepared essentially as described [16]. All procedures were conducted at 2--4°C. Briefly, excised roots were rinsed twice in distilled water and homogenized in an ice-cold mortar in 250 mM sorbitol, 25 mM Tris--2 N-morpholinoethanesulfonic acid (MES) (pH 7.8), 3 mM ethylene glycol bis(/3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM 2-mercaptoethanol and 5 mg/ml bovine serum albumin (BSA), strained through four layers of cheesecloth, and centrifuged at 13 000 × g for 15 min. The resulting supernatant was centrifuged at 80 000 • g for 30 min and the pellet was resuspended in 250 mM sorbitol, 2.5 mM Tris--MES (pH 7.2) plus 0.5 mM EGTA, layered over 10% (w/w) Dextran T-70 in the same buffer, and centrifuged at 70 000 × g for 90 min in a swinging bucket rotor. The interfacial band was collected and used as the source of sealed membrane vesicles. Phosphoryla tion of mere brane pro teins The m e t h o d used for the phosphorylation of membrane proteins was as described by Campbell and MacLennan [17] with slight modifications. The standard reaction mixture contained, in a 1-ml final volume, 250 mM sorbitol, 20 mM Tris--MES buffer (pH 7.0), 10 mM MgCl~, 0.25 mM EGTA, 10 mM NaF, 1- 2 mg of protein vesicles and, where present, 0.5 mM CaCl2 nad/or 5 t~g/ml calmodulin. After a preincubation of 2 min at 25°C, the reaction was started by the addition of 1 mM ATP and allowed to proceed for 5 min at the same temperature. The reaction was terminated by diluting the mixture 10-fold with ice-cold buffer (250 mM sorbitol, 2.5 mM Tris--MES, pH 7.2). Membrane vesicles were reisolated by centrifugation at 80 000 × g for 30 min. Pellets were dissolved in the dilution buffer and used for the ATPase assay. In experiments done to determine 32P-incorporation vesicles were incubated in the same reaction

medium except that 0.1 mM labelled [~/_32p]_ ATP (300 to 500 c.p.m./pmol) was used and then processed as above. Radioactivity was determined by pipetting 100-ul aliquots of resuspended pellets in 10 ml of Lumagel (Lumac) in a liquid scintillation counter (Beckman LS 7500). For the time-course analysis, 100-pl aliquots of the reaction mixture were withdrawn at different intervals and pipetted into 2 volumes of ice-cold 10% trichloroacetic acid (TCA), 20 mM Na-pyrophosphate and filtered through fiberglass GF/C filters. Filters were washed three times with 10 ml of the same solution, dried and the 32P-radioactivity determined in a liquid scintillation counter.

Dephosphorylation of membrane proteins Membrane proteins phosphorylated as above were used for the dephosphorylation step. The standard reaction mixture contained 250 mM sorbitol, 10 mM Tris--MES buffer (pH 7.5), 2 mM MgC12, the reisolated membrane vesicles (1--2 mg protein) and, where present, 10 pg/ml alkaline phosphatase. The incubation was for 15 min at 25°C and the reaction was terminated by diluting the mixture 10-fold with cold buffer (250 mM sorbitol, 2.5 mM Tris--MES, pH 7.2}. Membrane vesicles were reisolated by centrifugation at 80 000 × g for 30 min. Pellets were dissolved in the dilution buffer and used for the ATPase assay, and, where determined, for 32P-incorporation. A TPase activity ATPase activity was measured at pH 6.5 in a 0.5-ml volume containing 30 mM Tris-MES, 3 mM ATP (Tris salt), 3 mM MgSO4, and 50--100 pg protein vesicles at 27°C for 30 min. Released inorganic phosphate was determined by the Fiske and Subbarow m e t h o d [18]. Calmodulin preparation Calmodulin was prepared from calf brain according to Cocucci [ 19].

155

Protein determination Membrane proteins were determined by the method of Bradford [20] using the BioRad protein assay dye-reagent and bovine gamma globulin as standard.

Squibb and Sons Inc. All other chemicals were reagent-grade. Results

The time course of 32P-incorporation from [~/-32P]ATP into microsomal membrane vesicles isolated from corn roots and incubated in the presence of Ca 2÷ (chloride}, calf brain CaM, or Ca 2÷ plus calmodulin is shown in Fig. 1. Maximum phosphorylation is achieved when both calcium and calmodulin were present, indicating that the process is Ca2+/calmodulin dependent. Experiments

Chemicals Dextran T-70 was purchased from Pharmacia. [~-32P]ATP (3000 Ci/mmol) from NEN. ATP, carbonylcyanide m-chlorophenylhydrazone (CCCP), nigericin and alkaline phosphatase were obtained from Boehringer. DCCD and DES were from Sigma. Fluphenazine • 2 HC1 was kindly provided by E.R.

2000

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O. a 1000 LLI

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(2. nO (.) Z

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7 4

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OL 0

I 4

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I 8 TIME

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8 (rntn)

I 12

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Fig. 1. T i m e course o f m e m b r a n e protein p h o s p h o r y l a t i o n at 25°C. Sealed m e m b r a n e vesicles isolated from corn roots were incubated in 2 5 0 mM sorbitol, 20 mM Tris--MES (pH 7.0), 10 mM MgCl2, 0.25 mM E G T A , 10 mM NaF, 0.1 mM [~-32P]ATP ( 3 0 0 - - 5 0 0 c . p . m . / p m o l ) (+) and w h e r e present, 0.5 mM CaCl: ( v ) , 5 u g / m l CaM (=), or Ca 2+ plus CaM (=). Samples were w i t h d r a w n at different intervals and 32P-incorporated d e t e r m i n e d as described in Materials and m e t h o d s . Inset: ATPase activity (=) as a f u n c t i o n o f p h o s p h o r y l a t i o n degree. Conditions for p h o s p h o r y l a t i o n were as above in the presence o f CaCI 2 plus CaM. ATPase activity is e x p r e s s e d as u m o l P l / m g protein • h.

156

using cyclic AMP, an activator of certain animal protein kinases [21], showed no effect on the phosphorylation activity of this fraction (not shown). The inset in Fig. 1 shows the decrease in ATPase activity as a function of the phosphorylation time, performed under the most favourable conditions (i.e. in the presence of Ca 2÷ plus calmodulin). Maximum inhibition was achieved within 5 min, mirroring protein phosphorylation that is almost complete after the first 4 or 5 min. Table ! shows the effect of phosphorylation treatment on the ATPase activity which can be uncoupled by means of the protonophore CCCP, i.e. the activity that may be ascribed to a coupled H*-ATPase. Phosphorylation causes both a decrease in ATPase activity {see also inset in Fig. 1} and a loss of CCCP-stimulating capacity (see also Table III in Ref. 14). In the presence of 50 mM K ~, nigericin appears to uncouple the ATPase activity of phosphorylated membranes to a higher extent than non-phosphorylated vesicles; + 80% vs. + 38%, respectively. The effect of K ÷ and various inhibitors of microsomal ATPase activity on phosphorylated and non-phosphorylated membranes is compared in Table II. Membranes lose

Table I. Effect of CCCP on microsomal ATPase activity of membranes with different degrees of phosphorylation. Vesicles were treated to obtain maximum protein phosphorylation in the presence of Ca 2÷ plus CaM reisolated and tested for ATPase activity and 32P-incorporation as described in Materials and methods. Addition: ±10 uM CCCP. The values are the average of three experiments having S.E. not exceeding 5%. 2P_incorporated (pmol/mg protein)

--

700 1050 1700

ATPase activity (umol Pi/mg protein • h) Control

+ CCCP

4.6 3.5 3.1 2.3

5.8 4.3 3.6 2.4

II. Effect of inhibitors on the ATPase activities of microsomal vesicles. ATPase activity was measured at 27°C in 30 mM Tris--MES, 3 mM MgSO,, 3 mM ATP--Tris, pH 6.5, in the presence of 0.5% ethanol. Results are the average of 3 to 5 experiments in triplicate. Values in parentheses represent the stimulated (+) or inhibited ( - ) ATPase activity. Table

Treatment

Control KC1,50mM NO~-, 50 mM Vanadate~ 100 uM DCCD, 50 uM DES, 50 pM Molybdate, 100 ~M NaF, 5 m M Oligomycin, 5 ug/ml

ATPase activity (umol Pi/mg protein • h) Aa

Bb

4.7 6.8(+2.1) 3.3 (--1.4) 3.2 (--1.5) 2.8 (--1.9) 3.3 (--1.4) 4.5 (--0.2) 4.5(--0.2} 4.4 (--0.3)

2.5 3.0 2.25 1.7 2.0 2.12 2.3 2.3 2.22

(+0.5) (--0.25) (--0.8) (--0.5) (--0.38} (--0.2) (--0.2} (--0.28)

bA,, non-phosphorylated vesicles. phosphorylated vesicles.

more than 70% of the K ÷ stimulation upon phosphorylation. Microsomal ATPase inhibitors are much less effective on the ATPase activity of phosphorylated membranes; infact, the ATPase activity inhibited by DCCD and DES is decreased by about 70%. Both NO3-and vanadate-sensitive ATPases are present in our vesicle preparation; phosphorylation decreases the NO3--sensitive ATPase activity more (--82%) than the vanadate-sensitive (--47%}. Moreover, Table II shows that the microsomal fraction used is not contaminated by mitochondrial ATPase or phosphatase activity since oligomycin and NaF or molybdate, respectively, are substantially without effect; phosphorylation has practically no effect on these activities. Antipsychotic drugs have been useful for counteracting the effects of CaM-mediated processes in animal and plant tissues [22]. Table III shows that the presence of fluphenazine during Ca2+/CaM-stimulated phosphorylation inhibits the phosphorylation process, decreasing the a m o u n t of 3:P-incorporated, and concurrently decreases the inhibition on

157 Table III. Effect of fluphenazine (FPZ) during the phosphorylation treatment on microsomal ATPase activity and 32P-incorporation. Vesicles were treated to obtain protein phosphorylation, reisolated and tested for ATPase activity and 3:P-incorporation as described in Materials and methods. Addition: _+50 aM FPZ. The values are the average of four experiments having S.E. not exceeding 5%.

Phosphorylation treatment

ATPase activity (umol P.~mg protein • h)

32P-incorporated (pmol/mg protein)

Ca 2+ + CaM ATP + Ca 2+ + CaM Ca ~÷ + CaM + FPZ ATP + Ca 2+ + CaM + FPZ

4.3 2.2 4.4 3.6

-1500 800

t h e A T P a s e activity. M o r e o v e r , T a b l e I I I s h o w s t h a t t h e e f f e c t e x e r t e d b y Ca2+-CaM o n t h e A T P a s e a c t i v i t y is n o t direct, b u t is mediated through the phosphorylation process. T h e decrease in activity o f t h e Mg:*dependent ATPase upon phosphorylation can be reversed if p h o s p h o r y l a t e d m e m b r a n e vesicles are a l l o w e d t o d e p h o s p h o r y l a t e . T a b l e IV s h o w s t h a t m e m b r a n e s p r e v i o u s l y phosphorylated and then incubated either in the a b s e n c e or p r e s e n c e o f e x o g e n o u s alkaline p h o s p h a t a s e r e c o v e r t h e i r A T P a s e activity a n d CCCP-sensitivity. T h e increase

in A T P h y d r o l y s i s is n o t d u e t o residual alkaline p h o s p h a t a s e in t h e f r a c t i o n , since little Pi release c o u l d be d e t e c t e d using p - n i t r o p h e n y l p h o s p h a t e (Table IV). T h e effectiveness of the d e p h o s p h o r y l a t i o a process is clearly s h o w n b y t h e r e d u c e d a m o u n t o f 32P-radioactivity f o u n d in t h e vesicles after treatment. Discussion

T o date, t h r e e specific e x a m p l e s o f p l a n t enzymes modulated by a phosphorylationdephosphorylation mechanism have been reported: pyruvate dehydrogenase [23], p y r u v a t e Pi dikinase f r o m C4 leaf tissues [ 2 4 l a n d the q u i n a t e - N A D o x i d o r e d u c t a s e [ 2 5 ] . This p a p e r p r o v i d e s f u r t h e r e v i d e n c e t h a t a Mg2+-dependent A T P a s e m a y be regulated through a phosphorylation-dephosp h o r y l a t i o n cycle o f m e m b r a n e p r o t e i n s as a l r e a d y r e p o r t e d [ 1 4 ] . O p t i m a l p h o s p h o r y l a t i o n requires b o t h c a l c i u m a n d c a l m o d u l i n (Fig. 1), v e r y similar t o t h e p r o t e i n kinase activity f o u n d in m e m b r a n e f r a c t i o n s isolated f r o m p e a s h o o t s [26] a n d zucchini h y p o c o t y l s [ 2 7 ] . Phosp h o r y l a t i o n in t h e p r e s e n c e o f c a l c i u m a n d calmodulin reduces the total ATPase activity and, in p a r t i c u l a r , t h e i o n o p h o r e - s t i m u l a t e d p a r t o f it {inset, Fig. 1, a n d T a b l e I). I n c o r p o r a t i o n o f 32p into c o n t r o l a n d in calcium-

Table IV. Effect of dephosphorylation treatment on ATPase activity of membranes previously phosphorylated. Phosphorylated membranes obtained as described were allowed to dephosphorylate in the presence or absence of 10 ug/ml alkaline phosphatase, reisolated and tested for ATPase activity and 32P-incorporation as described in Materials and methods. The values are the average of four experiments in triplicate having S.E. not exceeding 5%. Treatment

ATPase activity (umol Pi/mg protein • h) Control

None Phosphorylation Phosphorylation + dephosphorylation (--phosphatase) Phosphorylation + dephosphorylation (+phosphatase) aUsing p-nitrophenylphosphate as artificial substrate.

4.5 2.4 4.0 6.1

Phosphatase activity a (~molPi/mg protein • h)

32P-incorporated (pmol/mg protein)

6.0

--

F

2.6 4.9 9.2

--0.35

1400 975 350

+CCCP

158 or CaM-treated vesicles may reflect contamination by calcium and/or CaM not completely removed during the isolation of the microsomal fraction. On the other hand, calmodulin has been found to be a subunit of a protein kinase [21]. All the ATPase inhibitors examined are more effective on the ATPase activity of non-phosphorylated relative to phosphorylated vesicles (Table II). It seems appropriate to c o m m e n t on the effect of NO3- and vanadate; such inhibitions suggest that the control mechanism exerted by the phosphorylationdephosphorylation cycle concerns the ATPase activities localized on both the plasmalemma and the tonoplast. The loss of NO3- sensitivity upon phosphorylation is greater than that of vanadate, suggesting the possibility that the ATPase activity on the tonoplast may be more sensitive to phosphorylation. A control mechanism on the vanadate-sensitive plasmalemma ATPase activity would support the hypothesis suggested by Hanson and Trewavas [28] in a discussion on plant growth as a consequence of H ÷ efflux pumping, that Ca2+-CaM may regulate the H ÷ATPase at the plasmalemma. Moreover, working on the effect of injury, Hanson and co-workers [14,29--32] suggested that the transient inhibition of H ÷ efflux pumping as a consequence of shock might be correlated with an increased cytoplasmic concentration of Ca 2÷, which, in turn, through a iink with calmodulin activates protein kinase(s) capable of phosphorylating membranes. Ca 2÷, conserving a large electrochemical gradient for influx [33,34], should be facilitated to enter the cell probably because of an increased membrane permeability due to injury [32]. Since the phosphorylation-dephosphorylation mechanism also controls the NO3--sensitive activity, it seems possible to apply these hypotheses to the tonoplast ATPase as well. The presence of a Ca2+-transport activity in corn root microsomal fraction [16] might induce one to think that the observed decrease in ATPase activity upon phosphory-

lation of membranes could be attributed to the build-up of a Ca 2÷ gradient during the phosphorylation treatment, thus counteracting any further H ÷ pumping. Conditions which certainly avoid such a gradient formation (i.e. the presence of the Ca 2÷ ionophore A-23187 during the phosphorylation treatment), does not lead to a preservation of the ATPase activity {data not shown). The reduction of 32p-incorporation into membrane proteins by the antipsychotic drug fluphenazine (Table III) suggests a direct involvement of CaM in the phosphorylation step. Treatment with exogenous alkaline phosphatase, which dephosphorylates the vesicles previously phosphorylated, leads to a recovery of the total and ionophore-stimulated ATPase activity over the control level (Table IV). Moreover, an endogenous phosphoprotein phosphatase activity can be detected, as preincubating the phosphorylated microsomal fraction in the absence of alkaline phosphatase leads to partial dephosphorylation and recovery of ATPase activity (Table IV), in agreement with an other report [26]. In conclusion the data reported herein suggest a control on the H*-pumping ATPase exerted by a phosphorylation-dephosphorylation mechanism and that the membranes phosphorylation step is catalyzed by a Ca 2÷dependent, calmodulin-stimulated protein kinase(s).

Acknowledgements This work was financially supported by a grant from the Italian Ministry of Education (60%). I wish to thank Professor S. Cocucci for his valuable discussion and advice during this work and Professor J.B. Hanson for the critical reading of the manuscript.

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