Planta 9 Springer-Verlag 1988

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1 Planta (1988)176: Planta 9 Springer-Verlag 1988 A guansine 5'-triphsphate-dependent prtein kinase is lcalized in the uter envelpe membrane f pea chlrplasts Jiirgen Sil 1, Irene Fischer 1, and Kenneth Keegstra 2 1 Btanisches Institut der Universitfit Miinchen, Menzinger Strasse 67, D-S000 Mfinchen 19, FRG, and 2 Btany Department, University f Wiscnsin, Madisn, Wiscnsin 53706, USA Abstract. A guansine 5'-triphsphate (GTP)-dependent prtein kinase was detected in preparatins f uter chlrplast envelpe membranes f pea (Pisum sativum L.) chlrplasts. The prteinkinase activity was capable f phsphrylating several envelpe-membrane prteins. The majr phsphrylated prducts were 23- and 32.5-kildaltn prteins f the uter envelpe membrane. Several ther envelpe prteins were labeled t a lesser extent. Fllwing acid hydrlysis f the labeled prteins, mst f the label was detected as phsphserine with nly minr amunts detected as phsphthrenine. Several criteria were used t distinguish the GTP-dependent prtein kinase frm an ATP-dependent kinase als present in the uter envelpe membrane. The ATP-dependent kinase phsphrylated a very different set f envelpe-membrane prteins. Heparin inhibited the GTP-dependent kinase but had little effect upn the ATP-dependent enzyme. The GTP-dependent enzyme accepted phsvitin as an external prtein substrate whereas the ATP-dependent enzyme did nt. The uter membrane f the chlrplast envelpe als cntained a phsphtransferase capable f transferring labeled phsphate frm [7-32p]GTP t ADP t yield (7-32p]ATP. Cnsequently, additin f ADP t a GTP-dependent prtein-kinase assay resulted in a switch in the pattern f labeled prducts frm that seen with GTP t that typically seen with ATP. Key wrds: Casein kinase II - Chlrplast envelpe - Guansine triphsphate-adp phsphtransferase - Guansine triphsphate-prtein kinase Pisum (prtein kinase)- Prtein phsphrylatin Abbreviatins: GDP (GMP, GTP)=guansine 5'-diphsphate (mn-, tri-); kda-kildaltn ; S.5 = cncentratin f substrate supprting half-maximal velcity; SDS-PAG = sdium ddecyl sulfate-plyacrylamide gel electrphresis Tricine=N-(2- hydrxy-l,l-bis(hydrxymethyl)ethyl)glycine Intrductin Prtein kinases are widely distributed in eukarytic cells. The physilgical rles f prtein kinases have been studied mst extensively in animal cells where prtein phsphrylatin/dephsphrylatin cascades play an imprtant rle in regulating the activity f many sluble enzymes. In additin, membrane-bund prtein kinases play imprtant rles in signal transductin acrss bilgical membranes (delman et al. 1987). Numerus prtein kinases have been identified in plant cells. Prtein kinases f chlrplasts have received cnsiderable attentin, with several chlrplast cmpartments cntaining prtein-kinase activities (Laing and Christeller 1984). Distinct ATP-dependent prtein kinases and phsphprreins are lcated in the uter envelpe membrane (Sil and Buchanan 1983) and in the inner membrane (Sil 1985). The sluble chlrplast-prtein fractin was nt nly demnstrated t have ATPdependent prtein-kinase activity (Fyer 1984; Budde and Chllet 1986), but als ADP-dependent prtein-kinase activity (Ashtn and Hatch 1983). Mst extensively studied is the phsphrylatin f prteins in the thylakid membranes by different prtein kinases (Allen and Bennett 1981 ; Lin et al. 1982; Cughlan and Hind 1986; Bhalla and Bennett 1987). Prtein kinases als using GTP as phsphryldnr are mst cmmn enzymes f the casein kinase II type (Hathaway and Traugh 1983). They have been detected in a wide variety f plant and animal tissues (Ventimiglia and Wl 1974; rdmann et al. 1982; Gwda and Pillay 1982; Yan and Ta 1982; Glver et al. 1983). Prtein kinases f this type have nt s far been fund t depend n any f the well-studied secnd messengers like camp, cgmp, Ca 2 + r diacylglycerl. The majr exgenus prtein substrate fr casein kinase II

2 J. Sll et al. : GTP-dependent prtein kinase 489 are acidic prteins like phsvitin and casein but nt histnes. Thugh detailed studies f this prtein-kinase class are available even with purified enzymes (fr references, see abve) n definite physilgical rle r regulatry functin culd be ascribed t prtein kinases f the casein kinase II type until tday. Despite the reprts f prtein-kinase activities in plant cells (fr a review see Ranjeva and Budet 1987), knwledge f their rles remains incmplete. Fr thse prtein kinases that are fund assciated with membranes f plant cells, it is tempting t speculate that they are invlved in signal transductin at these membranes. Fr example, a thylakid prtein kinase has been implicated in regulating the distributin f light-harvesting capacity between the tw phtsystems (Allen and Bennett 1981). Alternatively, it has been suggested that the prtein kinases f the chlrplast envelpe may have a rle in the ATP-dependent transprt f cytplasmically synthesized prteins int chlrplasts (Schindler et al. 1987). During attempts t understand further the rle f prtein phsphrylatin at the chlrplast envelpe a GTP-dependent prtein kinase was detected. This enzyme was distinct in its bichemical prperties and endgenus prtein substrates frm the ATP-dependent prtein kinase described earlier (Sll 1985). Fig. 1. Purified envelpe membranes frm pea chlrplasts were analyzed by SDS-PAG. Lane A, mixed envelpe membranes; lane B, separated inner envelpe membranes; lane C, purified uter envelpe membranes. After SDS-PAG the gels were stained with Cmassie Brilliant blue. Numbers indicate mlecular weights in kda Material and methds Plant material. Pea plants (Pisum sativum L., cv. Prgress N. 9) were grwn in a grwth chamber under a 12-h light-dark regime and 20 ~ C cnstant temperature. Chemicals. [7-32p]GTP (370GBq.mm1-1) in 50% ethanl and [7-3ap]ATP (ll0tbq-mm1-1) were frm Amersham (Braunschweig, FRG). Catmdulin antagnist W-5 (N-(6- aminhexyl)-l-naphthatene sulfnamide hydrchlride) and prtein-kinase inhibitr H-9 (N-(2-aminethyl)-5-isquinlinesulfnamide dihydrchlride) were frm Seikagaku America Inc., St. Petersburg, Fla., USA. Prteinaeus prtein-kinase inhibitr frm bvine heart was frm Sigma, St. Luis, M., USA. Guansine 5'-triphsphate was used as lithium salt and stred like GDP at -80 ~ C. All ther chemicals were f reagent grade and used withut further purificatin. Islatin f chlrplast cmpnents. Outer and inner chlrplast envelpes were islated frm 14-d-ld pea plants (Fig. 1 ; Keegstra and Yusif 1986). Chlrplasts were prtease-treated with thermlysin at 200 gg prtease, mg ~ chlrphyll (Jyard et al. 1983; Cline et al. 1984) and separated inner and uter envelpe membranes were purified frm these plastids (Keegstra and Yusif 1986). Sluble chlrplast prteins were islated after hyptnic lysis f purified intact chlrplasts and subjected t centrifugatin at g fr 30 min. Thylakids were islated after hyptnic lysis and centrifugatin f 2000-g fr 2 min frm the same batch f chlrplasts. Thylakids were washed three times with 10mml.1 -~ N-[2-hydrxy-l,1- bis(hydrxymethyl)ethyl]glycine (Tricine)-KOH ph 7.9 at 100 gg chlrphyll.ml-1 (Arnn 1949) and centrifugatin f 2000.g fr 2 min. Prtein-kinase assay. The ethanl was remved frm an aliqut f (7-32P]GTP by evapratin under a stream f N2 t less than half the riginal vlume and additin f HzO back t the riginal vlume. A prtein-kinase assay cntained, if nt therwise defined: 8 pml.1 1 [y-3zp]gtp (370 GBqmml 1), 5 mml-1-1 MgC12, 2 mml.1-1 MnC12, 25 mml- 1- x Tricine-KOH ph 7.9 and abut 2.5 gg envelpe-membrane prteins in a final vlume f 25 gl. Reactins were initiated by the additin f membranes and carried ut at 20~ fr 5 15 min. Assays with ATP kinase were dne under the cnditins described in Sil (1985). The incubatin was terminated by the additin f I vl. f twfld-cncentrated slubilizatin buffer (Laemmli 1970). Analysis f the phsphrylatin reactin. Samples were analyzed by sdium ddecyl sulfate-plyacrylamide gel electrphresis (SDS-PAG; Laemmli 1970) using a 10% running gel. Gels were stained with Cmassie Brilliant blue and destained in the presence f 20 retl. 1-1KH2PO4. Dried gels were expsed t film vernight (Kdak X-AR 5) at - 80 ~ C, using an intensifying screen. Bands f 32P-phsphprtein were subsequently lcalized n the gel, cut ut, rehydrated with 500 lal HzO and the radiactivity quantitated by liquid scintillatin cunting. Phsphamin-acid analysis. The prtein-kinase assay was stpped by the additin f 10 vl. f 10% trichlracetic acid (w/v) and washed three times with 5% trichlracetic acid prir

3 490 J. Sll et al. : GTP-dependent prtein kinase t hydrlysis at 110~ fr 1-2 h in 6 N HC1. Twenty ~tg f the unlabeled phsphamin-acids P-tyrsine P-threnine and P-serine were added t the reactin. The mixture was dried under a stream f N2, disslved in 20 gl H20 and sptted nt a precated Silica-gel thin-layer chrmatgraphy plate (Merck, Darmstadt, FRG; Kieselgel 60) and subjected t electrphresis under water cling fr 4 h at 1000 V using acetic acid: frmic acid: H20 (78:25:897; by vl.) as develping slvent. The psitin f phsphamin acids was lcated by ninhydrin staining and radiactivity was lcalized and detected by autradigraphy. Results During effrts t characterize the prtein-kinase activity present in preparatins f uter envelpe membranes derived frm pea chlrplasts (Sll 1985), the nucletide specifity f the kinase was examined. In these studies, it was bserved that [y-32p]gtp culd serve as phsphryl dnr, but the pattern f labeled prteins was appreciably different frm that bserved when [7-32p]ATP was used (Fig. 2, lanes 11 12). In rder t gain a mre cmplete picture f the presence f GTP-dependent prtein-kinase activities in chlrplasts, the prtein-kinase activities f all chlrplast fractins were measured, using bth [7-32p]ATP and [7-32p]GTP. The results f these analyses are shwn in Fig. 2, lanes The extent f 32p incrpratin varied t sme degree frm preparatin t preparatin fr prteins which were nt the majr phsphrylatin prducts. The results f SDS- PAG f purified envelpe membranes are shwn in Fig. 1 and demnstrate the purity f the respective membrane ppulatin and the typical distributin f envelpe marker prteins (Cline et al. 1984). As can be seen in lanes 1-5, when [7-32p]ATP is used as the phsphryl dnr, each f the chlrplast fractins yields a distinctive pattern f labeled plypeptides. On the ther hand, when [7-32p]GTP is used, nly preparatins f the thylakid membrane and the uter envelpe membrane shw appreciable labeling. The labeling patterns bserved with [7-32P]ATP and [7-32p]GTP labeling f thylakids (Fig. 2, lanes 1 and 10, respectively) lk similar and mst likely result frm the actin f the same prtein kinase. Hwever, the patterns bserved fllwing [7-32]ATP and [7-32p]GTP labeling f uter envelpe membranes are distinctly different frm each ther (Fig. 2, lanes 11 and 12). The simplest interpretatin is that these different patterns result frm the actin f tw separate prtein kinases, ne specific fr ATP and ne specific fr GTP. In an effrt t evaluate this hypthesis, bth the ATP-dependent and GTP-dependent activities were subjected t further characterizatin. The tw prtein-kinase activities f the uter envelpe membrane display dramatically different sensitivities t inhibitin by heparin (Hathaway et al. 1980). As shwn in Fig. 3, the phsphrylatin f envelpe-membrane prteins by [7-32P]GTP is severely inhibited by heparin. The labeling f the 23-kDa prtein is inhibited 50% by 0.15 mu.m1-1 (0.88 ng.m1-1) heparin and the labeling f the 32.5-kDa prtein is inhibited 50% by 0.2mU.m1-1 (1.2ng.m1-1) heparin. On the ther hand, inhibitin f labeling with [7-32p]ATP is variable. Several prteins shw n decrease in labeling at the highest level f heparin examined. Hwever, the 23- and 32.5-kildaltn (kda) prteins shw inhibitin f labeling similar t that bserved with [7-32p]GTP. These bservatins supprt the cnclusin that there are tw different prtein kinases in the uter envelpe membrane. Further evidence fr the presence f tw enzymes cmes frm analysis f the inhibitry effects f nucleside diphsphates. It was demnstrated previusly that ADP acts as an inhibitr f the ATP-dependent kinase (Sll 1985). The data in Fig. 4 demnstrate that GDP serves as an inhibitr f the GTP-dependent kinase. ffrts t examine the effects f ADP n the GTP-dependent prtein kinase led t unexpected results. Rather than cause an inhibitin f the GTP-dependent prtein kinase, additin f ADP caused a switch in the pattern f phsphrylated prducts (Fig. 5). Additin f 10 gml.1-1 ADP t a labeling reactin using [7-32p]GTP resulted in a shift in the pattern f labeled plypeptides t that bserved when labeling was cnducted with [7-32p]ATP (cmpare lanes 1, 2 and 3 in Fig. 5). Additin f 100 gml. 1-1 ADP als resulted in the ATP labeling pattern, but als caused a reductin in the amunt f labeling, presumably because f the inhibitry effect f ADP (Fig. 5, lane 4; Sll 1985). Additin f 10 gml.1-1 r 100 gml.1-1 GDP caused the expected inhibitin f labeling (Fig. 5, lanes 5 and 6). This unexpected result f ADP additin culd be explained if the preparatin frm the uter envelpe membrane als cntained a nucletidephsphtransferase activity that culd transfer a phsphate frm GTP t ADP t yield ATP and GDP. Assays were cnducted t measure fr such an activity in an effrt t prvide direct evidence t supprt this explanatin. As shwn in Fig. 6, uter envelpe membranes d cntain an activity which cnverts [7-32P]GTP t radiactive ATP when ADP is present. T prvide evidence that the switch in the phsphrylatin pattern was caused by the actin f

4 J. Sll et al. : GTP-dependent prtein kinase 491 Fig. 2. Prtein phsphrylatin in pea chlrplast cmpartments using [7-32p]ATP and [7-3Zp]GTP. Left panel, lanes 1-5 shw prtein-phsphrylatin patterns f thylakids (1), chlrplast-sluble prteins (2), inner envelpe (3), uter envelpe (4) and thermlysin-treated uter envelpe (5) using [7-32p]ATP. Lanes 6-10 shw prtein-phsphrylatin pattern f thermlysin-treated uter envelpe (6), uter envelpe (7), inner envelpe (8), chlrplast-sluble prteins (9) and thylakids (10) using [7-32p]GTP. qual amunts f prteins were incubated with [7-32p]ATP and [7-32p]GTP (8 gml. 1-1) f the same specific activity (370 GBq. mml ~) fr 5 rain and further analyzed by SDS-PAG as described in Material and methds. The right panel shws a mre direct cmparisn f prtein phsphrylatin in the uter chlrplast envelpe using [7-32p]GTP (lane 11) and [7-32P]ATP (lane 12). All ther cnditins were as described abve. Numbers between panel A and B indicate mlecular weights in kda. Bth panels shw autradigraphs the nucletide phsphtransferase and nt be the actin f adenylate kinase (Murakami and Strtmann 1978; Birkenhead et al. 1982), labeling was perfrmed in the presence f PI, P5-di(adensine- 5')pentaphsphate, a knwn inhibitr f adenylate kinase (Lienhard and Secemski 1973). When the experiment shwn in lanes 1, 3 and 4 f Fig. 5 was repeated in the presence f P1, P5-di(adensine-5')pentaphsphate (frm 4 t 60 gml-1-1) the switch in patterns shwn in Fig. 5 still ccurred (data nt shwn). Thus we tentatively cnclude that the change in labeling pattern is nt due t the actin f adenylate kinase and is mst likely due t the actin f the nucletide phsphtransferase. Further supprt fr this cnclusin will require a mre detailed characterizatin f the nucletide-phsphtransferase activity. These results indicate the presence f tw distinct prtein-kinase activities in the uter chlrplast envelpe. Further discriminatin between the tw kinases was made pssible by their specificity with externally added prtein substrates. Phsvitin

5 492 J. Sll et al. : GTP-dependent prtein kinase 1.00 I I ~' I 7e lo, I d_ 0.50 O Q ~~ ~176 B ] ~, I I ( 035 0s0 I [ I I GDP M3 Fig. 4. Inhibitin f GTP-dependent prtein phsphrylatin by GDP in uter envelpe membranes f pea chlrplasts. Phsphrylatin prducts were analysed by SDS-PAG and incrpratin rates were quantified by tiquid scintillatin cunting as described abve. Incrpratin rates were calculated as the sum f 32P-label in the 23-plus 32.5-kDa prteins "~ 0.25 t t [ I 2 3 Heparin [mu] Fig. 3A, B. Inhibitin by heparin (mu.m1-1) f uter-envelpe-bund GTP and ATP prtein kinases frm pea chlrplasts. Discriminatin between the ATP and GTP kinase was made pssible by their different respnses t heparin. A The respnse t heparin in the presence f D,-3=P]GTP; 23-( x - x ), 32.5-( ), and 74-kDa (A--A) prteins. B The inhibitin f prtein phsphrylatin by heparin in the presence f [7-32P]ATP; 23-( x ), 32.5-(e), 58-(11), 74-(v), and 86-kDa (A) prteins. The insert in A shws the autradigram f the data quantitated in A and B. Heparin inhibitin is demnstrated fr GTP (left) and ATP (right). Heparin cncentratins increase frm the center t the utsides (1 mg. ml-1) an acidic prtein is accepted as external prtein substrate using [7-32p]GTP (data nt shwn). Bth the membrane-bund (Sll 1985) and als the purified ATP-dependent kinase d nt use this acidic prtein as phsphate acceptr, but uses Histne IIIs as preferred acceptr prtein (Sll 1988). Phsphrylatin f envelpe prteins by GTP shws a temperature ptimum f 20~ while phsphrylatin in the presence f ATP exhibits a temperature ptimum between 30 and 37 ~ C. Bth enzymes are still active at lw temperature and have residual activities f abut 30% and 15% at 0 and -12 ~ C, respectively (assay in presence f 25% glycerl; data nt shwn). Radilabelled phsphrus frm [7-32p]GTP was incrprated int membrane prteins in a time- and prtein-cncentratin-dependent manner. Incrpratin rates were linear up t 15 rain and up t a final membrane prtein cncentratin f 1 mg.m1-1 (data nt shwn). We therefre chse a time between 5 and 15 min and a prtein cncentratin f 0.1 rag. ml- 1 fr ur standard incubatin. The uter-envelpe-bund GTP kinase has a high affinity fr its substrate GTP, and an S0.s (cncentratin f substrate supprting halfmaximal velcity) f 1.5 pml. 1-1 was determined frm (Fig. 7). Maximal incrpratin rates were abut 1 5 pml. (mg prtein)- 1. min- 1. The GTP kinase had a brad ph ptimum between ph 7 and 8 with steep decreases in activity at mre acidic r alkaline phs (Fig. 7, insert). Analysis f the phsphrylated amin acids shwed that mst (90%) f the label was present in phsphserine

6 J. Sil et al. : GTP-dependent prtein kinase 493 Fig. 5. ffect f ADP n the [7-32p] GTP-dependent phsphrylatin f plypeptides in the uter envelpe membranes f pea chlrplasts. Lane 1, phsphrylatin by [,/.3 zp] GTP (16 laml. 1-1, 185GBq.mml-1), lane 2, phsphrylatin by [y- ~2P]ATP (i61aml.1 i, 185 GBq.mml 1); lane 3, [~- 32P]GTP + 10 I-tml. 1-1 ADP; lane 4, [7-3 zp] GTP laml. 1 -~ ADP; lane 5, [y-32p]gtp+10 gml.1-1 GDP; lane 6, [y- 32P]GTP+100 laml.l -~ GDP. Samples were analyzed n a 121/2% running gel; all ther cnditins were as described in Material and methds Fig. 6. Detectin f a nucletide triphsphate-nucletide diphsphate phsphtransferase in the uter-envelpe membrane fractin f pea chlrplasts. The presence f a phsphtransferase activity was evaluated directly using thin-layer-chrmatgraphy analysis f the reactin prducts. A phsphtransferase reactin was cnducted in 20 mml.1-1 Tricine-KOH ph 7.9, 4 mml.l-' MgC12, 60 laml.1-1 [7-32P]NTP, 60 gml.1 1 NDP, 10 lag prtein, fr 10 min at 20~ in a final vlume f 25 lal. A 1-lal aliqut was sptted nt precated plyethylene imine cellulse plates (G 1440 PI; Schleicher & Schiill, Dassel, FRG) and develped in 1 ml-1 -x KH2PO4. Authentic [7-32p]ATP and [7-32p]GTP were run as internal standards while little (10%) was detected in phsphthrenine (data nt shwn). Many prtein kinases depend n the presence f divalent catins fr maximal activity. In ur hands, the GTP-dependent kinase required bth Mg 2 and Mn 2 + fr maximal activity. N incrpratin was detected in the absence f divalent catins (Fig. 8). Maximal activity was bserved at 10mml.1-1 Mg 2+ and 2mml.1-1 Mn 2+. Increasing the Mg 2+ cncentratin up t 15 retl. 1-1 in the absence f Mn 2 + resulted in a cntinual increase in enzyme activity with bth endgenus membrane prtein substrates (Fig. 8). Increasing the Mn 2+ cncentratin prduced different respnses fr the phsphrylatin f the 23- and 32.5-kDa prteins (Fig. 8): 32p-label in the 23-kDa prtein increased up t 15 mml.1- ~ Mn ~+ while maximal incrpratin int the 32.5-kDa prtein was btained at 2 mml.1-1 Mn2+; higher Mn 2+ cncentratin resulted in an inhibitin. The inhibitin was vercme by the simultaneus inclusin f Mg 2+ (Fig. 8). The presence f Ca 2- (5-500gml.1-1) r Ca 2+ plus calmdulin (10U, 0.3 gg) had n effect upn enzyme activity. Assays were dne in the presence f I mml- 1 - ~ ethylene diaminetetraacetic acid (DTA); the Ca z cncentratins used abve represent the unchelated amunt f Ca 2 in the assay. Furthermre the calmdulin antagnist W-5, which causes 50% inhibitin f calmdulin-dependent kinases when pres-

7 494 J. Sll et al. : GTP-dependent prtein kinase ~'r ~ _ 0.25 I I I I l.... / 0 /... i f.~.~ ~ ~ ~ ~ O ~ GTP [pm] I /\ ph I O O I Fig. 7. Influence f GTP cncentratin upn the activity f GTP-dependent prtein kinase frm uter envelpe membranes f pea chlrplasts. Incrpratin rates were calculated as the sum f 32p label in the 23 plus kda prteins, which were excised frm the gel and quantitated as described in Material and methds. Bth prteins shw the same respnse t increasing substrate cncentratins. Insert ph-dependence f 32p incrpratin int uter-envelpe plypeptides frm [7-3ZP]GTP. The buffer used in the acidic range was 50 mml.1-1 [bis(2- hydrxyethyl)imintris(hydrxymethyl)methane]-hc1 (zx-zx) whereas 50 mml.1- ~ Tricine- KOH (-) was used in the alkaline range 12-9 t-- A 5 mm Mn=" [] 2 ~ Mn =+ O n Mn = kda ent at 250 gml.1-1 (Chafuleas et al. 1982), had n effect n the GTP-dependent kinase at 2 mml. l- 1 final cncentratin. N effect n the GTP-dependent kinase was bserved with GMP (0-0.4 mml. l- 1), cgmp ( mml.l-1) r the prtein kinase inhibitr H-9 (0-20 gml.l-1). The drug H-9 has been shwn t inhibit cgmp dependent prtein kinase (Ki 0.87 pml.l-1; Hidaka et al. 1984). The prteineaus prtein kinase inhibitr (delmann et al. 1987) caused less than 50% inhibitin at a final cncentratin f 40 gg inhibitr per ml. r / A 5mMMn =+ 25 kda 9 [] 2 mn Mn =+ 0 n Mn =+ X n Mg =" ~ A A""'- _ 0 5 to 15 Mn 2" r Mg 2" [rnm] Fig. 8. ffect f divalent catins rl the GTP-dependent phsphrylatin f plypeptides frm the uter envelpe membranes f pea chlrplasts. nzyme activity was measured by keeping either the Mn 2 + r Mg 2 + cncentratin cnstant while the ther was varied. The upper part f the figure shws the results btained fr the 32,5-kDa prtein, the lwer part shws the results fr the 23-kDa plypeptide -- Discussin The present study prvides evidence fr the existence f a GTP-dependent prtein kinase f the casein kinase II type (Hathaway and Traugh 1979, 1983; delman et al. 1987) in the uter envelpe membrane frm pea chlrplasts. The reasning fr this classificatin is the fllwing: (i) the prtein-kinase activity is independent f cgmp, camp, Ca 2+ r calmdulin; (ii) the S,s fr GTP is very lw (1.5 gml.1-1 while fr casein kinase I the Km fr GTP is generally high (1 mml. 1-1), and fr ther casein type II enzymes the S.s fr GTP is abut fld higher than that reprted here (Hathaway and Traugh 1983; delman et al. 1987); (iii) the prtein kinase accepts phsvitin as an acidic external prtein substrate in the presence f [7-32p]GTP; (iiii) the GTP-dependent prtein kinase is strngly inhibited by heparin (Table 1; Hathaway et al. 1980). Prtein kinases f this type

8 J. Sll et al. : GTP-dependent prtein kinase 495 Table 1. Cmparisn f uter-envelpe-bund prtein-kinase activities using [7-32p]GTP and [7-32P]ATP Prtein phsphrylatin GTP ATP in the presence f: Sensitive t heparin yes n Phsvitin as exgenus substrate yes n Temperature ptimum 20 ~ C ~ C S.s f the 1.5 gm GTP 37 gm ATP membrane-bund enzyme Preferentially labeled prteins 32.5 kda, 86 kda, 23 kda 18 kda Highly dependent yes n n divalent catins have been described frm wheat germ (Yan and Ta 1982), islated nuclei f tbacc (rdmann et al. 1982) and sybean ctyledns (Gwda and Pillay 1982). In mst cases the subcellular lcalizatin was nt determined. The uter envelpe f pea chlrplasts cntains at least tw prtein-kinase activities, an ATP-dependent prtein kinase (Sll and Buchanan 1983; Sll 1985) and the GTPdependent kinase described abve. Several criteria were used as evidence that the tw activities are nt a result f the same enzyme. First the GTP-dependent enzyme phsphrylates a different set f endgenus membrane prteins (Fig. 2). Secnd, the tw enzymes differ in their respnse t heparin. The GTP-dependent prtein kinase is inhibited by heparin whereas the ATPdependent activity generally is nt. The GTP-dependent prtein kinase may als use [7-32P]ATP as a substrate. This tentative cnclusin is derived frm the bservatin that heparin exerts an inhibitry effect n ATP-dependent phsphrylatin f nly the 23-kDa and the 32.5-kDa plypeptides, whereas the phsphrylatin f ther uter envelpe prteins is nt affected (Fig. 3). Third, the tw activities differ in a number f bichemical parameters. Fr example, their respnse t divalent metal ins is quite different. The GTP-dependent enzyme abslutely requires Mn 2 + r Mg 2+, whereas the uter-envelpe-bund ATP-dependent prtein kinase is nt cmpletely dependent n divalent catins and shws nly 50% stimulatin in the presence f 2 mml.1 1 Mg2+ (Sll 1985). Interestingly, the simultaneus inclusin f ADP and [7-32p]GTP in the phsphrylatin assay gave rise t a typical [y-3zp]atp phsphrylatin pattern, indicating that a very active nucletide phsphtransferase is present in the membrane fractin used. Further evidence fr this was prvided by an assay which directly measures this enzyme. Preliminary bservatins indicate that the additin f unlabeled ATP t the phsphrylatin mixture including radiactive [~/-32p]GTP results in a shift f the labeling pattern similar t that bserved with unlabeled ADP (data nt shwn). Similar phsphtransferase activities were reprted t exist in crude extracts f swine heart and several rat tissues (Ku 1974). The chlrplasts are strictly cmpartmentalized rganelles, having three separate membrane systems (uter envelpe, inner envelpe and thylakids) and three separate sluble spaces (envelpe lumen, strma and thylakid lumen). Tday, data are available fr prtein-phsphrylatin activities in all f these cmpartments except the thylakid lumen (Ranjeva and Budet 1987; Sll and Bennett 1988). As can be seen frm Fig. 2, thylakid plypeptides and in particular the light-harvesting cmplex prtein shw the highest ttal 32P-incrpratin rates. Hwever, envelpe membranes shw strng incrpratin rates in cmparisn with strma prteins and a larger number f labeled prteins in cmparisn with strma and thylakids. Assuming that the envelpe-membrane prteins accunt fr abut 1% f ttal chlrplast prteins (Duce et al. 1984), the verall 32p-incrpratin int envelpe prteins is lw cmpared with thylakid prtein phsphrylatin but prbably accunts fr mre than 5% f the ttal label present in all chlrplast phsphprteins. In-rganell labeling, using intact pea chlrplasts and [7-32p]ATP in the dark (30 nml.1-1 at 4 ~ C fr 60 s), shwed that under these cnditins abut 90% f the radiactive label was present in envelpe-membrane plypeptides (Sll and Bennett 1988). The expsed lcalizatin f the envelpe-membrane cmpartment as a barrier between the rganelle and the cell as well as its multiple enzymatic functins make it mst likely that phsphrylatin at this membrane is a majr regulatry phenmenn. This wrk was supprted by grants frm the Deutsche Frschungsgemeinschaft and frm the U.S. Natinal Science Fundatin. References Allen, J.F., Bennett, J. (I 981) Phtsynthetic prtein phsphrylatin in intact chlrplasts. FBS Lett. 123, Arnn, D.J. (1949) Cpper enzymes in islated chirplasts. Plyphentxidase in Beta vulgaris. Plant Physil. 24, 1-15 Ashtn, A.R., Hatch, M.D. (1983) Regulatin f Ca phtsynthesis: regulatin f pyruvate, Pi dikinase by ADP-dependent phsphryiatin and dephspbrylatin. Bichem~ Biphys. Res. Cmmun. 115, Bhalla, P., Bennett, J. (1987) Chlrplast phsphprteins:

9 496 J. Sll et al. : GTP-dependent prtein kinase phsphrylatin f a 12 kda strmal prtein by the redx cntrlled kinase f thylakid membranes. Arch. Bichem. Biphys. 252, Birkenhead, K., Walker, D., Fyer, C. (1982) The intracellular distributin f adenylate kinase in the leaves f spinach, wheat and barley. Planta 156, Budde, R.J.H., Chllet, R. (1986) In vitr phsphrylatin f maize leaf phsphenlpyruvate carbxylase. Plant Physil. 82, Chafuleas, J.G., BaRn, W.., Hidaka, H., Byd, A.., Means, A.R. (1982) Cahndulin and cell cycle: Invlvement in regulatin f cell-cycle prgressin. Cell 28, Cline, K., Werner-Washburne, M., Andrews, J., Keegstra, K. (1984) Thermlysin is a suitable prtease fr prbing the surface f intact pea chlrplasts. Plant Physil. 75, Cughlan, S.J., Hind, G. (1986) Prtein kinases f the thylakid membrane. J. Bil. Chem. 261, Duce, R.A., Blck, M.A., Drne, A.J., Jyard, J. (1984) The plastid envelpe membranes: Their structure, cmpsitin and rle in chlrplast bigenesis. Subcell. Bichem. 10, 1-84 delman, A.M., Blumenthal, O.K., Krebs,.G. (1987) Prtein serine/threnine kinases. Annu. Rev. Bichem. 56, rdmann, H., B6cher, M., Wagner, K.G. (1982) Tw prtein kinases frm nuclei f cultured tbacc cells with prperties similar t the cyclic nucletide-independent enzymes (N I and NII) frm animal tissue. FBS Lett. 137, Fyer, C. (1984) Phsphrylatin f a strmal enzyme prtein in maize (Zea mays) mesphyll chlrplasts. Bichem. J. 222, Gwda, S., Pillay, D.T.N. (1982) Cyclic AMP independent prtein kinases frm sybean ctyledns (Glycine max L.). Plant Sci. Lett. 25, Glver, C.V.C., Sheltn,.R., Brutlag, D.L. (1983) Purificatin and characterizatin f a type II casein kinase frm Drsphila melangaster. J. Bil. Chem. 258, Hathaway, G.M., Lubben, T.H., Traugh, J.A. (1980) Inhibitin f caseinkinase II by heparin. J. Bil. Chem. 225, Hathaway, G.M., Traugh, J.A. (1979) Cyclic nucletide independent prtein kinase frm rabbit reticulcytes. J. Bil. Chem. 254, Hathaway, G.M., Traugh, J.A. (1983) Casein kinase II. Methds nzyml. 99, Hidaka, H., Inagaki, M., Kawamt, S., Sasaki, Y. (1984) Isquinlinesulfnamides, nvel and ptent inhibitrs f cyclic nucletide dependent prtein kinase and prtein kinase C. Bichemistry 23, Jyard, J., Billeccq, A., Bartlett, S.G., Blck, M.A., Chua, N.H., Duce, R. (1983) Lcalizatin f plypeptides t the cytslic side f the uter envelpe membrane f spinach chlrplasts. J. Bil. Chem. 258, Keegstra, K., Yusif, A.. (1986) Islatin and characterizatin f chlrplast envelpe membranes. Methds nzyml. 118, Ku, J.F. (1974) On the questin f the existence f a guansine adensine triphsphate specific prtein kinase activated by adensine 3':5-mnphsphate dependent prtein kinase. J Bil. Chem. 249, Laemmli, U.K. (1970) Cleavage f structural prteins during the assembly f the head f bacteriphage T4. Nature 227, Laing, W.A., Christeller, J.T. (1984) Chlrplasts phsphprteins: distributin f phsphprteins within spinach chlrplasts. Plant Sci. Lett. 36, Lienhard, G.., Secemski, J.J. (1973) Pl,PS-Di(adensine- 5')pentaphsphate, a ptent multisubstrate inhibitr f adenylate kinase. J. Bil. Chem. 248, Lin, Z.F., Luzer, H.A., Racker,. (1982) Prtein kinases frm spinach chlrplasts. J. Bil. Chem. 257, Murakami, S., Strtmann, H. (1978) Adenylate kinase bund t the envelpe membranes f spinach chlrplasts. Arch. Bichem. Biphys. 185, Ranjeva, R., Budet, A.M. (1987) Phsphrylatin f prteins in plants: Regulatry effects and ptential invlvement in stimulus/respnse cupling. Annu. Rev. Plant. Physil. 38, Schindler, C., Hracky, R., Sil, J. (1987) Prtein transprt in chlrplasts: ATP is prerequisit. Z. Naturfrsch. 42c, Sil, J. (1985) Phsphprteins and prtein kinase activity in islated envelpes f pea (Pisum sativum L.) chlrplasts. Planta 166, Sil, J. (1988) Purificatin and characterizatin f a chlrplast uter envelpe bund ATP-dependent prtein kinase. Plant Physil. 87, Sil, J., Bennett, J. (1988) Lcalizatin f a 64 kda phsphprtein in the lumen between the uter and inner envelpes f pea chlrplasts. ur. J. Bichem. 175, Sll, J., Buchanan, B.B. (1983) Phsphrylafin f chlrplast ribulse biphsphate carbxylase/xygenase small subunit by an envelpe-bund prtein kinase in situ. J. Bil. Chem. 258, Ventimiglia, F.A., Wl, I.G. (1974) A kinase that transfers the 7-phsphryl grup f GTP t prteins f eukarytic 40S ribsmal subunits. Prc. Natl. Acad. Sci. USA 71, Yan, T.F_J., Ta, M. (1982) Purificatin and characterizatin f a wheat germ prtein kinase. J. Bil. Chem. 257, Received 5 May; accepted 8 August 1988