c FEUS 1992 (Received August 27/December 3, 1991) - EJB

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1 Eur. J Biohem. 24, (1992) FEUS 1992 Protein phosphorylation and + Mg2 influene light harvesting and eletron transport in hloroplast thylakoid membrane material ontaining only the hlorophyll+binding lightharvesting omplex of photosystem I1 and photosystem I Mihael A. HARRISON and John F. ALLEN Department of Pure and Applied Biology, University of Leeds, England (Reeived August 27/Deember 3, 1991) EJB A material ontaining only photosystem I (PSI) and the hlorophylla/bbinding lightharvesting omplex of PSII (LHC11) has been isolated from the hloroplast thylakoid membrane by solubilization with Triton X1. Fluoresene spetrosopy shows that, within the material, LHC11 is oupled to PSI for exitationenergy transfer and that this oupling is dereased by the presene of Mg2+, whih also dereased PSI eletron transport speifially at limiting light intensity. Inlusion of phosphorylated LHCI1 within the material did not alter its struture, but gave dereased energy transfer to PSI and inhibition of eletron transport whih was independent of light intensity, implying effets of phosphorylation on both light harvesting and diretly on eletron transport. Inlusion of Mg2+ within the phosphorylated material gave dereased energy transfer, but slightly inreased PSI eletron transport. A ationindued diret promotion of PSI eletron transport was also observed in isolated PSI partiles. The PSI/LHCI1 material represents a model system for examining protein interations during lightstate adaptations and the possibility that LHCI1 an ontribute to the antenna of PSI in light state 2 in vivo is disussed. The two types of photosystem operating within the hloroplast thylakoid membrane are onneted in series for nonyli photosyntheti eletron transport and must therefore turn over at equal rates. Photosynthesis will be at its most effiient when the two photosystems reeive exitation energy at equal rates. Photosyntheti organisms have evolved an adaptation mehanism, termed state 1 state 2 transitions, that balanes the transfer of exitation energy to the two photosystems, despite variations in light regime. Preferential exitation of photosystem I1 (PSII) gives rise to state 2 and a derease in energy transfer to PSII relative to photosystem I (PSI). Preferentizl exitation of PSI gives rise to state 1 and an inrease in exitationenergy transfer to PSII relative to PSI [l]. The transition to state 2 in higherplant hloroplasts ours as a result of over redution of plastoquinone by preferential exitation of PSII. This gives rise to a derease in the absorption rosssetion of PSII, whih results in a derease in the PSII fluoresene yield at room temperature [231 and in a derease in PSII eletron transport at limiting light intensity [4]. These effets result from phosphorylation of LHCI1 by a protein kinase, the ativity of whih may be oupled to the Correspondene to M. A. Harrison, Department of Biohemistry and Moleular Biology, Univrsity of Leeds, Leeds LS2 YJT, West Yorks., England Ahhreviations. Chl, hlorophyll; C121nd, 2,6dihlorophenol indophenol; DCMU, 3(3,4 dihlorophenyl)l.ldimethylurea; Hepes, N2hydroxythylpiperazineN 2ethanesulphoni aid; LHCI, hlorophyll u/dbinding lightharvesting omplex of PSI: LHC11, hlorophyll a/hbinding lightharvesting omplx of PSI1 ; PSI, photosystem I; PSII, photosystem 11. redox state of plastoquinone [3, 51 or to a omponent of the ytohrome b6[fomplex [6]. Phosphorylation of LHCI1 auses its migration away from PSII entres in the granal membranes and into the PSIenrihed stroma lamellae or grana margins [7, 81. Phosphorylation of LHC11, the%.e f ore, alters the proteinprotein interations within the PSII antenna omplex. If the role of state transitions in eukaryoti systems is to maximize the quantum effiieny of photosynthesis, despite unfavourable hanges in environmental onditions, then the transition to state 2 should involve a omplementary inrease in exitation energy transfer to PSI. Data from lowtemperature fluoresene spetrosopy has indeed demonstrated an absolute inrease in the fluoresene yield from PSI in state 2, both in vitro 19,11 and in vivo [lo]. This would tend to indiate an inrease in the PSI absorption rosssetion in state 2. However, attempts to determine the implied inrease in PSI photohemistry have proven to be inonlusive, with either a very small inrease [1133] or zero hange [1417] in the rate of PSI photohemistry under state 2 onditions. There remains, therefore, some ontroversy surrounding the possibility that LHC11, speifially in its phosphorylated form, an assoiate with PSI. It does appear to be the as that PSI and phospholhci1 do beome ompartmentalized together in the stroma lamellae under state 2 onditions 171, but the possibility of funtional assoiation remains an open question. Changes in ation onentration may also influene exitationenergy distribution [I An earlier mehanisti model for the regulation of exitationenergy distribution involved inreased exitation of PSI in state 2. as a result of

2 118 inreased 'spillover' of exitation from PSII [21, 221. This 'spillover' was proposed to our as a result of ation fluxes at the membrane surfae, resulting in partial destaking of the grana; a derease in Mg2+ would lead to a derease in membrane lateral heterogeneity, enabling PSII and PSI to assoiate for energy transfer. A material whih ontains only the hlorophyllprotein omplexes PSI and LHC11, has been isolated from the hloroplast thylakoid membrane after washing with alkaline Tris/glyine and solubilization with the detergent Triton X I [23]. This material, similar in polypeptide omposition to a omplex isolated from barley [24], has been shown by freezefrature eletron mirosopy to be omposed of rystalline sheets of partiles approximately 8 nm in diameter and interspersed with larger partiles of diameter 1317 nm [23]. The ordered sheets of 8nm partiles were similar in morphology to the 2dimensional rystals of purified LHCI1 [25]. The larger partiles were presumed by the authors to be PSI partiles, as desribed in [26]. Analysis of the spetrosopi and eletrontransport properties of this PSI/LHC11enrihed material may provide an insight into possible interations of these two hlorophyllprotein omplexes in vivn in state 2. We have examined the effet of ations on both fluoresene and eletron transport in the PSI/LHCI1 material and also upon the photohemistry of isolated PSI partiles. In addition, the effet of inluding phosphorylated LHCI1 in the LHCI1 arrays of the material was determined. Assoiation of phospholhci1 with PSI is a requirement of the omplementary absorption rosssetionhange model for state transitions. The PSI/phosphoLHCI1 material should therefore provide a suitable model system in vitro for the situation proposed to our in vivo. MATERIALS AND METHODS Isolation of thylakoid membranes Thylakoid membranes were isolated from 12 dold pea seedlings (var. Feltham First) by homogenization in ieold buffer ontaining.33 M sorbitol, 5 mm MgC12, 1 mm NaC1, 2 mm EDTA and 5 mm Hepes/NaOH, ph 7.6. The homogenate was filtered through muslin and the hloroplasts reovered by entrifugation at 8 x g for 2 min. Chloroplasts were lysed in buffer ontaining 5 mm MgCI2 and 5 mm HepesjNaOH, ph 7.6 and the thylakoid membranes reovered by entrifugation at 8 x g for 5 min. Thylakoids were resuspended to a hlorophyll onentration of.5 mg. ml' in 1 mm sorbitol, 5 mm MgCI2, 5 mm NaCl, 5 mm Hepes/ NaOH, ph 7.6, and maintained on ie in the dark until required. Chlorophyll onentrations were determined using the equations desribed in [27]. A single preparation of thylakoid membranes was divided into two equal bathes. One bath was inubated under dim white light ( 5 pe. m2. s') in the presene of 4 pm ATP and 1 mm sodium fluoride for 1 min, whereas the seond was maintained in the dark in the absene of sodium fluoride. The former onditions give a high degree of phosphorylation of thylakoid proteins, whereas the latter result in dephosphorylation of LHCI1 [28]. All inubations were performed in a irulating water bath at 15 C. The ompetene of the thylakoid membranes for performing protein phosphorylation and dephosphorylation reations was onfirmed by a onurrent radiolabelling experiment with [y3'p] ATP at a speifi ativity of 1 nci. (nmol ATP)' [29]. Isolation of hlorophyllprotein omplexes The PSI/LHCI1 material was isolated from the two bathes of thylakoid membranes by the method desribed in [23], in whih membranes were washed in 5 mm sorbitol ontaining 5 mm EDTA, ph 7.8, followed by two washes with buffer ontaining 6.2 mm Tris/HCl and 48 mm glyine, ph 8.3. The resulting pellet was resuspended to a hlorophyll onentration of.5 mg. ml' in 25 mm Tris/HCI, ph 8.3. Triton X1 was added to a final onentration of.5% (massivol.) and the suspension inubated at room temperature for 3 min with gentle stirring. Sodium fluoride (5 mm) was inluded in all buffers to prevent dephosphorylation of LHC 11. The material ontaining aggregated PSI and LHCI1 was harvested by entrifugation at 3 x g for 3 min at 4"C, resuspended in 25 mm Tris/HCl, ph 8.3, and stored in the dark at 4 C. The material was found to be stable under these onditions, with no loss of eletron transport apaity or degradation of hlorophyll evident after several weeks of storage. PSI partiles and LHC11 were isolated by the methods desribed in [3] and [31], respetively. Freezefrature eletron mirosopy Freezefrature eletron mirosopy was performed on samples of the PSI/LHCI1 material whih were prepared by freezing in liquid nitrogen slush and fraturing at 12 C. The frature surfae was shadowed with platinum and oated with arbon. Replias were leaned in aid at a graduated series of onentrations, washed and examined in a Philips EM2 eletron mirosope. Eletrontransport measurements PSImediated eletron transport was measured as oxygen uptake via a Mehler reation with asorbateredued 2,6 dihlorophenol indophenol (C1,Ind) as eletron donor (2 mm asorbate and 1 pm C1,Ind) and methyl viologen as eletron aeptor (1 pm). Sodium azide (5 mm ) was inluded to inhibit endogenous atalase ativity. Measurements were made in a Hansateh DW2 oxygen eletrode at 15"C, with eletron transport driven by white light supplied by fibreopti able and varied in intensity by the insertion of neutraldensity filters between fibre opti able and eletrode hamber. For eletron transport measurements, the PSI/LHCI1 material was diluted to a hlorophyll onentration of 3 pg. ml' in 25 mm Tris/HCI, ph 8.3. The material was found not to deompose during these measurements; it ould be reovered by lowspeed entrifugation with no disernible loss of hlorophyll. Polyarylamidegel eletrophoresis Samples of hlorophyllprotein omplexes and PSIjLHC I1 material were prepared for SDSIPAGE by solubilization in 4% SDS, 4 M urea, 5% 2meraptoethanol, 1% glyerol in 5 mm Tris/HCl, ph 6.8. Samples were inubated at room temperature for 15 min. SDSjPAGE was performed on % gradient gels with the buffer system of [32] and inlusion of 4 M urea. Eletrophoresis was performed at 4 C with a onstant urrent of 15 ma. Gels were stained in Coomassie brilliant blue, destained and airdried onto ellophane. The polypeptide omposition of the PSI/LHCI1 material inorporating either phosphorylated or nonphosphorylated LHCI1 was analysed by densitometry of dried gels with a

3 119 a b 1 Fig. I. SDS/PACE analysis of hlorophyllprotein omplexes. SDS/ PAGE gel lanes show thylakoid membranes [l], isolated PSI [2], isolated LHCI1 [3], PSI/LHCI1 material ontaining nonphosphorylated LHCI1 [4] and PSI/LHCI1 material ontaining phosphorylated LHC11 [5]. Positions and moleular masses of marker proteins (M; kda) are indiated. Pharmaia Ultrosan XL laser densitometer. Absorbane profiles of individual gel lanes were ompared using Pharmaia proprietary dataanalysis programs. Lowtemperature fluoresene spetrosopy Fluoresene spetrosopy at 77 K was performed using a PerkinElmer LS5 sanning spetrofluorimeter with a low temperature aessory. Samples were diluted to a hlorophyll onentration of 5 pg. ml' in Tris/HCl, ph 8.3, and rapidly frozen in liquid nitrogen. Samples were maintained in the dark and exposed to the exitation beam for 1 min prior to reording of spetra. RESULTS AND DISCUSSION The TritonX1solubilization proedure produes material enrihed in PSI and LHCI1 polypeptides, but ontaining no PSII. Fig. 1 shows SDS/PAGE analysis of PSI/LHC I1 omplexes derived from phosphorylated and nonphosphorylated thylakoid membranes, with isolated PSI and LHC I1 omplexes for omparison. The reationentre polypeptide of PSI is learly present in the PSI/LHCI1 material as a monomer with an approximate moleular mass of 65 kda and as a dimer of approximate moleular mass 13 kda. LHCI polypeptides in the mass range 25 2 kda are also evident [24, 331, indiating that the material represents an LHC11/ LHCI/PSI reationentre assoiation. It is lear that LHC I1 polypeptides in the range 2825 kda make up a large proportion of the overall assembly. In order that valid and informative omparisons an be made between the funtional properties of phosphorylated and nonphosphorylated PSI/LHCI1 isolates, it is imperative that they be otherwise idential in omposition. Fig. 2 shows the profiles of absorbane measurements at 633 nm, resulting from densitometri sans of the gel lanes from Fig. 1 whih ontain polypeptides from the two isolated forms of the PSI/ a b Fig. 2. Densitometri sans of SDS/PAGEanalysed PSI/LHC11 material. Lanes from Coomassiebrilliantbluestained gels were sanned for absorbane at 633 nm. (a) PSI/LHCI1 material ontaining nonphosphorylated LHCI1 (Fig. I, lane 4). (b) PSI/LHCI1 material ontaining phosphorylated LHC11 (Fig. 1, lane 5). () Differene profile resulting from the subtration of san (b) from san (a), indiating differenes in polypeptide omposition betwn phosphorylated and nonphosphorylated PSI/LHCI1 isolates. Sans are plotted to the same sale. LHCI1 material. Subtration of one san profile from the other provides a differene spetrum whih reflets variations in the polypeptide omposition of eah form of the material, and from Fig. 2 it is evident that the phosphorylated and nonphosphorylated forms of the material are essentially idential in protein omposition. In addition, both forms of material were found to have the same hlorophyll a/b ratio (2.61 and 2.63 for nonphosphorylated and phosphorylated forms, respetively). It is reasonable to onlude, therefore, that the only differene between the two forms of PSI/LHCI1 material is the presene of phosphate groups, bound ovalently to the LHCI1 omponent of one of the two types. Although the possibility of onstitutive phosphorylation of LHCI1 within either form of PSI/LHCI1 omplex annot be eliminated, it is ertainly the ase that the material derived from membranes exposed to the phosphorylating onditions desribed will ontain a muh higher proportion of phosphorylated LHC11. Valid omparisons, based on the extent of phosphorylation, may therefore still be made. The LHCI1 population within the thylakoid membrane is subdivided into disrete subpopulations, eah ontained within different membrane ompartments [7, 81. However, it appears likely that the LHCI1 ontained within the PSI/LHC 11 material is representative of the whole population, rather than simply representative of the subpopulation whih is ontained within the stroma lamellae. This onlusion is based on the observation that there are no differenes in the relative proportions of different LHCI1 polypeptides in the phosphorylated and nonphosphorylated forms of the material (Fig. 2). Note also that the yields of PSI/LHCI1 material from phosphorylated and nonphosphorylated membranes were idential (5% total membrane hlorophyll), suggesting that a relatively high proportion of total membrane LHCI1 is inorporated into the material. If the PSI/LHCI1 material originated solely from the stroma lamellae, the phos

4 111 phorylatd sample would be antiipated to ontain both a higher relative proportion of those LHCI I polypeptides whih are abundant within the rapidly phosphorylating 'mobile' pool [8] and a higher overall LHCII/PSI ratio. The LHC I1 ontained within the PSI/LHC11enrihed material also showed a polypeptide omposition idential to that of the purified LHC11, whih is ertainly representative of the whole membrane LHCI1 population (Fig. 1). If PSI entres are assoiated with 21 moleules of hlorophyll u and b, in a ratio of 6. [24] and LHCI1 binds hlorophylls u and hlpolypeptide in a ratio of 1.1 [34], then a LHCII/PSI polypeptide stoihiometry of 12 an be alulated for an assembly ontaining PSI and LHC11 and having a hlorophyll u/b ratio of 2.6. This would equate to a LHC11/ PSI stoihiometry of 4 when the trimeri assembly of LHCI1 is onsidered. Fig. 3 shows freezefrature eletron mirographs of phosphorylated (Fig. 3 a) and nonphosphorylated (Fig. 3 b and ) PSI/LHCI1 material. As previously reported [23], the material omprises sheets of rystalline arrays of partiles heterogeneously distributed and estimated from this study to be 1 nm and 152 nm in diameter. The ratio of 2 nin partile/lo nm partile is approximately 4, onsistent with the onlusion that the small partiles represent LHCI1 triniers and the larger partile represent PSI. The presene of divalent ations was previously reported not to alter the fine struture of the partile arrays, nor was there any evidene from this study to indiate that phosphorylation of the LHC 11 omponent of the assembly altered the overall struture. Although the presene of residual membrane lipid annot be eliminated, note that the lose paking of partiles and the similarity of LHC11enrihed regions to the 2dimensional rystals of LHCI1 [25] suggest that signifiant amounts of lipid are not present. It is likely that the solubilisation onditions used give rise to formation of the material by allowing dirt interation between hydrophobi protein domains. Fluoresene spetrosopy at 77 K shows that LHCI1 and PSI are oupled for exitationenergy transfer in both the phosphorylated and nonphosphorylated forms of PSI/LHC I I material. Fluoreseneemission spetra, reorded for both lypes of material, are shown in Fig. 4. Emission peaks at 685 nm and 735 nm are seen with exitation through hlorophyll h, and therefore prinipally through the LHCI1 omponent of the material. The 685nm fluoresene arises from LHC11, whereas the 735nm emission arises from LHCI [35, 361. The fluoresene spetra therefore indiate oupling for exitation energy transfer between LHC11 and LHCI. Addition of 1 mm MgC12 dereases the fluoresene at 735 nm relative to that at 685 nm in both the phosphorylated and nonphosphorylated forms of the material. However, even in the absene of ations the ratio F735/F685 for the phosphorylated material is lower than it is in the nonphosphorylated form, and the extent to whih ations lower F73s/ F685 for phosphorylated material is not as great as it is in nonphosphorylated material. Fluoreseneemission data are summarized in Table 1. These data an be interpreted in terms of inreased nonradiative deay of LHC11absorbed light in the absene of ations or under nonphosdhorvlated onditions. or inreased energy transfer from 'LHCI1 to PSI under these onditions. Fig. 3. Freezefrature eletron mirographs of the PSI/LHC11 The inrease in fluoresene at 735 nm is not due to enrihed material. Freezefrature lxtron mirographs ofpsi/lhca diret ation effet at PSI, Sine the presene Of ations did 11 material derived from (a) phosphorylated and (b, C) nonphosnot alter fluoresene emission from isolated PSI partiles phorylatd thylakoid membranes by solubilization with Triton X (data not shown, but see [23]). 1. Bars drawn on eah figure represent.5 pm.

5 mm Emission wavelength [nm] Fig. 4.77K fluoresene emission spetra for PSI/LHCI1 material. 77 K fluoreseneemission spetra for PSI/LHC11 material, inluding (a) nonphosphorylated LHCI1 and (b) phosphorylated LHC11, in the presene (broken lines) or absne (solid lines) of 1 mm Mg2+. Exitation was through hlorophyll h at 474 nm. Spetra were normalized to the emission at 685 nm. Table 1. Summary of the 77K fluoresene emission spetra for PSI/ LHCI1 material. Fluoresene emission spetra were reorded for PSI/LHCI1 material with or without phosphorylated LHCI1 and in the presene or absene of 1 mm MgZf. Values for the ratio F735/ Fhss are derived from the spetra shown in Fig. 4. Fluoresene emission ratio F73s/F68685 Mgz+ + Mgz+ Nonphosphorylated Phosphorylated Table 2. Summary of the 77 K fluoreseneexitation spetra for PSI/ LHCI1 material. The relative ontributions of exitation through horophyll a at 435 nm and through hlorophyll h at 474 nm were determined for fluoresene emission at 735 nni for PSI/LHCI1 material with or without phosphorylated LHC11, in the presene or absene of I mm MgZ '. Values were derived from the spetra shown in Fig. 5. Exitation ratio Fex=435/Fex=474 for emission at 735 nm Mg2+ +Mg2+ Nonphosphorylated Phosphorylated r f 1 8 : ON Q Y m P 3 2 Q n x * o U Light intensity, pe m? s' Fig. 6. Lightdependene urves for PSImediated eletron transport reations in the PSI/LHCIl material. Oxygen uptake by the Mehler reation was determined for PSI/LHCIT isolates ontaining nonphosphorylated (irles) or phosphorylated (triangles) LHC11, in the presene (losed symbols) or absene (open symbols) of 1 mm Mg". Data points are the means of four separate measurements. mm Exitation wavelength lnml Fig K fluoresene exitation spetra for PSI/LHCI1 material. 77 K exitation spetra for PSI fluoresene emission at 735 nm were reorded for PSI/LHCI1 isolates inluding (a) nonphosphorylated LHCI1 and (b) phosphorylated LHC11, in the presene (broken lines) or absene (solid lines) of 1 mm Mgz+. Fluoresene emission arising from LHCI was deteted at 735 nm. Sptra were normalized to the hlorophylla exitation peak at 435 nm. Fluoresene exitation spetra onfirm that, in the PSI/ LHCI1 material ontaining phosphorylated LHC11, PSI and LHCI1 are onneted to a lesser extent than they are in the nonphosphorylated material (Fig. 5). The relative ontribution of light absorbed through hlorophyll b at 474 nm to PSI fluoresene at 735 nm is lower in the phosphorylated material than it is in the nonphosphorylated material, and this ontribution is further dereased by the addition of Mg2 '. These ations also derease the relative ontribution of hlorophyll b light to PSI fluoresene in the nonphosphorylated material. The exitationspetra data are summarized in Table 2. The fluoresene spetrosopy data strongly suggest that phosphorylation of LHCI1 renders this omplex less able to interat with PSI in the PSI/LHCI1 arrays for the purpose of energy transfer. This may be due to dereased ooperativity between adjaent LHCI1 trimers, or dereased onnetivity between LHCI1 and the LHCI omponent of PSI, or both. Inlusion of Mg2+ also appears to derease the interation of LHC11 with PSI. Sine ations indue aggregation of isolated LHCI1 in vitro [31], it might be expeted that inlusion of Mg2+ would give loser assoiation of LHCII trimers at the expense of hydrophobi interation with LHCI. PSImediated oxygen uptake via a Mehler reation, with asorbateredued ClJnd as eletron donor, was measured for both phosphorylated and nonphosphorylated forms of PSI/LHCI1 material, in the presene or absene of ations.

6 > 9 1 m L. 6 x m CI a 3 " VII VII Fig. 7. EadieHofstee plots for PSImediated eletron transport reations in the PSI/LHC11 material. EadieHofstee plots of rate (V) of oxygen uptake by Mehler reation against V/I for PSI/LHCI1 material inluding (a) nonphosphorylated LHCI1 (irles) and (b) phosphorylated LHCI1 (triangles) in the presene (losed symbols) or absene (open symbols) of 1 mm Mg2+. Rate V is expressed as pmolo2. mg Chl. h '. 1 is photon flux density in pe. m. s '. Fig. 6 shows lightdependene urves for PSI eletron transport in the PSl/LHCI1 material under the four onditions. From the fluoresene spetrosopy data it would be antiipated that addition of Mgz+ to nonphosphorylated material would give rise to inhibition of PSI eletron transport. From Fig. 5 it is lear that this is the ase. Kineti analysis of the eletrontransport rates for nonphosphorylated material (plus or minus Mg2+) as EadieHofstee plots (Fig. 7a) shows that the rates tend towards the same V,,, (126 pmol O2. mg Chl. ht'), indiating lightsaturation of the ationindued inhibition. The inhibitory effet of Mgz+ on nonphosphorylated PSI/LHCI1 matrial an therefore be asribed to an ation at the level of light harvesting by LHC11, onsistent with the idea that Mg2+ indue dissoiation between LHCI1 and LHCI. It is also antiipated from the spetrosopi data that inorporation of phospholhci1 within the PSI/LHCI1 material would give rise to inhibition of PSI photohemistry and that this inhibition would be inreased by the presene of MgZ ', sine both LHCI1 phosphorylation and addition of ations derease energy transfer from LHCI1 to PSI in the PSI/LHCI1 material. Both inhibitory effets should be eliminated at high light intensity if their ation is solely at the level of light harvesting. From Fig. 6, it is lear that phosphorylation of LHCI1 within the PSI/LHCI1 material does give rise to inhibition of PSI eletron transport, but from omparison of th kineti plots in Figs 7a and b, it is also evident that this inhibition is not abolished at saturating light intensity. It is probable, therefore, that phosphorylation of the LHCI1 omponent of the material has two distint effets; partial inhibition of light harvesting (as implied by the spetrosopi data) and a diret effet on PSI eletron transport. This seond, diret effet ould plausibly be due to the high density of LHC11bound phosphate groups whih ould interfere with binding of eletrontransport mediators at PSI. Addition of Mg2+ to the PSI/LHCI1 material ontaining phosphorylated LHC11 resulted in a partial alleviation of the inhibition of PSI eletron transport (Fig. 6). This onflits with the fluoresene data whih learly showed that addition of Mgz+ to phosphorylated material resulted in a derease in exitation energy transfer to PSI, in addition to that attribu table to phosphorylation of LHC11. This seemingly ontraditory situation, whereby + MgZ appears to inhibit energy transfer between LHCI1 and PSI in the phosphorylated material, but enhanes PSI eletron transport, may be explained L z ul E 6 ON E, 4 x g 2 a C ul : z o > 2 Y Q Q 3 I I I I I I I i Light intensity, 1IE.m? s' ' 7 VII I 1 Fig. 8. Lightdependene of oxygen uptake by Mehler reation for isolated PSI partiles. (a) Lightdependene urves of oxygen uptake and (b) EadieHofstee plots of rate V (pmol O2. mg Chl. h ') against VjI for the data in (a), in the presene (losed symbols) or absene (open symbols) of 1 mm Mgz+. Estimated V,,, for the rates plus and minus Mg2* are 72 pmol O2. mg Chl'. h' and 57 pmol 2. mg Ch1l h', respetively. The estimated K,,, is 17 WE. m'. s'. by the observation that Mg2+ have a diret stimulatory effet on PSI eletron transport in purified PSI partiles. Fig. 8a and b show the lightdependene urves and derived Eadie Hofstee plots for oxygen uptake via a Mehler reation for Triton X1solubilized PSI partiles in the presene or absene of 1 mm MgZ '. The presene ofations in this reation mixture promoted eletron transport (Fig. 8a) and this effet was independent of light intensity (Fig. 8b). The effet of I I 14

7 MgZ on isolated PSI partiles is, therefore, to diretly promote eletron transport reations. It should be noted that the erfet or ations on PSI eletron transport in vitro in hloroplasts poisoned with 3(3,4 dih1orophenyl)1,i dimethylurea is reported to be lightindependent, but inhibitory [37]. The reason for this disrepany is not known. The presene of + Mg2 in PSI/LHCI1 whih also ontains phosphorylated LHC11, may at at several levels; an inhbition of exitation energy transfer to PSI through LHCI1 (indiated by the fluoresene spetra), a diret stimulation of PSI eletron transport reations and a partial alleviation of the inhibitory effet of LHC11bound phosphate groups. This latter effet ould be postulated to ome about through a hargeneutralization mehanism. The net result is a small inrease in eletron transport rates at limiting light intensity and above those observed in the absene of ations. The inrase in fluoresene arising from PSI at 735 nm, obsrved in ationdepleted PSI/LHCI1 material is analogous to the spillover effets observed with ationdepleted thylakoid membranes. However, the model for state transitions inorporating omplementary absorption rosssetion hange requires the speifi assoiation of phosphorylated LHCI1 with PSI in stale 2. The situation ourring in the PSI/LHC11 material whih inludes phospholhci1 is more likely to mirror the state2 situation in vivo. It is important to not, therefore, that phospholhc11 appears intrinsially less able to at as an antenna for PSI than does nonphosphorylated LHC11. It an be argued that, if phosphorylation an indue spatial separation of LHCI1 omplexes whih are losely aligned for exitation energy transfer at PSII [7], either by eletrostati repulsion [38] or by a onformational hange mehanism, then it is likely that phosphorylated LHCI1 omplexes will remain unable to reassoiate and ontribute to the antenna of PSI. The phosphorylationindued effets are likely to be aentuated in state 2 in vivo, sine the mobile, phosphorylated LHC I1 population within the thylakoid membrane in state 2 will have a higher phosphate/protein stoihiometry than does the LHCI1 within th PSI/LHCI1 material. The LHCI1 within the detergentsolubilized PSI/LHCI1 material will be representative of the whole LHCI1 population of the thylakoid membrane from whih it was derived. The effet of MgZt on LHCI1 within the nonphosphorylated PSI/LHCI1 material has been explained by a mehanism in whih the ations sreen negative harges in the Nterminal hydrophili domains of LHC11 polypeptides, inreasing LHCII/LHCI1 interations at th expense of assoiation with LHCI [23]. Although no disernibl hanges in the ultrastruture of the PSI/LHCI1 assembly were brought about by ationbinding or phosphorylation, only small hanges in the protein onformation would be required to alter exitation energy transfer, and these may be below the level of resolution of the eletron mirosopy in Fig. 3. Changes in the protein struture are known to our as a result of protein phosphorylation and to give rise to large hanges in the funtional ativity [39]. It has been proposed that the absolute inrease in PSI fluoresene yield observed at 77 K in state 2 [9, lo], whih does not appear to be aompanied by a onomitant inrease in PSI photohemistry [14171, may be due to an inreased exiton density in a pigment bed assoiated with PSI, to whih LHCI1 ould ontribute If these exitons had insuffiient energy for photohemial trapping, a situation ould be nvisaged in whih an inrease in longwavelength fluoresene from this pigment bed ould our at ryogeni temperatures 1113 without any inrease in PSI photohemistry. It is interesting to note that, superfiially at least, the result of removing ations from the phosphorylated PSI/LHCI1 material is a derease in PSI eletron transport at limiting light intensity, whilst giving rise to inreased fluoresene emission at 735 nm. However, if these effets observed in vitro are to be applied as an explanation for the inreased PSI fluoresene observed in state 2, it must be postulated that protein phosphorylation and ation flux effet PSI funtion in vivo. This has not been shown to be the ase. Irrespetive of any diret effets on eletron transport, the lear finding of this study is that phosphorylated LHC11, ontained within detergentsolubilized PSI/LHCI1 material, is less able to interat with LHCI than is the nonphosphorylated form of the protein. If this finding is extrapolated to the proteinprotein interation ourring in the thylakoid membrane, then any inreased exitation of PSI is unlikely to arise as a result of assoiation with LHC11. The results of this study tend to favour the idea that the transition to state 2 in eukaryoti photosyntheti systems is primarily a protetive response to prevent exessive exitation of PSII. 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