H+ antiport activities in the plasma membrane of the marine alga Platymonas viridis

Volume 309,number 3, 333-336 FEBS 1152"/ © 1992 Federationof EuropeanBiochcmi~dSo=ictlcs0014S?OMg'~J$$,00

~ptcmtmr 1992

H+-translocating ATPase and Na+/H + antiport activities in the plasma membrane of the marine alga Platymonas viridis Larisa G . Popova and Yurts V. Balnokin Labaratory of Salt ~xehaage attd &tit Tolerance, P/trot Plo'sialagy Institute. Russian Academy of Science. Batanichedia)'a 35, 127276 Moscow. Russia Received 1"7 July 1992 Proton transport by the vanadate.~nsitive ATPatm in plasma membrane (PM) ~ i g l ~ from the marine uni~llular mi~roalp Ptatynmnas vlrldls

was investiltat¢d, The ATPMe~ndent generation of.apH acrof,.s the membran~ of PM vesicl~ was followed by the changml in the abmrbgn~ of the aminoacridine probe. Acridine orange, Nit" caused the d~ay ofdpH Ilenemted by the ATPas¢. the at0.=of the decay b¢iall del~ndent on the concentrations of Na" added, The phenomenon was Sla~ciflc for Na', Amiloride inhibited Na'-deI~ndent ,dpH d~.ay. The exi~rimcnts support the idea of a Na'-¢xtruding mechanism (H'.tranfloeatinil ATRts¢ plus Na'IH" antiporter) operating in the PM of marine alga PI, virldl~, H'.ATPa~; Na'/H" antiport; Marine al~a; Plasmalemma: Plao'm~mts vlridi~

1. INTRODUCTION The plasma membrane (PM) in most of the unicellular algae of saline habitats plays a crucial role in the regulation of cytoplasmic Na" concentrations. Nevertheless, the membrane ion transport systems of the halotolerant algae have been investigated poorly, To date there has been no reason to doubt that the H'-pump (H'-ATPas¢) operates in the PM of glycophytes and fresh water algae [1]. It is generally accepted that the energy ofd,ffH generated by the H'.pump in the PM of these organisms is utilized for the extrusion of Na" from the cells by means of a Na'/H" antiporter [2. 3], Preliminary investigations on the ion transport and the properties of the PM ATPas~ J~romthe halotolerant (marine) algae Ntt:schia [4], tIa[icistis [5]. and Acetobularia [6] have not demonstrated the H'-pump (H'-ATPase) in the PM of these organisms. A primary active Cl'-traasporting system (Cl'-ATPas¢) has been demonstrated to operate in the PM of Halicistis [5] and Acetobularia [6]. An ATPas¢ synergistically stimulated by Na" and K" was found in the PM-enriched membrane fractions from Nit:schia [41. On the other hand, subsequent investigations of the ion transport carried out on intact ceils of the halotolerant alga Dunaliella indicate an H °translocating system in the PM of this alga [7]. It has Carreq;ondence address: Yu,V, Balnokin, Plant Physiology Institute. Russian Academy of Science. Botanicheskaya, 35. 12"/276 Moscow, Russia. Fax: (7)(095)482 16B£ Abbreviati,,t,,: A/IH. electrochemical H" potential dil'rerenc¢: CCCP, m.chloro~rt~onylcyanide phenylhydrazone; DTT. dithiothr¢itol; EGTA, ethyl~neglycol.bi~.aminocthyl ether) N.N,N',N'.tetraac~ti¢ acid,

Published by Elsevier Scletlce Publishers B.V.

also been found in membrane fractions from algae of genus Dunaliel/a that the properties of the PM ATPase from these algae are similar to those of the common p.type ATPa~e from higher plants [8,9], Neverthel~s, there is no clear evidence as to whether this ATPa.~ in the PM of the halotolerant algae is an H'-tran~locating one, since all attempts to observe ATP-indu~l proton transport in PM v~icle~ from th¢-~ algae were unsuccessful up to now [8.10]. At the same time, the Na'/H" antiport activity has been found in the PM fractions isolated from Dunatielta cells [l 1]. We have previously reported the isolation of highly purified PM from the marine mieroalga Platymanas t'irhfi," and investigated the properties of its PM ATPase [12]. Here the ATP-dependent generation of the o H gradients across the plasma membrane v~icl~ and Na'-dependent .dpH decay in the PM vesicles from the marine alga PI. viridis are demonstrated with an Acrid. ine orange probe, Theseexperiments have revealed that a Na" e~tru,ion m~hanism consisting of an I-l'.transloeating ATPas¢ and a Na'/H" antiporter OlZ:rates in the PM of the marine alga Pt. viridis. 2. M A T E R I A L S

AND

METHODS

PI, riridi.¢cells were cultured in artificial sea water at 0.46 M NaCI a¢ d¢~:ribed previoufly [12]. The highly puriP.cd PM v~icl~ wcrc obtained according to a method pr¢~ntcd by as and d~cribcd in detail previously [1,]. This method i,, based on the partial proteolysis afda¢ Blycoprotein c¢11wall followed by mild hypo-osmotie shock and subsequent membrane partitioning by dilTercntial ccntfifugation in a diKontinuous sucro~ gradient. Here, we ¢mployed this method with slight modifications, namely, maanltol wa', u~d instead of #yccrol in all solutions. T-he final membrane i:n:~t was rc, ml:~ndCd ifi ~,hemedium consisting of 0,2 M mannitol, $mM Tris.MES (pH 7.2). i mM D'F'I'. 2 mM MgCl_. and 0.2 mM EGTA.

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The ATP.depend=nt formation and the Na'.induced dissipation of the interior-acid pH gradient across Ihc m¢mt~mnes in isolated PM ve~iel¢~of Pt. ~'tridis (H',pump activity and Na'IH" cxchan~ activity, accordingly) were assayed by monitoring the changes in absorbance of the amino-acridine prol~, Acrldin~ oared, recorded with a Hitachi SS? dual.wavelength sp¢ctrophotomatcr at 492-S40 nm [13]. "l he assay was performed in 2 ml of re.action mixture containing 0,2 M mannltol, I0 mM Tri,=.MES (pH 7.0), I mM D'I'F, 0.2 mM EGTA, 100 mM NO; (as Tris =iall). 10 mM MB..fiOo tl#M Acridine orange, 20.-.i0/all proteim The ~ i d c s were equilibrated with the medium for I S-20 rain I>=Foreadding ATP. ATP (buff=red with Tris to pH 't.0) at a final concentration of I m M wn~ added to initiate the formation of dpl'I. Various concentrations of Na" (as indicated in the figure klcnd~) wet= added to initial= Na'IH" ext'haalle. Th¢ initial rates of ab~rbanc¢ ¢hanlle¢ due to anliport activity were determined by drawing the tanl~nts of the recorded traccz obtained in the first l0 s after salt addition. Protein content was determined by the method of Simpson and Somme [14] with bovine strum albumin as a standard,

As shown in Fig, I, the addition of ATP to the reaction mixture containing the PI. riridia PM vesicles resuited in an appreciable decrease in Acridine orange absorbanee. The protonophorous uncoupler CCCP completely aboli,~hed the ATP-induced decrease in ab-

;

ATP

=e

3, RESULTS

ATP

September 1992

CCCP

;

cccP

- IL_

CCCP

I

¢r~szS°4'mm

~OOi ~ ¢

Fig. 2. Na'IH" antiport activity in isolated plasma membrane vcliclcs from PI, t'iridix, dpH gen¢rated by the addition of ATP to th¢ reaction mixture contained PM vcaicl¢, {first arrow) was dissipated by Na" (second arrow, indicated concentrations), CCCP (12 pM. third arrow) abolished the Pmaining proton i]radient,

sorbanc¢ indicating a dpl-Igeneration across the membranes of FM vesicles in the presence of ATP. A spontaneous decay of the generated zlpH was slow, showing a rdativ¢ly low permeability of the vesicles to proton,~. The additions of vanadute, an inhibitor of PM ATPasz or the plant and fungi cells, decreased the generated dpH to a considerable degree (Fig. 1). This indicates the involvement of the vanadate-sensitivc ATPase in the dpH generation across the Pt. vh.idis PM. Na" Ceither as chloride or as sulfate) eau,~ed the decay of,dpH generated by the ATPase (Fig, 2). The rate of Table i

R

Cation sl'g'cificity of Na'/H" antiport in PI. rieidi.¢ plasm;t membrane vesiclas Addition

Vanadate Fig. 1. ATP.dependent intmvcsicular acidification in isolated plasma membrane vesicles from PI. vie/dis monitoring by changes in the ab. sorbs_n~e ~f A ~ . oran=~+ The_~Lsssywas ~rrh.d out m d-=scribed in ~ction 2. ATP, baiT=red with Trig to pH 7,0, was added at the first arrow, At the ~cond arrow 12 ~M CCCP was added to dissipate proton gradient. Trace (A), standard assay; trace (B), standard assay with addition of 0,1 mM sodium vanadate,

334

Natl. I00 mM Na_,SO~, SO mM KC[. I00 m M CsCl, I00 m M LiCl, l_a0_mM

Activity (relative) I00 98 22 l0

Assays wcrc pzrform~ as d=scrib~'d in Fig, 2, The activities were determined us initialratcl of abscrbancc chansc~ (as described in section 2),

Volume 309, number 3

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September 1992

sp¢ific inhibitor of the Na'/H" antiport in various animal membranes [15] added prior to Na" also delayed ,4pH decay induced by Na" (Table ll). 4. DISCUSSION

10 >_

fly 0.4

Q2

/

Z

Q4

0.2

<1

I/INn' I, mM "1

5O

1OO 2OO

Na÷ CONCENTRATION, mM Fill, 3. Na" concentration depe~dcng¢ of,dpH decay. Data from Fi~, 2. The initial relative rat~ of ab~rbance changes were determined in section 2. (e). experimental curve: (o), theoretical Michaclis-Menten curve, I n . r | : Lineweaver-Burk plot of the theoretical curve Iiives the theorctig"al K. = 10 raM,

the decay depended on the concentrations of Na" added. The effect of Na" displayed a sigmoidal curve with increasing Na" concentrations (Fig. 3). It can be supposed that the sigmoidai nature of Na" effect is due to the superposition of two independent processes: (1) an acceleration of intra-vesicular acidification due to the stimulation of the ATPase by low Na" concentralions [12]. and (2) ,dpH decay due to Na'/H* antiport. Assuming there is no stimulation of the ATPase by Na °, this sigmoidal curve could be approximated by a Michaelis- Menten curve with apparent KM = 10 mM (Fig. 3). The Na'/H" antiport under consideration is specific for Na'. Other cations tested (K', Cs" and Li*) resulted in ,dpH decay at significantly lower rates (Table l), Accordingly, these cations are the potent competitive inhibitors of Na'/H" exchange (Table II). Amiloride. a

The vanadate-sensitive ATPas¢ activity has been identified recently as a component of the PM of Duna[. iella [8-10] and Platymo~zas [12] cells. Here we have shown the translocation of protons in the PM vesicles from PI, viridis to be initiated by addition of the ATP and sensitive to vanadate. This is apparently the first direct demonstration of a H'-translocating ATPas¢ in tl~¢ PM of the alga living under high salinity conditions. The dissipation of ,~pH generated by the ,~TP~e in response to an addition of Na" by a mechanism tidal exhibit~ saturation kinetics is specific for Na" and sensitive to amiloride, and indicates the functioning of the Na'/H" untiporter in the PM of PI. t.lridi~, H'-ATPas¢ and Na'/H" antiporter in the PM of PI, viridis probably form a mechanism extruding Na* from the cell interior and maintaining low intracellular concentrations of Na" under high salinity conditions. It remains to be elucidated whether this system (H'ATPase plus Na'/H" antiporter) is the only ion transporting system in the PM of salt.tolerant algae extruding Na" from the ceils. In this respect the curlier data on the unusual PM ATPascs in Nit:schia [4]. lfalio'sti~ [5]. and Acetohul, ria [6] are extremely inter~ting. It is worth mentioning here the organization of ion transport in the cytoplasmic membrane o f the halo- and alkalotolerant bacterium |:ihrio alginolyticus, in which the ATP-driven primary Ha" pump was di,~:overed [16]. A similar enzyme may function in the plasmalemma of halotolerant algae, So, one should study the possibility of ATP.driven transport of Na" in PM vesicles from marina algae, Positive results would demonstrate the operation of a Na'-transloc.ating ATPa~ in the plasmalemma of marine algae. Aek.owledx.-mr.ts: We would like to thank Dr, Yu. G, Molotkovsky and Dr. i.M, Andreyev for a~l'ul advi~ and for the u~¢ of their laboratory facilities during thit work, We would a h o like to thank Dr, N,A, Mya=~'dov for assiftan¢¢ in pla,ma membrane preparation and Dr', N.V, Obruchcva for critical reading of the manuscript.

Table II lnlzibition of Na'/H" antiport in PL viridus plasma membrane' vesicles Addition Natl. [00 mM +KCI, 1(30 mM *LiCI, 100 mM ~CsCt. 100 mM +amilorid¢, 0,2 mM

Activity (r=lative) 100 28 37 20 l0

Assays were performed a~ described in Fig. 2, The cations were added I~fore addition of Na'.

REFERENCES [l] Sz¢. H, (19l~4) Physiol, Plant. 61. 683-691, [2] Rather'. A, and Jaeoby. B, (1970) J. Exp, Rot. 27. ~3-~52. [3] Braun, Y., Hassidim, M., l..¢rner, H,R. and Reinhold, L. (198~) Plant, Physiol. 87. ["J4-108. [4] Sullivan, C.W, and Volcani. B,E, (1974) Prec. Nat. Acad. Sci, USA 71, 4376--43t10, IS] Graves, LS. and Gutknceht. J, (19771 J. Membr, Biol, 36. 65-~1, [6] Goldfarb, V., Sanders, D. a,~d Gradmann. D. (1984,) J. Exp, Bet. 35. 64S-6St~. [7] Balnokin, Yu, V, and Medvcdev, A,V, (1984) Soviet Plant Physiol, 31. 625-629,

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[8] Wei~s.M... Sekler. [..rid Pick. U. (1989) Biochim. Biophys. Acl;~ 974. 2.~1--2~. [9] Smahel. M.. Hztmann. A. amI Gradmanm D. (Ig~C) Planla 181. 4~(z.-.$(~4. [10] Gimmicr. H.. Schneider. L. -nd Ku~dcn. R. (Ig~9) Z. N.turl'or~h 44¢. 12tt-13ZI. [1 I] Katz. A.. Kaback. H.R. and Avron. M. (Ig~16) FEllS Lett. 202. 141-1.44. [12] Popovu. L.. llalnokin. Yu. V.. My.soedov. N.A.. Lapoofllkin. E.V. and Beiov. A.P. (1991) Dokl. Acad. Nauk USSR 317. 2111256 (i, R~fi~m}.

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[13] ~huldincr. $.. Ro||cnbcrll. H. zzr~dAvron. M. (1972) E,r. J. Bioch~m. 25. ~.1-70. [14] Simpson. [.A. and Somme. O. (1982) Anal. Biuchem. ! 19. 424427. [15] L'Allem,in. G.. Frunchi. E.. Grago¢. E. Jr. and Pon~ilur. J. (Ig~14) J. Biol. Chem. 259. 4313--4319. [16} Dibrov. P.A.. Skulachev. V.P.. Sokolov. M.V. and Verkho~k;Ly~. M.L. (igtllI) FEBS Lelt. 233. 3SS-358.