Polypeptide profiles of chlorophyll · protein complexes and thylakoid membranes of spinach chloroplasts

In addition to the major chlorophyll · protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll $\text{a}\text{b}$ · protein and from the reaction centre complex of Photosystem II.Data are presented on the polypeptide profiles of purified digitonin-subchloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll · protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Purification and some properties of an Mg2+-, Ca2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca²⁺, Mg²⁺ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by pI values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M_rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. TheK_m value for ATP was 0.45 mM. The activity was stimulated by Ca²⁺ and Mg²⁺, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.     

Isolation and characterization of Photosystem I and II membrane particles from the blue-green alga, Synechococcus cedrorum

Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10–30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose.The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 μmol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains β-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N₂ fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome $\text{b-559}$ ratio of 50 : 1. The Photosystem I particle is highly enriched in $\text{P-700}$, with a chlorophyll to $\text{P-700}$ ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.     

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point ($\text{p}\text{I}$), apparent molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP_132–135^4–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ but different $\text{p}\text{I}$ were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.     

Titles of related papers in other sections

Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide.The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ is closer to the in vivo form rather than the monomer.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Membrane-surface electric properties of Triton-fractionated spinach subchloroplast fragments

Surface charge density of subchloroplast fragments fractionated from spinach by Triton X-100 treatment was estimated from cation-induced quenching of chlorophyll fluorescence, with the premise that the fluorescence yield is dependent on the surface electric potential of the preparations. Application of the Gouy-Chapman theory of diffuse double layer to the subchloroplast preparations, or treating the surface of the preparations under electric charge regulation conditions yielded a result suggesting the Photosystem II reaction-center preparation (TSF-IIa) to be more negatively charged than the Photosystem I reaction-center preparation (TSF-I). Isoelectric points of the subchloroplast fragments were determined by measuring 90° light scattering and more directly by gel isoelectric focusing. Isoelectric points of TSF-I and -IIa were estimated to be 4.8 and 4.0 from light-scattering experiments, and 4.5 and 4.1 from gel electrophoresis, respectively. The TSF-II preparation that contains both a light-harvesting complex and the reaction-center (core) complex showed a small cation-induced quenching of chlorophyll fluorescence. This fluorescence quenching may be ascribed mostly to the regulation of energy transfer in the preparation (Yamamoto, Y. and Ke, B. (1980) Biochim. Biophys. Acta 592, 296–302). Furthermore, the TSF-II preparation showed a broad and indefinite peak in light scattering in the pH range 3–8, suggesting that the complex probably carries a small amount of charge in this pH range. The physiological role of the membrane surface charge of the subchloroplast preparations in membrane structure and cation regulated processes in chloroplast is discussed.     

Variable chlorophyll a fluorescence from P-700 enriched Photosystem I particles dependent on the redox state of the reaction centre

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence.2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs.3. EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite.4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles.5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P⁺X are more effective quenchers of chlorophyll fluorescence than PX^?, where P is P-700.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Purification of cytochrome b-559 from oxygen-evolving Photosystem II preparations of spinach and maize

A rapid and simple procedure is presented for the purification of chloroplast cytochrome b-559. The method is based on the protocol devised by Garewal and Wasserman (Garewal, H.S. and Wasserman, A.R. (1974) Biochemistry 13, 4063–4071), which we have modified to eliminate the requirement for a lengthy electrophoretic step. Novel features of our method include: the use of oxygen-evolving Photosystem II preparations (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539) as the starting material; isocratic elution of cytochrome b-559 from a DEAE-cellulose column (yielding the protein in a pure state); and a simple column procedure for removal of excess Triton X-100. The procedure has been applied to both spinach and maize (Zea mays L.). Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. Lithium dodecyl sulfate polyacrylamide gel electrophoresis of cytochrome b-559 from both spinach and maize reveals a major polypeptide band (apparent molecular mass = 9 kDa), and two minor bands (apparent molecular masses = 10 kDa and 6 kDa).     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.