Phosphorylation In Chloroplasts Research Articles

Working day and night: plastid casein kinase 2 catalyses phosphorylation of proteins with diverse functions in light- and dark-adapted plastids.

Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.

Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation.

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes.

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of light-harvesting complex stress-related protein3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-light harvesting complex I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.

Energetic and regulatory role of proton potential in chloroplasts

The review focuses on the energetic and regulatory role of proton potential in the activity of chloroplasts, the light energy-converting organelles of plant cells. Mechanisms of generation of the transmembrane difference of electrochemical potentials of hydrogen ions (Δµ(~)(H+)) in the chloroplast thylakoid membranes are considered. Methods for measuring the intrathylakoid pH in chloroplasts are described. It is shown that under conditions of phosphorylation in chloroplasts, the pH of the intrathylakoid space decreases moderately (pH(in) ≥ 6.0-6.2, at the stroma pH(out) ≈ 7.8-8.0), with a corresponding concentration component of Δµ(~)(H+) equal to ΔpH ≤ 1.6-2.0. On analyzing the energy and structural features of ATP synthase of chloroplasts, we conclude that the energy stored as the concentration component of the proton potential ΔpH is sufficient to sustain ATP synthesis. The mechanisms of pH-dependent regulation of electron transport in chloroplasts (photosynthetic control of electron transport, enhancement of non-photochemical quenching of chlorophyll excitation in the light-harvesting antenna, light-induced activation of the Calvin-Benson cycle reactions, activation of ATP synthase) are considered briefly.

Signal integration by chloroplast phosphorylation networks: an update

Forty years after the initial discovery of light-dependent protein phosphorylation at the thylakoid membrane system, we are now beginning to understand the roles of chloroplast phosphorylation networks in their function to decode and mediate information on the metabolic status of the organelle to long-term adaptations in plastid and nuclear gene expression. With the help of genetics and functional genomics tools, chloroplast kinases and several hundred phosphoproteins were identified that now await detailed functional characterization. The regulation and the target protein spectrum of some kinases are understood, but this information is fragmentary with respect to kinase and target protein crosstalk in a changing environment. In this review, we will highlight the most recent advances in the field and discuss approaches that might lead to a comprehensive understanding of plastid signal integration by protein phosphorylation.

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid.

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.

ADP-Dependent phosphorylation regulates association of a DNA-binding complex with the barley chloroplast psbD blue-light-responsive promoter.

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a -10 promoter element and an activating complex, AGF, that binds immediately upstream of -35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between -71 and -100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1-5 mM) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mM), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

Effect of encecalin, euparin and demethylencecalin on thylakoid electron transport and photophosphorylation in isolated spinach chloroplasts

The major phytotoxic compounds (encecalin, euparin and demethylencecalin) isolated from Helianthella quinquenervis (Hook) A Gray (Asteraceae) were evaluated on different photosynthetic activities in chloroplasts isolated from spinach leaves. ATP synthesis, proton uptake and electron flow (basal, phosphorylating and uncoupled) were inhibited by encecalin and demethylencecalin in a concentration dependent manner, therefore acting as Hill reaction inhibitors. Encecalin and demethylencecalin did not affect photosystem I (electron transport from diaminodurene to methylviologen), but they inhibited photosystem II (from water to 2,5-dibromo-3-methyl-6-isopropyl-1,4-p-benzoquinone). Since these compounds inhibited electron flow in the photosystem II partial reactions from water to silicomolybdate and from diphenylcarbazide to dichlorophenol-indophenol, the site of inhibition was located in the span from P680 to QA of the electron transport chain. Euparin, inhibited ATP synthesis, proton uptake and basal and phosphorylating electron transports, but it has not effect on uncoupled electron flow from water to methylviologen. Mg2+-ATPase activity from bound membrane thylakoid chloroplasts was also inhibited by this compound. These results suggested that euparin inhibited phosphorylation in chloroplasts, acting as an energy-transfer inhibitor. © 1998 Society of Chemical Industry.

5-O-?-d-galactopyranosyl-7-methoxy-3?,4?-dihydroxy-4-phenylcoumarin, an inhibitor of photophosphorylation in spinach chloroplasts

5-O-β-D-galactopyranosyl-7-methoxy-3',4'-dihydroxy-4-phenylcoumarin isolated from Exostema caribaeum (Rubiaceae) has been found to act as an energy-transfer inhibitor in spinach chloroplasts. ATP synthesis and phosphorylating (coupled) electron flow were inhibited by 89 and 72%, respectively, at a concentration of 400 μM. H(+)-uptake, basal and uncoupled electron transport were not affected by the coumarin. The light-activated Mg(+2)-ATPase activity from bound membrane thylakoid chloroplasts was slightly inhibited by the coumarin. Also, the heat-activated Ca(+2)-ATPase activity of the isolated coupling factor protein was insensitive to this compound. In chloroplasts partially stripped of coupling factor 1 by an EDTA treatment, the coumarin showed a restoration of the proton uptake process. These results suggest that the 4-phenylcoumarin under investigation inhibited phosphorylation in chloroplasts by specifically blocking the transport of protons through a membrane-bound component or a carrier channel (CFO) located in a hydrophobic region at or near the functional binding site for the coupling factor 1.

Effects of permeant buffers on the initiation of photosynchronous phosphorylation and postillumination phosphorylation in chloroplasts.

Under canonical chemiosmotic formulations, the development of a delocalized transmembrane proton gradient should precede and, in the absence of a membrane potential, should account for all the capacity of an energy transducing system to synthesize ATP. Furthermore, any agents, such as permeant proton-absorbing buffers, that slow down the kinetics of the development of this gradient should, consequently, delay ATP synthesis. We have studied the very early (0 through 1000 ms) steps of photosynthetic ATP synthesis utilizing real-time, rapid flow-quench techniques. We have investigated the effect(s) that permeant buffers exert on this process where these buffers show no uncoupling effects, and the transmembrane potential has been collapsed by valinomycin and K+. Experimentally this system was dissected into two ATP synthesizing components, as follows: synthesis of ATP strictly concomitant with light influx and unaffected by the addition of permeant buffers. We refer to this as photosynchronous phosphorylation and synthesis of ATP monitored after the light was extinguished and which was greatly diminished by the addition of proton-absorbing permeant buffers, thus exhibiting the characteristics of conventional postillumination phosphorylation, and we suggest that it represents part of capacitance phosphorylation. The potential for capacitance phosphorylation initiates very rapidly under light and gradually builds up to steady-state level, and it is governed by canonical chemiosmotic principles. We estimate that its contribution to overall ATP yield is minimal during the first few cycles of the system and that it increases gradually towards steady state when it contributes to the majority of ATP synthesized. Neither a delocalized transmembrane proton gradient nor a strictly localized intramembrane proton pathway can account for these observations so we have proposed that a gating mechanism exists which delivers intramembrane protons initially directly to the ATP synthetase complex but subsequently to the lumen as well, and thus, allows the lumen to act as a capacitor during the steady state. This study can reconcile the findings of Ort et al. (Ort, D. R., Dilley, R. A., and Good, N. E. (1976) Biochim. Biophys. Acta 449, 108-124) with the contrasting findings of Vinkler et al. (Vinkler, C., Avron, M., and Boyer, P. D. (1980) J. Biol. Chem. 255, 2263-2266) through the opposite effects which osmotic strength and KCl concentration exert on the two ATP synthetic phases (during and after illumination) of the rapid flash technique used in those studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Light and CO 2 limitation of photosynthesis and states of the reactions regenerating ribulose 1,5-bisphosphate or reducing 3-phosphoglycerate

Spinach leaves were illuminated at various temperatures or CO 2 concentrations until steady-state photosynthesis could be measured. Subsequently, they were frozen rapidly in liquid nitrogen and freeze-dried. From the dry material, chloroplasts were isolated in a mixture of organic solvents in which polar metabolites are insoluble. Metabolite levels were determined in the chloroplast fraction. From measured levels of dihydroxyacetone phosphate, fructose 6-phosphate (Fru-6-P), ribulose 1,5-bisphosphate (Rbu-1,5-P 2), ATP and ADP, mass-action ratios of the reaction dihydroxyacetone phosphate + 2 glyceraldehyde 3-phosphate + 3 ATP + Fru-6-P → 3 Rbu-1,5-P 2 + 3 ADP + P i were computed. They increased at constant light intensity with increasing CO 2 concentration or increasing temperature as photosynthetic flux increased. Surprisingly, however, mass action ratios decreased as flux increased with increasing light intensities. Moreover, mass-action ratios were linearly correlated to light-limitation coefficients which were obtained by computing the light limitation of photosynthesis from the slopes of light and CO 2 response curves and multiplying obtained values with that increment of photosynthesis which was measured on increasing the light intensity to saturation. The results are interpreted to indicate tight enzymic control of the formation of ribulose bisphosphate by light. As light intensities are increased, light-regulated enzymes are activated to an extent which permits a decrease in the mass action ratios instead of the increase expected to drive increased carbon flux. Since the reactions catalyzed by phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase are close to thermodynamic equilibrium even when photosynthetic fluxes are large, ratios of dihydroxyacetone phosphate to 3-phosphoglycerate indicated the state of chloroplast phosphorylation potentials and the redox state of NADP which together form the assimilatory power [ATP] · [ADP] −1 · [P i] −1 · [NADPH] · [NADP +] −1. Assimilatory power decreased as carbon flux increased with increasing light intensity and increasing CO 2 concentration, but increased as carbon flux increased with increasing temperature. Again this indicates a decrease in the flow resistance of the carbon cycle as light or CO 2 is increased. The decrease in the flow resistance is attributed to enzyme activation when light is increased, or to increased carboxylation when CO 2 is increased.

Light scattering, chlorophyll fluorescence and state of the adenylate system in illuminated spinach leaves

Scattering of green light and chlorophyll fluorescence by spinach leaves kept in a stream of air or nitrogen were compared with leaf adenylate levels during illumination with blue, red or far-red light. Energy charge and ATP-ADP ratios exhibited considerable variability in different leaves both in the dark and in the light. Variability is explained by different possible states of the reaction oxidizing triose phosphate or reducing 3-phosphoglycerate. Except when oxygen levels were low, there was an inverse relationship between light scattering and chlorophyll fluorescence during illumination with blue or red light. When CO 2 was added to a stream of CO 2-free air, chlorophyll fluorescence increased, sometimes after a transient decrease, and both light scattering and leaf ATP ADP ratios decreased. Similar observations were made when air was replaced by nitrogen under blue or high-intensity red light. Under these conditions, over-reduction caused inhibition of electron transport and phosphorylation in chloroplasts. However, when air was replaced by nitrogen during illumination with low-intensity red light or far-red light, light scattering increased instead of decreasing. Under these light conditions, ATP ADP ratios were maintained in the light. They decreased drastically only after darkening. Although ATP ADP ratios responded faster than light scattering or the slow secondary decline of chlorophyll fluorescence due to illumination, it appeared that in the steady state, light scattering and chlorophyll fluorescence are useful indicators of the phosphorylation state of the leaf adenylate system at least under aerobic conditions, when chloroplast and extrachloroplast adenylate systems can effectively communicate.

A demonstration of the physiological role of membrane phosphorylation in chloroplasts, using the bipartite and tripartite models of photosynthesis

Using the equations derived from the bipartite and tripartite models for photosynthetic organization in green plants, we have been able to characterize the effect of membrane phosphorylation on energy transduction. Phosphorylation reversibly increases α (the proportion of absorbed quanta going directly to Photosystem (PS) I). This increase in α we believe to be due to a decrease in the coupling between the PS II core and its associated light-harvesting complex [ΨT(3,2)·ΨT(2,3)]. Phosphorylation also reversibly increases the transfer of energy from PS II to PS I [ψT (II→I)]. We propose that membrane phosphorylation provides the in vivo control of α, ΨT(3,2)·ΨT(2,3) and ψT (II→I). From the data we present it is clear that the changes caused in energy distribution as a result of phosphorylation are large enough to induce real changes in electron-transfer reactions. The effects of phosphorylation on these parameters are distinct from those of Mg 2+ depletion. We have discussed changes in ΨT(3,2)·ΨT(2,3) (the coupling term) with respect to the ‘connected package’ model of photosynthetic units (Butler, W.L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4697–4701) and the proposed α- and β-centers of PS II (Melis, A. and Homann, P.H. (1976) Photochem. Photobiol. 23, 345–350). The demonstration of changes in reversible coupling [ΨT(3,2)·ΨT(2,3)] strongly supports a connected package model in which the degree of ‘connectivity’ is under physiological control.

A measurable consequence of the mitchell theory of phosphorylation

The phosphorylation reaction catalyzed by a membrane bound ATPase must be endothermic when driven by a transmembrane pH difference. This fundamental property has not been recognized in current discussions concerning the chemiosmotic mechanism of phosphorylation, although it can serve as a direct indicator of the participation of a pH gradient in the process. This note presents a description of the thermodynamics of a transmembranal electrochemical potential gradient either when it is dissipated by a non specific leakage, or when it drives phosphorylation. As an illustration, it is shown that a temperature decrease of 0.01°C is to be expected in a typical acid-base driven phosphorylation in chloroplasts.

Control of electron flow in intact chloroplasts by the intrathylakoid pH, not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH4Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH4Cl. However, at low concentrations, NH4Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH4Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH4Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.

Binding of modified adenine nucleotides to isolated coupling factor from chloroplasts as measured by polarization of fluorescence.

1 Fluorescent nucleotides (1,N6-ethenoadenosine diphosphate and triphosphate, ɛADP and ɛATP) replace the natural nucleotides rather efficiently (65–85%) in several chloroplast reactions (ADP inhibition of electron transport, ATP inhibition of electron transport, ATP stimulation of proton uptake, etc.). 2 ɛAMP does not bind to coupling factor protein. 3 The latent isolated coupling factor protein, which reconstitutes phosphorylation well when added to depleted chloroplasts, binds (in less than 1 min) both ɛADP and ɛATP at two divalent cation-sensitive sites each of similar binding affinity (dissociation constant Kd∼ 1–3 μM) although the ɛADP and ɛATP sites are not the same. 0.1 M NaCl increases Kd by 3–5-fold. 4 Polarization of fluorescence of nucleotides and coupling factor suggest that cation-induced changes of protein conformation exist. 5 Binding of coupling factor to nucleotides is maximal at pH 8 where phosphorylation in chloroplasts is also maximal, but changes (with isolated coupling factor) at other pH values are not directly related to the enzyme action. 6 Interaction of inorganic phosphate (Pi) with coupling factor in the presence of nucleotide occurs as monitored by a sizable increase in Kd for ɛADP-coupling factor and ɛATP-coupling factor binding sites, and by decrease in polarization of fluorescence of coupling factor; this suggests negative interaction of adenine nucleotides and Pi on coupling factor. 7 Interaction of coupling factor with ɛADP and ATP on the one hand, and with ɛATP and ADP on the other, occurs as indicated by sizable increases in the Kd for ɛADP-coupling factor and ATP-coupling factor, respectively; this suggests s negative interaction of ADP and ATP on coupling factor. 8 The kinetics of release of ɛADP and ɛATP from coupling factor when excess ADP or ATP were added in competition were relatively slow compared to the rate at which photophosphorylation proceeds in vivo; this suggests that coupling factor may have to be bound to the hydrophobic HFo protein and/or the thylakoid membrane for fully functional adenine nucleotide binding to be expressed. Already bound ɛATP was more difficult to remove, even with Dowex 2X, from coupling factor than already bound ɛADP suggesting a more ‘internal’ binding of the former.

Lack of site-specificity of spinach chloroplast coupling factor 1

The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1. Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.

Medium ADP and not ADP already tightly bound to phylakoid membranes forms the initial ATP in chloroplast phosphorylation

Measurements are reported on the source of the ADP and γ-phosphoryl moieties of the initial ATP formed when chloroplast thylakoid membranes are energized by an acid-base transition. Millisecond mixing and quenching experiments demonstrate that most or all of the initial ATP, formed in amounts considerably less than the amount of CF 1-ATPase present, arises from medium ADP and medium P i. With no or low amounts of added medium ADP, the tightly-bound ADP present in thylakoid membranes is released to the medium on energization, then subsequently forms ATP. These results rule out the possible conversion of the ADP tightly-bound to CF 1-ATPase to tightly-bound ATP as a step in the main pathway of chloroplast phosphorylation. Less complete experiments indicate a similar behavior of tightly-bound ADP of submitochondrial particles.

Biochemical Aspects of Electron Transport and Phosphorylation in Chloroplasts

Biochemical Aspects of Electron Transport and Phosphorylation in Chloroplasts

Inhibition of photosystem II-dependent phosphorylation in chloroplasts by mercurials

Photophosphorylation supported by the coupling site associated with Phostosystem II electron transport (coupling site II) is 50 to 60 times less sensitive to the energy transfer inhibitor HgCl 2 than phosphorylation supported by the coupling site associated with Photosystem I electron transport (coupling site I). Coupling site II phosphorylation is only about 2 times less sensitive to the lipophilic mercurial p-hydroxymercuribenzoate (PHMB), however. Both coupling sites are equally sensitive to CF 1 antiserum. These results suggest that a portion of the energy conserving apparatus associated with coupling site II is in a more hydrophobic environment than the corresponding apparatus associated with coupling site I.