Role of surface proteins in the manifestation of temperature effect on thylakoid membranes

Biochem. Physiol. Pflanzen 185, 1-10 (1989) VEB Gustav Fischer Verlag lena

Role of Surface Proteins in the Manifestation of Temperature Effect on Thylakoid Membranes BAL RAM SINGH and GAURI S. SINGHAL School of Life Sciences, lawaharlal Nehru University, New Delhi, India Key Term Index: absorption, difference spectra, phase transition, pheophytins, surface proteins, thylakoid membranes

Summary The role of surface proteins of thylakoid membranes in temperature induced spectral changes was examined. On removal of surface proteins by treating the thylakoid membranes with 6 M guanidine hydrochloride, a blue shift of 3-7 nm was observed in the absorption spectrum. Cooling and heating of these membranes showed the same basic pattern of spectral changes as untreated (normal) thylakoid membranes indicating that the basic membrane structure remained intact after removal of the surface proteins. However, the negative and positive I':!..A bands were observed at different wavelengths in treated and untreated membranes. A characteristic negative I':!..A band at 690 nm observed in untreated thylakoid membranes was not observed in surface protein-depleted thylakoids. Pheophytinization of chlorophyll molecules was observed in the guanidine HCI treated thylakoid membranes which was enhanced substantially upon heating. Results indicate that surface proteins have a significant role in maintaining the membrane organization of the thylakoids.

Introduction The role of thylakoid membrane proteins in determining the spectral characteristics of pigments has been extensively reviewed (LITVIN and SINESHCHEKOV 1975; KATZ et al. 1977). It has been concluded that membrane proteins are mainly responsible for the existence of various spectral forms of chlorophylls a and b and previous X-ray diffraction studies on bacterial reaction center complexes (DEISENHOFER et al. 1985) support the view that the spectral forms of the chlorophyll originate from the spatial orientation of the pigments which is determined by the protein framework of chlorophyll-proteins: Structural proteins of thylakoid membranes are considered to be mostly the pigment-protein complexes which represent the major intrinsic part of the thylakoid membrane proteins and form the core of PS I and PS II (MACHOLD 1975; Suss et al. 1976; THORNBER et al. 1977; ANDERSON 1980). The 3 main surface (extrinsic) pwteins, carboxydismutase, ATPase, and NADP reductase can be removed by washing the thylakoids with sodium pyrophosphate solution and chelating agents (STROTMAN et al. 1973; MCCARTY 1971). Some other proteins of chloroplasts, which are bound by hydrophilic and hYLlf()phobiclOrcesto the;!m.embrane, require chaotropic agents such as guanidine hydrochloride:(Gu~HCl) (MACHOLD 1975) or proteolysis combined with urea extraction (Suss et al. 1976Yfor their release. These proteins are termed as associated Abbreviations: ANS, anilinonaphthalene sulfonic acid; Gu-HCI, guanidine hydrochloride; PS I, photosystem I; PS II, photosystem II BPP 185 (1989) 112

proteins and are not well characterized. Removal of surface proteins by Gu-HClleaves the basic structure of the membrane intact (MACHOLD 1975). Treatment of chloroplasts with 6 M Gu-HCI is reported to abolish the antagonistic effect ofNa+ and Mg+2 ions on the fluorescence of chlorophyll a and the acid induced enhancement of bound ANS fluorescence (ANDLEY et al. 1981). It is believed that the Gu-HCl extractable proteins are involved in electron transport and photophosphorylation (MACHOLD 1975). The aim of the present study is to assess the possible role of the surface proteins in determining the spectral characteristics of the pigments. The temperature induced spectral changes in the normal thylakoid membranes (with associated proteins) are considered to be due to organizational changes in the pigments, proteins and lipids of the thylakoid membranes (BRODY and SINGHAL 1979; ARMOND et al. 1980; SINGH and SINGHAL 1984, 1985, 1989). These spectral changes in partially protein depleted thylakoid membranes (without associated proteins) will provide information about the role of associated proteins in the manifestation of temperature effects on the thylakoid membranes vis-a-vis spectral characteristics.

Materials and Methods Barley (Hordeum vulgare L. CVIB 65) seedlings were grown in the dark for 8 d before being brought into light for greening. Seedlings were harvested after 72-96 h of greening for chloroplast isolation . Chloroplasts were isolated according to SINGH and SINGHAL (1984). The leaves were cut into = I cm long pieces and kept in ice. The pieces were then crushed in a Waring blender using 0.4 M sucrose in 50 mM Na-phosphate buffer (PH 7.8) as grinding medium. The homogenate was squeezed through 8 layers of cheese cloth and centrifuged at 200 x g for 2 min to remove cell debris. The pellet was discarded and the supernatant centrifuged again at 1,000 x g for 10 min . The centrifugations were carried out at 4 D C. The chloroplast pellet was re-suspended in 50 mM Na-phosphate buffer (PH 7.8). The surface proteins were removed by treating the chloroplast membranes with 6 M Gu-HCl (Sigma) according to MACHOLD (1975) . Briefly, isolated chloroplasts were purified on a sucrose-glycerol density gradient to obtain stroma free thylakoid membranes. These membranes were slowly stirred with 6 M GuHCl in 50 mM Na-phosphate buffer (PH 8.9) for 65 hat 2°C. The soluble proteins were removed by centrifugation at 144,000 x g for 15 min. The precipitate was washed with water to remove any trace of Gu-HCI. The pellet was dissolved in 50 mM Na-phosphate buffer (PH 7.8) for the experiments . Absorption and difference absorption spectra were recorded as described previously (SINGH and SINGHAL 1989). The rate of heating was 0.5 °C min- 1 and average rate of cooling was 1 DC min- I. Temperature was stabilized for 5 - 10 min at each temperature of 6.A measurement.

Results The absorption spectra of normal (untreated) and partially protein depleted chloroplasts reveal that removal of surface proteins with 6 M Gu-HCl caused a significant blue shift in the absorption peaks both in red and blue regions (for example, a 7 nm blue shift of the Qy band; Table 1). The absorption spectrum of Gu-HCl treated chloroplasts showed a further blue shift of peaks (22.5 nm in red and 15 nm in blue regions) after temperature cycles (viz. ambient ~ cooling; cooling~ ambient; ambient~ heating; heating~ ambient cycle ). This was incontrastto a negligible (1-2 nm) red shift of the absorption peaks in untreated chloroplasts after temperature cycles (Table 1). The Gu-HCl treated chloroplasts, kept as reference for difference spectra measurements during different temperature cycles, also showed blue shifts of 20 and 13 nm red and blue peaks, respectively, of the absorption spectrum (spectrum not shown, see Table 1). 2

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Table 1. Absorption maxima of chloroplast membranes before and after temperature cycles. Chloroplast samples

Untreated chloroplasts

Gu-RCI-treated chloroplasts

Red peak

Blue peak

Red peak

Blue peak

Before temperature cycle (0 h)

677 ± 0.5nm

436 ± 0.5 nm

670 ± 0.5nm

433 ± 0.5 nm

After temperature cycles (6 h)

679 ± 0.5nm

437 ± 0.5nm

647 ± 0.5nm

418 ± 0.5 nm

At room temperature (6 h)

679 ± 0.5nm

437 ± 0.5 nm

650 ± 0.5nm

420 ± 0.5nm

During ambient---,) cooling ofGu-HCI treated chloroplasts (26 to 6°C), an increase occurred in absorbance giving rise to positive ft..A bands at 450 nm with a shoulder at 490 nm and at 678 (Fig. 1 a). Cooling of untreated chloroplasts from 23.3 °C (ambient) to 7 °C resulted in a progressive increase in absorbance showing positive ft..A bands at 435 and 495 nm with a shoulder at 470 nm and at 678 nm. A negative ft..A band at 690 nm was observed with a simultaneous increase in absorbance at 700-730 nm (Fig. 2a). Heating of the Gu-HCI treated and untreated chloroplasts back to ambient temperature resulted in progressive decrease in ft..A at all wavelengths (Figs. 1 a, b, and 2b) resulting in a negative ft..A at 663 nm in Gu-HCI treated chloroplasts. Heating of the samples above the ambient temperature resulted in a decrease in absorbance giving rise to negative ft..A bands at 440,470,480 and 675 nm in case the of Gu-HCI treated chloroplasts (Fig. 1 b and c) and at 440,495 and 680 nm in the case of untreated chloroplasts (Fig. 2c). A simultaneous increase in absorbance was observed between 690 and 735 nm both in treated and untreated chloroplasts. The negative ft..A band at 675 nm in treated chloroplasts seems to have resulted from a shifting of the negative ft..A band observed at 663 nm (at 24.7 °C). At the same time, a progressive increase in absorbance occurred giving rise to positive ft..A bands at 640-645 nm and 415 nm with a shoulder at 455 nm in Gu-HCI treated chloroplasts (Fig. 1 b and c). The positive ft..A band at 415 nm is notably very prominent. Cooling the Gu-HCI treated chloroplasts, in general, caused an increase in the positive ft..A bands at 640-645,450-455 and 415 nm and a decrease in the negative ft..A bands at 675 and 470-480 nm (Fig. I d). However, there was a decrease in the positive ft..A band at 690-735 nm. The untreated chloroplasts showed a progressive decrease in the negative ft..A bands which resulted in positive ft..A bands at 670 and 435 nm with a shoulder at 470 nm (Fig. I) during the heating ---,) ambient cycle. Discussion The temperature induced spectral changes are considered to be the consequence of organizational changes in the thylakoid membranes resulting from an alteration of the interactions among its components (ARMOND et al. 1980; SINGH and SINGHAL 1984, 1989). Involvement of protein conformation in determining the temperature induced spectral changes 1*

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of the chloroplasts membranes is well established (BRAND 1977, 1979; SINGH and SINGHAL 1989) but so far the nature of the involved proteins is not specified. The data presented here indicate that the surface proteins are involved not only in electron transport and photophosphorylation as suggested by MACHOLD (1975) but can also influence the spectral characteristics and temperature induced spectral changes of photosynthetic pigments within the membranes. This contention is supported by (1) the blue shifted absorption peaks in Gu-HCI treated chloroplasts, (2) the non-observation of negative LlA band at 690 nm in the Gu-HCl treated chloroplasts during ambient ~ cooling cycle, (3) the observation of LlA bands at BPP 185 (1989) 112

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different wavelengths during cooling and heating cycles of treated chloroplasts relative to untreated chloroplasts. It is possible that a trace amount of Gu-Helleft with thylakoid membranes might interact directly with lipid head groups and lor proteins but it is believed that such effect will be negligible on our results as Gu-Hel is quite soluble at 6 M concentration and thus it would be washed out. The shifts in the absorption and the i1A peaks on Gu-Hel treatment and subsequently on cooling or heating of chloroplasts indicate involvement of surface proteins, directly or indirectly, in maintaining the micro-environment ofthe chromophores. Spectral changes were also observed when the surface proteins of thylakoids were removed by proteolysis (SOss and BRECHT 1980). The fact that the red peak is more blue shifted than the blue peak on Gu-Hel treatment (Table 1) indicates that the X and Y transitions of chlorophyll molecules are sensitive differentially to the removal of surface proteins. The appearenceof a very prominent i1A peak at415 nm during ambient~ heating~ ambient cycles of Gu-Hel treated chloroplasts indicates formation of pheophytins. Heating does not appear to be the cause of pheophytinization in treated chloroplasts as the blue shifted peaks (corresponding to the pheophytins) were observed in treated chloroplasts kept at room temperature also (Table 1). But heating certainly promotes the pheophytinization as indicated by the appearence of a positive i1A band at 415 nm during ambient ~ heating ~ ambient temperature cycle and a more blue shifted absorption peak of treated chloroplasts after temperature cycle. The spectral characteristics of pheophytins are not observed in the untreated chloroplasts (Table 1). This indicates that the surface proteins of the thylakoid membranes influence the protection of the chlorophyll molecules from pheophytinization. The observation of positive i1A bands on cooling and heating of Gu-Hel treated chloroplasts as also observed in untreated chloroplasts supports the fact that even after removal of surface proteins, thylakoid membranes maintain the basic membrane structure as suggested by MACHOLD (1975). The Gu-Hel treatment of thylakoid membranes is known to cause a loss of about 40 % of membrane proteins (ANDLEY et al. 1981). As it is assumed that certain Gu-HCl extractable proteins penetrate the lipid bilayer (ANDLEY et al. 1981), the removal of surface proteins is likely to alter the balancing forces which maintain the functional state of thylakoid membranes . This is more relevant in view of the reports that a part oflight harvesting chlorophyll-proteins is exposed to the surface of thylakoid membranes (STEINBACK et al. 1979). Furthermore, removal of the surface (extrinsic) proteins is likely to alter the charge distribution on the surface of the membranes and is known to cause un stacking of the grana structure which changes the topographical distribution of the pigment-protein complexes within the chloroplast membranes (MACHOLD et al. 1977). Since some of the surface proteins penetrate into the lipid bilayers, their removal will possibly change the polarity of the inner core of the thylakoid membranes . Any such change in the polarity of the membrane core, which includes the integral proteins and lipids , will influence the transition dipole moments of the embedded chromophores resulting in the spectral shifts observed in our results. The random distribution of the pigment-protein complexes would change the orientation of bound pigments which is likely to affect their spectral characteristics. The loss of chlorophyll a fluorescence at 686 nm after treatment with Gu-Hel (ANDLEY et al. 1981) supports the desaggregation of the pigment-protein complexes . Additionally, the removal of the surface proteins including a part of the light harvesting 8

BPP 185 (1989) 112

pigment-protein complexes could influence the polypeptide folding of the integral proteins resulting into an altered spectral characteristics of the associated pigments. The fluidity of the membrane might be affected by removal of the surface proteins as the protein-lipid interactions are altered due to the loss of surface proteins . This will influence the interactions between pigments and lipids because the chromophores being hydrophobic in nature are likely to be located towards the hydrophobic core of the membrane bilayer. The Xray crystallographic studies of bacterial reaction centers have revealed that the pigments are located in the central hydrophobic region of the proteins which are also in contact with the nonpolar domain of the membrane lipids (DEISENHOFER et al. 1985) . Though it is not possible to specify the changes with a particular factor with the data presented, the observations certainly open clues for further research to determine the role of specific proteins in temperature induced spectral changes in chloroplast membranes.

Acknowledgements This work was partially supported by a grant from the US Department of Agriculture (FG-IN-574 to GSS). One of us (BRS) would like to thank Council of Scientific and Industrial Research, India, for the award of junior and senior research fellowships during this work.

References ANDLEY, U. P . , SINGHAL, G . S., and MOHANTY, P. K .: The effect of treatment of chloroplast membrane with guanidine-HCI and aqueous acetone on the fluorescence of bound ANS and chlorophyll-a. Photochem . Photobiol. 33, 235-242 (1981). ANDERSON, J. M.: P-700 content and polypeptide profile of chlorophyll-protein complexes of spinach and barley chloroplasts. Biochim . Biophys. Acta 591, 113-126 (1980). ARMOND, P . A., BJORKMAN, 0., and STAEHELIN, L. A.: Dissociation of supramolecular complexes in chloroplast membranes. A m anifestation of heat damage to photosynthetic apparatus. Biochim. Biophys . Acta 601,433-442 (1980) . BHARDWAJ, R., and SINGHAL, G. S.: Spectral distribution of fluorescence at 77 OK of heat-treated and developing water-stressed chloroplasts . Proc . Fifth IntI. Congress Photosynthesis (Ed. AKOYUNOGLU, G.). Vol. V , pp . 407-416 . Balban IntI. Sci. Services, Philadelphia 1981. BRAND, J.: Spectral changes in Anacystis nidulans by chilling. Plant Physiol. 59,970-973 (1977) . BRAND, J.: Spectral changes in membrane fragments and artificial liposomes of Anacystis induced by chilling . Arch. Biochem. Biophys . 193,385-391 (1979). BRODY , S . S., and SINGHAL , G . S.: Spectral properties of chloroplast membranes as a function of physiological temperatures. Biochem. Biophys. Res. Commun. 89 , 542-546 (1979). DEISENHOFER, J., Epp, 0., MIKI, K., HUBER, R., and MICHEL, H.: Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3 AD resolution. Nature 318, 618-624 (1985). KATZ , J. J. , NORRIS, J. R., and SHIPMAN , L. L.: Models for reaction center and antenna chlorophyll. Brookhaven Symposia in Biology 28 , 16-55 (1977). LITVIN , F. F ., and SINESCHKOV , V. A. :Molecular organization of chlorophyll and energetics of the initial stages in photosynthesis . In : Bioenergetics of Photosynthesis (Ed . GOVINDJEE) . pp. 619-661 , Academic Press, New York 1975. MACHOLD, 0. : On the molecular nature of chloroplast membranes . Biochim. Biophys . Acta 382, 494-505 (1975). MACHOLD, 0., SIMPSON, D. J., and HOYER-HANSEN, G.: Correlation between freeze fracture appearance and polypeptide composition of thylakoid membranes in barley. Carlsberg Res. Commun. 42,499-516 (1977). BPP 185 (1989) 112

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MCCARTY, R. E.: Chloroplast preparation deficient in coupling factor 1. Methods in Enzymology 23, 251-253 (1971). SINGH, B. R., and SINGHAL, G. S.: Effect of aliphatic alcohol on the temperature-induced absorption changes in barley chloroplasts. Photobiochem. Photobiophys. 8, 73-84 (1984). SINGH, B. R., and SINGHAL, G. S.: Temperature-induced absorbance changes in developing barley chloroplasts. Physiol. Plant. 65, 294-298 (1985). SINGH, B. R., and SINGHAL, G. S.: Temperature-dependent spectral changes of chloroplasts. Biochem. Physiol. Pflanzen 184, 193-203 (1989). STEINBACK, K. E., BURK, J. J., and ARNTZEN, C. J.: Evidence for the role of surface-exposed segments of light-harvesting complex in cation-mediated control of chloroplast structure-function. Arch. Biochem. Biophys. 195,546-557 (1979). STROTMAN, H., HESSE, H., and EDELMAN, K.: Quantitative determination of coupling factor CF 1 of chloroplasts. Biochim. Biophys. Acta 314,202-210 (1973). SDss, K.-H., and BRECHT, E.: Polypeptide composition and spectral properties of light-harvesting chlorophyll alb-protein complexes from intact and trypsin-treated thylakoid membranes. Biochim. Biophys. Acta 592,369-374 (1980). SDss, K.-H., SCHMIDT, 0., and MACHOLD,. 0.: The action of proteolytic enzymes on chloroplast thylakoid membranes. Biochim. Biophys. Acta 448, 103-113 (1976). THORNBER, J. P., ALBERTE, R. S., HUNTER, F. A., SHIOZAWA, 1. A., and KAN, K. S.: The organization of chlorophyll in the plant photosynthetic unit. Brookhaven Symposia in Biology 28, 132-148 (1977).

Received August 1, 1988; revisedform accepted November 9, 1988 Authors' addresses: B. R. SINGH (correspondence), Food Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI 53706, U.S.A.; G. S. SINGHAL, School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110067, India.

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BPP 185 (1989) 112