Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Transcription

1 Plant Cell Physil. 40(12): (1999) JSPP 1999 Phsphprteins a Prtein Kinase Activities Intrinsic t Inner Membranes f Ptat Tuber Mitchria Are Struglics 1, Kenneth M. Fredlu 2-3, Ian M. Mailer 2 a Jhn F. Allen Plant Cell Bilgy, Lu University, Bx 7007, S Lu, Sweden 2 Plant Physilgy, Lu University, Bx 117, S Lu, Sweden Inside-ut submitchrial particles (IO-SMP) were islated a purified frm ptat (Slarium tubersum L. cv.) tubers. When these IO-SMP were incubated with [y- 32 P]ATP mre then 20 prteins became labelled as a result f phsphrylatin. The 32 P incrpratin was stimulated by the xidising reagent ferricyanide. Except fr a 17 kda prtein which was phsphrylated nly in the absence f divalent catins, the prtein phsphrylatin required Mg 2+. The time fr half-maximum 32 P incrpratin was 4 min fr the 22 kda phsph-f, d'-subunit a 2 min fr the 28 kda phsph-f b-subunit f the prtn-atpase. The K m fr ATP fr the detected phsphprteins was between 65 fim a 110 /<M. The ph ptimum fr prtein phsphrylatin in inner membranes was between ph 6 a 8, a fr the F t d'-subunit a the F b-subunit the ph ptima were a ph 8, respectively. A 37 kda phsphprtein was phsphrylated n a histidine residue while the remaier f the inner membrane prteins were phsphrylated n serine r threnine residues. Tw autphsphrylated putative kinases were identified: ne at 16.5 kda required divalent catins fr autphsphrylatin, while anther at 30 kda did nt. A 110 kda prtein was labelled nly with [a- 32 P]ATP, suggesting adenylylatin. Key wrds: Autphsphrylatin F 0 F,-ATPase Inner membrane Mitchria Ptat (Slarium tubersum L.) tubers Prtein phsphrylatin. Pst-translatinal mdificatin by reversible prtein phsphrylatin is a ubiquitus regulatry mechanism in cellular bichemistry, implicated in such prcesses as cn- Abbreviatins: Bis-tris, Bis(2-hydrxyethyl)amin-tris(hydrxymethyl)methane; IO-SMP, inside-ut submitchrial particles; MOPS, 3-Mrphlinprpanesulfnic acid; NDPK, nucleside diphsphate kinase; t l/2, time fr half-maximum 32 P incrpratin; Tricine, A/-[tris(hydrxymethyl)methyl]glycine; TLC, thin layer chrmatgraphy; Tween 20, Plyxyethylene srbitanmnlaurate. 3 Present address; Nvartis Seeds AB, Bx 302, S Laskrna, Sweden. 4 Crresping authr: , jhn.allen@plantcell.lu.se; Fax, trl f enzyme activity, prtein degradatin, gene expressin a signal transductin (McEntyre 1994). In mammalian mitchria abut 10 phsphprteins have been detected by SDS-PAGE a autradigraphy (Ferrari et al. 1990, Technikva-Dbrva et al. 1993). Plant mitchria may be enriched in phsphprteins cmpared with mammalian mitchria, a abut 30 phsphprteins have been detected in intact plant mitchria using SDS-PAGE a autradigraphy (Dank a Markwell 1985, Smmarin et al. 1990, Pike et al. 1991). The regulatin by reversible phsphrylatin f the pyruvate dehydrgenase cmplex a the branched-chain a-ketacid dehydrgenase cmplex are the nly well-characterised mitchrial prtein phsphrylatin-dephsphrylatin events (Bradfrd a Yeamen 1986, Raall et al. 1989). Previus studies n prtein phsphrylatin in plant mitchria have been carried ut with intact mitchria, where inner membrane phsphprteins are nt easily distinguished frm uter membrane phsphprteins r frm the strngly labelled a-subunit f pyruvate dehydrgenase lcated in the matrix cmpartment (Petit et al. 1990, Smmarin et al. 1990). With the exceptin f the highly active pyruvate dehydrgenase kinase, mst prtein kinase activities identified in intact plant mitchria are lcalised in the uter membrane (Pical. et al. 1993). T date, nly tw inner membrane phsphprteins have been identified in plant mitchria, the 22 kda t5'-subunit a the 28 kda b-subunit f the F F,-ATPase (Struglics et al. 1998). In mammalian mitchria an additinal tw inner membrane phsphprteins have been identified, the 18 kda (IP) AQDQ subunit f cmplex I (Papa et al. 1996), a the 17 kda subunit IV f cytchrme c xidase (Steenaart a Shre 1997). The general prperties f prtein phsphrylatin in the inner mitchrial membrane are unknwn as is the number a lcatin f the prtein kinases invlved. Here we describe the kinetics, ATP-depeency a ph ptima f prtein phsphrylatin fr the 22 kda 5'- subunit a the 28 kda b-subunit f FFpATPase as well as fr ther, as yet unidentified, phsphprteins in the inner membranes f ptat tuber mitchria. We als reprt autphsphrylatin f inner membrane prteins, suggesting the presence f a nvel inner membrane prtein kinase in plant mitchria. Dwnladed frm at UCL Library Services n Nvember 1,

2 1272 Inner mitchrial membrane prtein phsphrylatin Materials a Methds Islatin f mitchria a inner mitchrial membranes Highly purified mitchria free frm plastids (amylplasts) a perxismes were islated frm ptat tubers (Slanum tubersum L. cv. Bintje r Ukama) as described in Struglics et al. (1993). Inside-ut submitchrial particles (IO-SMP) were islated accrding t Pical et al. (1993). Freshly prepared inner membranes were used fr all experiments. In vitr phsphrylatin The staard in vitr prtein phsphrylatin assay was carried ut in a vlume f 50 /ul cntaining fig, inner membrane prteins. Final cncentratins in the phsphrylatin buffer (1 x R buffer) were; 0.3 M sucrse, 50 mm HEPES-KOH, ph 7.5, 5 mm MgCl 2, 0.1 mm CaCl 2) 8-12//Ci [y- 32 P]ATP (AA0068, Amersham) a 0.2 mm ATP. In Figure 1 we als used [a- 32 P]ATP (PB10200, Amersham) with the same specific activity as fr [y- 32 P]ATP. Phsphrylatin experiments were carried ut at rm temperature (22 C) with incubatin times frm 20 s t 30 min as iicated in the text. The reactins were terminated by additin f sample buffer (Laemmli 1970) cntaining 0.1% SDS a then heat denaturated at 100 C fr 3 min. Reactin kinetics a A TP depeency Fr the kinetic studies IO-SMP prteins (3 mg) were incubated in 1 x R buffer in the presence f 210 nc\ [y- 32 P]ATP a 200 ^im ATP in a ttal vlume f 950^1. SOfA samples were withdrawn at different time pints, a the reactins were stpped as described abve. T examine ATP cncentratin depeency, IO-SMP prteins (140 ^g) were incubated in 1 x R buffer fr 2 min in a ttal vlume f 20^1 in the presence f different ATP cncentratins: specific activity was maintained at 15^Cinml~' ATP. The reactins were stpped as described abve. In situ phsphrylatin With a few mdificatins, the in situ phsphrylatin assay is adapted frm the methd f San Agustin a Witman (1995). IO-SMP prteins (2.6 mg) were slubilised in SDS-cntaining sample buffer (Laemmli 1970), laded in a 50 mm brad well, a separated by SDS-PAGE. The prteins were electrphretically transferred t a PVDF membrane (PALL) as described in Struglics et al. (1998). The membrane was cut int 1 cm strips a incubated at rm temperature fr 15 min in 50 mm HEPES-KOH, 140 mm NaCl, ph 7.5 prir t incubatin fr 12 h at 8 C in renaturatin buffer (10 mm HEPES-KOH, 140 mm NaCl, 2 mm EDTA, 2 mm DTT, 1% w/v BSA, 0.1% v/v Tween 20, ph 7.5). Fllwing renaturatin, the membrane strips were washed fr 5 min in 50 mm HEPES-KOH, ph 7.5 a then incubated at rm temperature fr 45 min (up t 8 h) in 5 ml phsphrylatin slutin cntaining: (a) 50 mm HEPES-KOH, ph 7.5, 10 mm MgCl 2 a 0.2 mm CaCl 2 r (b) 50 mm HEPES-KOH, ph 7.5 a 10 mm EDTA; bth supplemented with 200 /Ci [y- 32 P]ATP r {a- 32 P]ATP. After in situ phsphrylatin the PVDF membranes were washed as fllws: 1x5 min then 2 x 20 min in 50 mm HEPES-KOH, ph 7.5; 1 x 10 min in 50 mm HEPES, 1 M KOH, 0.05% v/v Tween 20; 2 x 10 min in 50 mm HEPES-KOH, ph 7.5; 1 x 10 min in 1 M KOH a 1 x 10 min in distilled H 2 O. The PVDF membranes were briefly stained with Cmassie blue (R250) a dried befre expsure t either autradigraphic film r phsphrlmager plates. Acid a alkaline treatment f phsphrylated prteins The acid-alkaline treatment f phsphrylated IO-SMP prteins is bradly based n the methd f Oda a Hasunuma (1994). IO-SMP (3.2 mg prtein) were phsphrylated accrding t the staard in vitr phsphrylatin assay in a ttal vlume f 625 fa. The prteins were laded in a 70 mm brad well, a separated by SDS-PAGE a electrphretically transferred t a Hyb- PVDF membrane (Amersham) as described in Struglics et al. (1998). The membrane was cut int 2 cm strips, incubated in 0.5 M HC1 r 0.5 M NaOH r 50 mm HEPES-KOH ph 7.5 at 65 C fr 2 h, dried a expsed t autradigraphic film r phsphrlmager plates. SDS-PAGE a detectin f phsphprteins SDS-PAGE was perfrmed accrding t Laemmli (1970), n a 10-15% T (C = 2.7%) gradient gel with Prtean II xi Slab Cell (BiRad) apparatus. Gels were stained with a Cmassie blue slutin (0.4% w/v Cmassie Brilliant blue [R250], 50% v/v methanl, 10% v/v acetic acid), a destained in 35% v/v methanl, 15% v/v acetic acid befre drying. Phsphprteins were visualised by expsure fr 3 t 4 d t a Phsphrlmager plate (Mlecular Dynamics) r t autradigraphic film (Hyper film MP, Amersham). Other analytical methds Prtein cncentratin was determined accrding t Bradfrd (1976), using IgG as a staard. Fr quantificatin analysis, the stained a dried gels were expsed t Phsphrlmager plates. The data were analysed with ImageQuant sftware (versin 1.2), a the quantificatin was based upn ttal pixels in prtein bas crrected fr backgru signals. Results In vitr labelling using [y- 32 PJA TP a [a- 32 P]A TP as phsphate dnrs In prtein phsphrylatin catalysed by prtein kinases, the y-phsphate frm ATP r GTP is transferred t the prtein substrates. T determine whether prtein radilabelling in inner mitchrial membranes results frm cvalent biing f Pi (phsphrylatin) r if, instead, ATP (r ADP a AMP) is bu t the inner membrane prteins, we used [y- 32 P]ATP as a phsphate dnr a cmpared the result with labelling btained with [a- 32 P]ATP. Figure 1A shws SDS-PAGE separated inner membrane prteins stained with Cmassie blue. Figure IB shws the crresping Phsphrlmage where inner membrane (IO-SMP) prteins have been labelled with [y- 32 P]ATP r [a- 32 P]ATP. At least 20 labelled bas were detected when IO-SMP were incubated with [y- 32 P]ATP (Fig. IB). Since nne f these prteins were labelled when [a- 32 P]ATP was used, we cnclude that the labelling results frm cvalent biing f y-phsphate grups t the prteins (Fig. IB). Replacement f [y- 32 P]ATP with [y- 32 P]- GTP resulted in weak labelling a n specific GTP-depeent prtein phsphrylatin was bserved in IO-SMP (data nt shwn). A 16 kda prtein was the mst heavily labelled phsphprtein in the IO-SMP, a the psitins fr the phsphrylated 22 a 28 kda F 0 F, subunits are als marked in the figure. Incubatin f IO-SMP prteins with [a- 32 P]ATP resulted in labelling f nly ne prtein, f 110 kda (Fig. IB). Since the 110 kda prtein is nt labelled with [y- 32 P]ATP, the label prbably riginates frm direct biing (adenylylatin) between [a- 32 P]AMP r [a- 32 P]-ADP a the prtein. Effect f ferricyanide The 32 P incrpratin int the IO-SMP prteins was higher in the presence f the xidis-

3 Inner mitchrial membrane prtein phsphrylatin kda kda -22 kda kda I Fig. 2 IO-SMP prteins phsphrylated in the presence f redx reagents. Autradigraph f IO-SMP prteins (250 fig) labelled with [y-32p]atp/200 /im ATP in 1 x R buffer in the ab32 Fig. 1 Radi-labelling f IO-SMP prteins by [y- P]ATP r [asence f redx reagents (cntrl), r in the presence f 5 mm 32 P]ATP. Inner mitchrial membranes (140/ig prtein) isptassium ferricyanide, r in the presence f 5 mm sdium di32 lated frm ptat tubers were incubated with either [y- P] ATP r [a-32p] ATP in the presence f 200 [*M ATP a 1 x R buffer fr 10 thinite. Prir t additin f ATP the samples were preincubated fr 3 min in the respective redx reagent. The phsphrylatin min. The reactins were terminated by biling in sample buffer reactins were terminated after 1 min by biling in sample buffer a prteins separated by SDS-PAGE. (A) Cmassie bluea the prteins were separated by SDS-PAGE. The psitins f stained IO-SMP prteins. (B) Phsphrlmage f radi labelled the 22 kda F, (5'-subunit, the 28 kda F b-subunit discussed in the IO-SMP prteins. The psitins f the 22 kda F! <5'-subunit, the text are marked by arrws a the psitins f mlecular mass 28 kda F b-subunit, the 16 kda phsphprtein as well as a 110 markers (kda) are iicated. kda prtein discussed in the text are marked by arrws a the psitins f mlecular mass markers (kda) are iicated. A B ing reagent ferricyanide cmpared t in the presence f the reducing reagent dithinite r in the absence (called cntrl) f any redx reagent (Fig. 2). The phsphrylatin was fu t be stimulated by lw cncentratins f ferricyanide (1 mm) a n further increase in 32P incrpratin culd be detected at higher cncentratins f ferricyanide, up t 10 mm (data nt shwn). In these experiments (Fig. 2) prtein phsphrylatin was carried ut fr nly 1 min, which is within the linear phase f IO-SMP prtein phsphrylatin kinetics (see Fig. 4). Effects f divalent catins Figure 3 shws an autradigraph f inner membrane prteins phsphrylated in the presence f EDTA r divalent catins. As a cmparisn with phsphrylatin in the presence f Mg2"1" r Ca 2+ we als shw in Figure 3 the staard assay fr inner membrane prtein phsphrylatin (5 mm MgCl mm CaCl2). Phsphrylatin in the presence f EDTA ablished 32 P incrpratin int all prteins, with the exceptin f a 17 kda prtein (Fig. 3). Phsphrylatin f the 17 kda prtein was suppressed by divalent catins (Fig. 3). Except fr phsphrylatin f the 17 kda prtein, inner membrane kinase activity required bth Mg2"1" as shwn in Fig. 3 a Ca 2+ as shwn by Pical et al. (1993). Mn 2+ culd replace Mg 2+ (bth tested ver a range f 1 t 10 mm) withut lss f kinase activity, but Zn 2+ (5 mm) ablished all phsphrylatin (data nt shwn). When phsphrylatin was carried ut in the presence f lw Ca 2+ cncentratins (^0.2 mm), the 32P incrpratin int a 41 kda prtein was similar t that in the presence f Mg 2+ (Fig. 3). At higher Ca 2+ cncentratins (>0.2 mm), the 32P incrpratin int the 41 kda prtein was suppressed, as fr the rest f inner membrane prteins (results nt shwn). Time-curse As an example f time-depeent prtein phsphrylatin in IO-SMP, we shw the kinetics fr fur prteins. The half-maximum time f Pj incrpratin (t1/2) was apprximately 4 min fr the 16 kda prtein a the Fi d'-subunit (Fig. 4A), while it was 2 min fr the 25 kda prtein a the F b-subunit (Fig. 4B). The tu2 fr the different IO-SMP prteins varied between 0.5 a 5 min (data nt shwn). Maximal labelling was bserved after min (Fig. 4).

4 Inner mitchrial membrane prtein phsphrylatin O «ynq time (min) - -* 41 kda -28 kda kda -17 kda -16 kda Fig. 3 Effect f divalent catins n prtein phsphrylatin in IO-SMP. Autradigraph f inner membrane prteins, labelled with [y-32p]atp/200 nm ATP. Prir t additin f ATP, 180/jg IO-SMP prtein was incubated fr 3 min in 0.3 M sucrse, 50 mm HEPES-KOH ph 7.5 in the presence f either 5 mm EDTA, 10 mm MgCl2, 0.2 mm CaCl2 r 5 mm MgCl2 a 0.1 mm CaCl2. The phsphrylatin reactins were terminated after 5 min by biling in sample buffer a the prteins were separated by SDSPAGE. Prteins discussed in text are marked by arrws a the psitins f mlecular mass markers (kda) are iicated. Effect f A TP cncentratin The apparent Km fr ATP f the prtein kinase(s) respnsible fr phsphrylatin f inner membrane prteins was between 65 t 110 //M ATP fr all phsphprteins including the 22 kda Fi 8subunit a the 28 kda F b-subunit (Fig. 5). These results were btained by phsphrylatin fr 2 min (Fig. 5) r fr 12 min (data nt shwn). We have used 200^M ATP as a staard in ur phsphrylatin assays, which will give clse t maximum rates f IO-SMP prtein phsphrylatin. ph depeence Prtein phsphrylatin in inner mitchrial membranes shwed a brad ph ptimum f ph 6.5 t 8, which is exemplified in Figure 6 by the phdepeent phsphrylatin f the 22 kda F, <5'-subunit. The highly labelled 16 kda had a sharper ph ptimum at ph 6, a the 28 kda F b-subunit shwed a ph ptimum at ph 8 (Fig. 6). The ph in the cytsl f plant cells is aru 7.5, a the ph in the mitchrial matrix is thught t be slightly higher (Kurkdjian a Guern 1989). The ph f 7.5 in ur staard assay was chsen t reflect physilgical citins, a as a cmprmise between the bserved ph ptima f phsphrylatin fr varius inner membrane pr time (min) Fig. 4 Time-depeent phsphrylatin f IO-SMP prteins. IO-SMP (3 mg prtein) was incubated in 1 x R buffer in the presence f [y-32p]atp/200 /JM ATP. At different time pints, samples f 50/JI (160 fig prtein) were withdrawn a the reactins were terminated by biling in sample buffer. IO-SMP prteins were separated by SDS-PAGE a the gel was Cmassie stained, dried a expsed t a Phsphrlmager plate. The phsphprteins were analysed using ImageQuant sftware, a relative units f 32P incrpratin was calculated where the incrpratin after 30 min was set t 1. A, titratin f phsphrylatin f the 16 kda a 22 kda (F, <5'-subunit) prteins. B, titratin f phsphrylatin f the 25 a 28 kda (F b-subunit) prteins. The brken line shw the estimated f ti /2 values. teins (Fig. 6). Autphsphrylatin f inner membrane prteins The renaturatin a in situ phsphrylatin f prteins separated by SDS-PAGE is a well established methd fr the detectin f prtein kinases (Geahlen et al. 1986, Ferrell a Martin 1991, Hutchcrft et al. 1991, San Agustin a Witman 1995, Race a Hi 1996), but it has never previusly been used n plant mitchria. In the inner membranes, tw autphsphrylated prteins with mlecular mass f 16.5 a 30 kda were identified by [y-32p]atp phsphrylatin after renaturatin f prteins immbilised n PVDF membranes (Fig. 7, lane 2 a 3). Fr cmparisn with the migratin f the 16.5 a 30 kda prteins in SDS-gels, the psitin f the highly labelled 16 kda phsphprtein a the phs-

5 Inner mitchrial membrane prtein phsphrylatin S A «a J3-0.2 *?* / 16 kda O 22 kda 25 kda 28 kda A 36 kda A 39 kda x 41 kda + 50 kda «65 kda a 71 kda 88 kda ATP cncentratin (]im) Fig. S ATP cncentratin-depeent phsphrylatin f IO- SMP prteins. IO-SMP (140/xg prtein) were incubated fr 2 min in 1 x R buffer in the presence f different ATP cncentratins where the specific activity was maintained at 15 /jci nml" 1 ATP. The reactins were terminated by biling in sample buffer a prteins were separated by SDS-PAGE. After Cmassie staining, drying a expsure t a Phsphrlmager plate, the phsphprteins were analysed using ImageQuant sftware. Relative units f 32 P incrpratin were calculated where the incrpratin either at 200 r 350 /JM ATP was set t 1. phrylated 22 a 28 kda F 0 F r ATPase subunits are als iicated in the figure (Fig. 7, lane 1). The 16.5 a 30 kda prteins were nt labelled when phsphrylatin was carried ut in vitr using [y- 32 P]ATP as a phsphate dnr (Fig. 7, lane 1), which can be explained by rapid phsphate transfer frm the enzymes (putative kinases) t available substrates. The 16.5 kda prtein was nt labelled in situ when [a- 32 P]ATP was used as phsphate dnr a nly a trace amunt f istpe was assciated with the 30 kda prtein (Fig. 7 lane 4). This iicates that these prteins have cvalently bu phsphate grups. A difference in the in situ phsphrylatin f the tw prteins was bserved in their requirement fr divalent catins. The 16.5 kda prtein required divalent catins fr autphsphrylatin (Fig. 7, lane 3), while the 30 kda prtein shwed autphsphrylatin als in the presence f EDTA (Fig. 7, lane 2). Pical et al. (1993), shwed that the ttal rate f 32 P- incrpratin int phsphprteins f IO-SMP was stimulated when in vitr phsphrylatin was carried ut in the presence f histnes (histne HI). In ur in situ phsphrylatin assay, the 32 P incrpratin int the 16.5 a 30 kda prteins were neither stimulated nr inhibited when labelling was carried ut in the presence f histnes r inner membrane peptides prepared by trypsin digestin (data nt shwn) Fig. 6 ph-depeent phsphrylatin f the 22 kda F, <5'-subunit; the 16 kda prtein a the 28 kda F b-subunit. IO-SMP prteins (140 pig) were phsphrylated with [y- 32 P]ATP/200 ^M ATP fr 5 min in the presence f 0.3 M sucrse, 5 mm MgCl 2, 0.1 mm CaCl 2 a 50 mm f the fllwing buffers at different ph:, MES-KOH, ph5.5 a 6; O, Bis-tris-HCl, ph 6 a 6.5;, MOPS-KOH, ph 6.5, 7 a ph 7.5; D, TES-KOH, ph 7, 7.5 a ph 8; *, HEPES-KOH, ph 7.5 a 8; A, Tris-HCl, ph 8, 8.5 a ph 9; x, Tricine-KOH, ph 8.5 a 9. The reactins were terminated by biling in presence f sample buffer a prteins were separated by SDS-PAGE. Dried Cmassie stained gels were expsed t a Phsphrlmager plate. The phsphprteins were analysed using ImageQuant sftware, a relative units f 32 P incrpratin were calculated where the maximum label was set t 1. Acid/alkaline stability f phsphamin acids in inner membrane prteins The stability f the cvalent b between phsphate a prtein depes n which amin acid is phsphrylated. This can be analysed by acid-alkali treatments f the phsphprteins (Ducls et al. 1991). When acid-alkali stability/lability measurements were applied t bltted 32 P-labelled inner membrane prteins (Fig. 8), nne f the inner membrane phsphprteins was stable uer bth acidic a alkaline citins, which wuld have iicated phsphrylatin n tyrsine residues PH

6 Inner mitchrial membrane prtein phsphrylatin Z; W c - _ O UJ X a Z I I kda28 kda22 kda kda kda kda kda 20.1 Fig. 7 Autphsphrylatin f IO-SMP prteins. PhsphrImage f IO-SMP prteins bltted t PVDF membranes, phsphrylated either in vitr r in situ. Lane 1, IO-SMP prteins in lxr buffer were phsphrylated fr 10 min with [y-32p]atp/atp accrding t the staard in vitr assay prir t SDS-PAGE a transfer t membrane; Lanes 2 t 4, IO-SMP prteins renaturated a phsphrylated in situ n PVDF membranes. Lanes 2 a 3, prteins incubated with [y-32p]atp. Lane 4, prteins incubated with [a-32p]atp. The in situ labelling was carried ut fr 8 h in 50 mm HEPES-KOH, ph 7.5 buffer supplemented with either 10 mm EDTA (lane 2) r 10 mm MgCl mm CaCl2 (lane 3 a 4). Phsphprteins were visualised by Phsphrlmaging. Prteins discussed in the text are marked by arrws a the psitins f mlecular mass markers (kda) are iicated. (Ducls et al. 1991). A 37 kda phsphprtein was acidlabile a alkali-stable, suggesting phsphrylatin n histidine residues. The remaining phsphprteins in the inner membrane shwed alkaline lability a acid stability (Fig. 8), iicating phsphrylatin n serine r threnine residues. Discussin The phsphprteins a kinase(s) are inner membrane prteins facing the matrix IO-SMP prepared frm ptat tuber mitchria cnsist f inner membrane vesicles depleted f matrix prteins (Liden et al. 1987, Pical et al. 1993). Thus, prtein phsphrylatin in these membranes is catalysed by egenus membrane-bu kinase(s) a all the substrates are membrane-bu prteins. Bth the kinase substrates a the active site f the prtein kinase(s) are lcated n the matrix surface f the inner membranes since (1) the SMP are inside-ut a sealed (Rasmussn a Meller 1991); (2) there is n ADP inside the IO-SMP vesicles t exchange with [y-32p]atp n the ATP-ADP carrier. The prtein phsphrylatin pattern f IO-SMPs -28 kda kda Fig. 8 Acid a alkali treatment f IO-SMP phsphprteins. Phsphrlmage f 16 min in vitr phsphrylated IO-SMP prteins separated by SDS-PAGE a transferred t PVDF membranes. Except fr the PVDF membrane marked cntrl, which was dried immediately, the rest f the PVDF membranes were incubated at 65 C fr 2 h in either f the fllwing slutins; 50 mm HEPES-KOH, ph 7.5 r 0.5 M HC1 r 0.5 M NaOH. After incubatin, the membranes were dried a expsed t a Phsphrlmager plate. Prteins discussed in the text are marked by arrws, a the psitins f mlecular mass markers (kda) are iicated. (Fig. IB) shwed n similarity with the phsphrylatin pattern f intact mitchria, mitchrial matrix prteins r mitchrial uter membrane prteins (Pical et al. 1993; ur unpublished results). Phsphrylatin with [y-32p]atp in intact mitchria r IO-SMP in the presence f ligmycin (which inhibits bth the synthesis a hydrlysis f ATP by the ATPase) changed neither the amunt f 32P incrpratin nr the phsphrylatin pattern cmpared with the cntrl in intact mitchria (Petit et al. 1990), r in the inner membranes (results nt shwn). Prperties f inner membrane prtein phsphrylatin Althugh camp-depeent prtein kinases have been reprted in mammalian mitchria (Papa et al a ref. therein), n such prtein kinases have been detected when phsphrylatin was carried using intact plant mitchria (Dank a Markwell 1995), r IO-SMP (ur unpublished results). The phsphrylatin f the inner membrane prteins requires divalent catins. A ntable exceptin is a 17 kda prtein which is phsphrylated nly in their absence (Fig. 3). The phsphrylatin takes min t reach

7 Inner mitchrial membrane prtein phsphrylatin 1277 maximum 32 P incrpratin (Fig. 4), much lnger than the 2-3 min reprted fr intact mitchria (Smmarin et al. 1990). The K m (ATP) varied between 65 a 110 ^M fr the varius phsphprteins a maximum 32 P incrpratin was reached at an ATP cncentratin abve 200 fim (Fig. 5). These results shuld nt be cmpared directly with results btained with intact mitchria, where the activities f uter membrane a matrix kinases predminate (Pical et al. 1993). Ttal 32 P incrpratin int IO-SMP prteins had the same brad ptimum at ph (Fig. 6) as bserved with intact mitchria (Dank a Markwell 1985, Smmarin et al. 1990), whereas the ptimum ph fr labelling iividual phsphprteins varied frm ph 6.0 t ph 8.0 (Fig. 6). As a cmparisn tw mammalian membranebu prtein kinases, purified frm bvine heart mitchria (Kitagawa a Racker 1982), a a inner membrane prtein kinase(s) prepared frm muse liver (Vardanis 1976) shwed sharp ph ptima at ph 9, ph 7.5 a ph 8.5, respectively. Prtein phsphrylatin f IO-SMP prteins, including the 22 a 28 kda F 0 F! subunits, was stimulated by the xidising reagent ferricyanide (Fig. 2). Additin f ferricyanide t inner membranes lacking reduced substrates will xidise the quinne pl, a this may trigger, directly r iirectly, an inner membrane kinase leading t an increased phsphrylatin f inner membrane prteins. This is ppsite t the effect f ferricyanide n prtein phsphrylatin in chlrplast thylakid membranes (Allen et al. 1981). Except fr the 37 kda phsphhistidine prtein, the inner membrane phsphprteins shwed 32 P incrpratin int serine a threnine residues (Fig. 8), which was als cnfirmed by TLC electrphresis f the 22 a 28 kda F 0 F]-subunit prteins (results nt shwn). Genistein inhibited tyrsine kinases in transfrmed plant rts (Rdriguez-Zapata a Hernaez-Stmayr 1998) a in chlrplast thylakids whereas it had n effect n mitchrial prtein phsphrylatin (Tullberg et al. 1998). Nne f the inner membrane phsphprteins was stable uer bth acidic a alkaline citins (Fig. 8), a their phsphrylatin was unaffected by genistein (data nt shwn). All f this suggests that the detected mitchrial inner membrane phsphprteins are nt labelled n tyrsine residues. The iividual phsphprteins The prperties f a number f the phsphprteins, identified by apparent mlecular mass, are summarised in Table 1. The 22 a the 28 kda FJFrSubunits -The 22 kda F,-ATPase <5'-subunit a the 28 kda F 0 -ATPase b-subunit are knwn phsphprteins (Struglics et al. 1998). The F 0 F r subunits were bth mdified by phsphmnester linkages (n serine r threnine), but the t\n, a the ph ptima fr phsphrylatin f these prteins differ (see Table 1). This may mean that they are phsphrylated by tw different prtein kinases. Alternatively, ne prtein kinase has different affinities fr the tw F 0 F r subunits. The 16.5 a 30 kda autphsphrylated prteins Sluble autphsphrylated prteins have been identified in the matrix f mitchria frm mammals (Lambeth et al. 1997, Bradfrd a Yeamen 1986) a plants (Rubin a Raall 1977, Miernyk et al. 1992). One third f all detected/identified plant prtein kinases shwed autphsphrylatin (Budde a Raall 1990). We suggest Table 1 Features f 32 P labelling f specific inner membrane prteins f mitchria Mw [a- 32 P]ATP [y- 32 P]ATP Mg 2+ EDTA ti /2 (min) ph" Ser/Thr-P His-P Cmments yes n strngest labelled prtein in IO-SMP * autphsphrylated, putative kinase* putative NDP K* yes n Fi <5'-subunit yes n F b-subunit autphsphrylated, putative prtein kinase 6 n yes acid labile prtein n n adenylylated prtein c Summary f 32 P labelling f inner membrane prteins investigated in this study. +, stimulatin a increased labelling;, inhibitin r n labelling;, nt determined; Mw, apparent mlecular mass in kda. " Optimum ph fr labelling. 4 See als text. c AMP r ADP biing prtein.

8 1278 Inner mitchrial membrane prtein phsphrylatin that the inner membrane 30 kda autphsphrylated prtein (Fig. 7) is a prtein kinase, althugh it may als be the catalytic subunit f a larger multisubunit prtein kinase. This wuld be cnsistent with the bservatin that the catalytic subunit f many prtein kinases is apprximately kda (Hanks et al. 1988). The 16.5 kda prtein is autphsphrylated a therefre a putative kinase (Fig. 7). It is t small t agree with the current uerstaing f the size limitatins f prtein kinases (Hanks et al. 1988), a may therefre be a kinase fr anther class f substrate. The 37 kda phsphprtein The 32 P-labelled IO- SMP cntain a 37 kda phsphhistidine prtein, as cncluded frm the acid-alkali stability measurements (Fig. 8). A 36 kda phsphhistidine prtein frm rat mitchria (Backer et al. 1986) a a 37 kda phsphhistidine prtein frm pea leaf mitchria (Hakanssn a Allen 1995) have als been reprted. Since phsphhistidine prteins are ften invlved in signal transductin pathways, a have been predicted t be present in the inner mitchrial membrane (Allen 1993a), the 37 kda inner membrane bu phsphhistidine prtein may be a sensr kinase f the bacterial type tw-cmpnent signal transductin pathway (Parkinsn a Kfid 1992). The 17 kda phsphprtein The 17 kda inner membrane phsphprtein was phsphrylated nly in the absence f catins (Fig. 3). Niman a Shaul (1995) shwed that a 17 kda NDPK prtein was heavily phsphrylated in the presence f EDTA a Muhnen a Lambeth (1995) suggested that NDPK in rat liver mitchria is membrane-bu. It is therefre pssible that the 17 kda EDTA-stimulated phsphprtein (shwn in Fig. 3) is a plant mitchrial inner membrane bu NDPK. The 16 kda phsphprtein The 16 kda phsphprtein was the mst heavily labelled prtein in ptat tuber inner mitchrial membranes (Fig. 1). We can nt exclude the pssibility that this prtein, in its unphsphrylated frm, n SDS-PAGE migrates t an apparent size f 16.5 kda a it may therefre be identical t the autphsphrylated 16.5 kda prtein (Fig. 7). It is interesting t nte that Steenaart a Shre (1997) identified the mst heavily labelled prtein in their mitchrial membrane fractin, a 17 kda phsphprtein, as cytchrme c xidase subunit IV. Perspectives Prtein phsphrylatin a dephsphrylatin are ften invlved in ne r mre steps in signal transductin pathways. These reactins are als ubiquitus as regulatry mechanisms which change the prperties f target enzymes. The present paper describes the prperties f a number f nvel prtein phsphrylatin reactins in the inner membrane f plant mitchria. The next step is t identify a characterize the kinases a phsphatases respnsible fr the phsphrylatin/dephsphrylatin. In parallel, the rle f prtein phsphrylatin shuld be investigated by identifying the phsphprteins a lking at pssible changes in their prperties r in the prperties f the cmplexes in which they are subunits (e.g. the F 0 F r ATPase subunits). Since the prtein phsphrylatin described here is redx-regulated (Fig. 2), it is tempting t speculate that sme f the phsphprteins are part f the respiratry chain a that electrn transprt is mdified by the phsphrylatin. Plant a fungal mitchria cntain specific enzymes, including the alternative xidase (Vanlerberghe a Mclntsh 1997) a alternative NAD(P)H dehydrgenases (Meller a Rasmussn 1998), a it wuld be f interest t knw whether the prtein phsphrylatin events described here are specific t plant mitchria, r f general imprtance in eukarytic cells. In prkarytes, tw-cmpnent signal transductin pathways invlve phsphrylatin f a sensr kinase n histidine, a f a respnse regulatr n aspartate (Parkinsn a Kfid 1992). The persistence, in evlutin, f tw-cmpnent redx signalling pathways (Allen 1993b) may be essential fr the functin f the genetic systems f mitchria a chlrplasts (Allen 1993a, Race et al. 1999). The 37 kda phsphhistidine prtein identified here may be part f such a signal transductin pathway, allwing the respiratry chain t exert regulatry cntrl ver gene expressin a ther mitchrial prcesses. Prtein phsphrylatin reactins such as thse described here may thus be crucial fr the integratin f functin, assembly a bigenesis f mitchria. We are grateful t Mrs Christina Nilssn a Miss Ineke de Jng fr excellent technical assistance. This study was supprted by grants frm the Swedish Cuncil fr Planning a Crdinatin f Research (J.F.A.), the Swedish Natural Science Research Cuncil (J.F.A. a I.M.M.) a The Ryal Swedish Academy f Science (A.S.). The experiments were carried ut by A.S. in cllabratin with K.M.F. All authrs prvided ideas a interpreted results. 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