Sean J. Coughlan

Chloroplast thylakoid protein phosphorylation is influenced by mutations in the cytochrome bf complex

The effects were studied of the plastoquinone analogs 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the 1,3-dinitrophenylether of iodonitrothymol (DNP-INT) on thylakoid protein phosphorylation activated either by light or by reduction in the presence of duroquinol or reduced ferredoxin. Light-activated phosphorylation in the presence of methyl viologen was increased by 0.5–1.0 μM DBMIB or DNP-INT as the reoxidation of plastoquinone and net linear electron flow were inhibited. The phosphorylations of the light-harvesting complex of Photosystem II, of a 32 kDa polypeptide and of the kinase itself (autophosphorylation) were progressively and selectively inhibited at tenfold higher concentrations of either inhibitory analog. Ascorbate, but not duroquinol or reduced ferredoxin, potentiated these selective inhibitions. If the membranes were reductively activated in the dark, however, DBMIB or DNP-INT progressively inhibited the phosphorylation of all thylakoid proteins (I50 = 10–15 μM). These observations appear to preclude a simple and direct regulation of protein kinase activity by the cytochrome bf complex at its plastoquinol oxidation (QZ) site. Mutants of Zea and Lemna lacking the cytochrome bf complex were tested for the ability to phosphorylate thylakoid proteins. In both cases, the redox-sensitive phosphorylation of light-harvesting chlorophyll ab protein complex of Photosystem II (LHC-II) was abolished, whereas other PS II peptides were phosphorylated as in the wild types. Immune blot analysis showed that this lesion was not due to the absence of the 64-kDa protein kinase. Nor did the mutants possess defective LHC-II, as shown by its utilization as a phosphorylation substrate in a heterogeneous reconstitution assay.

Loss of membrane proteins from thylakoids during freezing

Abstract Slow freezing of thylakoids (about 0.5 K/min) in media containing an excess of cryotoxic solutes results in the release of membrane proteins and in the rapid inactivation of photophosphorylation and other processes dependent on structural intactness of membranes. Although loss of photophosphorylation was observed before protein release became extensive, both processes were dependent on the ratio of cryotoxic to cryoprotective solutes in the membrane suspensions. Moreover, inactivation of photophosphorylation and protein release during freezing were similarly influenced by different cryotoxic solutes. Cryotoxicity of different anions increased with increasing Stokes law hydrated anion radius ( I > Br > Cl > F ). Divalent cations were more cryotoxic than monovalent alkali cations. Cryotoxicity thus followed a Hofmeister lyotropic power series. Two opposing effects of low temperature influenced protein release. Lowering the temperature increased accumulation of cryotoxic solutes in the membrane vicinity which promoted protein dissociation. However, rates of protein dissociation increased with increasing temperature. While the freezing temperature unequivocally determined the concentration of solutes in a solution coexisting with ice, both the extent of membrane inactivation and protein release depended on the initial concentration of solutes and on membrane concentration. Apparently, the volumes of the unfrozen hydrophilic phase and of the membranes are also important factors in freezing injury. The polypeptide pattern of released proteins differed drastically from that of thylakoids, but the same main proteins were released during freezing of membranes in the presence of different cryotoxic solutes. More than 35 polypeptide bands were observed in SDS-polyacrylamide gel electrophoretograms of released proteins. While the amount of protein released differed depending on freezing conditions and the composition of the suspending medium, more than 10% of the total membrane protein could be solubilized during freezing in the presence of sodium bromide or sodium iodide. Protein release during freezing is thought to be caused primarily by a suppression of intramembrane electrostatic interactions and ion competition although solute effects on water structure may also play a role.

The differential effects of short-time glutaraldehyde treatments on light-induced thylakoid membrane conformational changes, proton pumping and electron transport properties

Abstract A rapid quench technique utilizing the addition of excess buffer containing free amine groups (Tris, glycylglycine) to the reaction medium has enabled a detailed study of the time-course of glutaraldehyde inactivation on the spinach thylakoid membrane to be undertaken. The following light-induced parameters were inactivated in the sequence: slow transmittance changes (0–5 s) > coupling factor activity (5–20 s) > narrow angle 90° scattering changes (30–60 s). About 20% of PS II activity was lost by this treatment. No effect on activity, proton pumping and proton gradient formation was observed over the time-course studied. A consideration of these effects led to the proposal that the slow, light-induced transmittance changes reflect reversible thylakoid structural changes (unstacking, membrane flattening) in response to electron transport and the consequent proton pumping. The narrow angle 90° scattering changes were considered to reflect directly microconformational structural changes in response to the light-driven proton translocation as previously concluded from other workers.

Purification of membrane-bound ferredoxin: NADP+ oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids

A method is described for the isolation and purification of ferredoxin-NADP+ oxidoreductase (FNR, E.C. 1.18.1.2) and plastocyanin from spinach thylakoids. FNR is recovered from pools which are loosely and tightly bound to the membrane, with minimal disruption of pigment-protein complexes; yields can thus be higher than from procedures which extract only the loosely bound enzyme.Washed thylakoid membranes were incubated with the dipolar ionic detergent CHAPS (3-(3-cholamidopropyl-dimethylammonio)-1-propane-sulfonate). This provided an extract containing FNR and PC as its principal protein components, which could be rapidly separated from one another by chromatography on an anion-exchange column. FNR was purified to homogeneity (as judged from sodium dodecyl sulfate gel electrophoresis and the ratio between protein and flavin absorption maxima), using chromatography on phosphocellulose followed by batchwise adsorption to, and elution from hydroxylapatite. Plastocyanin was further purified on a Sephadex G-75 molecular sieve column.A typical yield, obtained in 3–4 days from 1 kg of deveined spinach leaves, was 7 mg of pure FNR (a single protein of Mr=37,000) and 3.5 mg of plastocyanin.

ABSORBANCE TRANSIENTS OF FERREDOXIN:NADP+ REDUCTASE IN ISOLATED THYLAKOID MEMBRANES

The reduction of NADP+ by ferredoxin: NADP+ reductase (FNR, EC 1.18.1.2) is the terminal step in the photosynthetic generation of strong reductant in chloroplasts (1). FNR, an FAD containing enzyme, also catalyzes electron flow from NADPH to ferredoxin (Fd), which supplies electrons for reduction of nitrite and sulfite (2), and may facilitate the redox poising of cyclic electron transport around PS1 (3).

Reconstitution of LHC Phosphorylation by a Protein Kinase Isolated from Spinach Thylakoids

Protein kinase activity is responsible for phosphorylating LHC (light-harvesting chlorophyll a/b protein complex of photosystem II), leading to its migration in the thylakoid membrane, the fractional redistribution of excitation energy between photosystems II and I, and the phenomenon of State transition. Early reports (1,2) in which the partial purification of two membrane-bound kinases was claimed, stated that these enzymes could not utilise isolated LHC as a substrate. We have confirmed (3) that neither protein recognized by Lin et al, (1) can be responsible for in situ LHC phosphorylation. Their preparation undoubtedly contained a protein kinase, however, and the activity of a similar preparation has been partially characterized using lysine-rich histone as a substrate (4). Subsequent work from this laboratory (5) described the purification to homogeneity of a thylakoid protein kinase which catalyzes the phosphorylation of isolated LHC, albeit at only 1–10% of a rate estimated for this enzyme and substrate when resident together in the thylakoid membrane. in this communication, we report rates of LHC phosphorylation that are close to physiological, in a system comprised of isolated, purified protein kinase (LHCK) and native LHC.

Ferredoxin-NADP+ Oxidoreductase: Studies on the Thylakoid Membrane Bound Enzyme

FNR has+been portrayed as a stromal enzyme in agreement with its function in NADP reduction for CO2. fixation. However, experimental evidence suggested that a part of the total FNR in chloroplasts may be bound to the thylakoid membrane. for example, only a part of the total FNR pool can be removed in extensive washes [l,3], and the enzyme has furthermore been retained in bf-complex preparations [4,5]. Vallejos et al. identified a 17 kDa FNR binding protein [6,7]. The possible role of a linker protein was discussed by Shin and coworkers [8]. A possible functional role in diverting electrons towards NADP+ reduction or cyclic electron transfer around photosystem I was suggested [9] absorbance changes at 530 nm in flash spectroscopic studies of cyclic electron transfer were assigned to FNR [10, G. Garab, personal communication]. The work presented here aimed for the total removal of FNR from spinach thylakoid membranes and in situ study of its binding site.

Purification and Characterization of a Thylakoid Protein Kinase

Although protein kinases are intimately involved in the control of metabolism in animal cells, little is known about the properties and functions of such enzymes in plants. The one exception is the well documented control of State transitions in the thylakoid by reversible phosphorylation of LHC-II (the light-harvesting chlorophyll a/b protein complex of photosystem II). The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool (1).