Assocation of the 33 kDa extrinsic polypeptide (water ... - Springer Link

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Photosynthesis Research 13:69 80 (1987) © Martinus Nijhoff Publishers, Dordrecht-- Printed in the Netherlands

Regular paper

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction E D I T H L. C A M M , 1 B E V E R L E Y ANDREW STAEHELIN 3

R. G R E E N , ~ D A V I D

R. A L L R E D 2 & L.

lDepartment of Botany, University of British Columbia, Vancouver, B.C. Canada, V6T 2B1," :Department of Infectious Diseases, Coll. of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA; SDepartment of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA Received 10 December 1986; accepted in revised form 17 February 1987 Key words: H20-splitting activity, membrane extraction, O2-evolving activity, photosystem II, 33 kDa extrinsic protein Abstract. Various washing procedures were tested on Trifon-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16raM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30raM octyi glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCI2 was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaC1 and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.

Abbreviations: Chl chlorophyll; DCPIP dichlorophenolindophenol; DPC - - diphenolcarbazide; DTT dithiothreitol; HEPES - - N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MES 2(N-morpholino)ethanesulfonic acid; SDS sodium dodecyl sulfate; Tris - - Tris(hydroxymethyl)aminomethane.

Introduction

In the p a s t few years, a p i c t u r e has b e e n d e v e l o p i n g o f the P h o t o s y s t e m II (PS II) c o m p l e x , its o r g a n i z a t i o n a n d its f u n c t i o n s . A r e c e n t r e v i e w [13] s u m m a r i z e s o n e c u r r e n t v i e w t h a t t h e PS II c o r e c o m p l e x consists o f 5 intrinsic p o l y p e p t i d e s a n d t h a t the w a t e r s p l i t t i n g s y s t e m is a d e t a c h a b l e e x t r i n s i c c o m p l e x o f 3 p o l y p e p t i d e s . A m o n g the c o m p o n e n t s o f these c o m p l e x e s are several p o l y p e p tides w i t h m o l e c u l a r w e i g h t s o f a r o u n d 3 2 - 3 4 k D a w h i c h are s o m e w h a t difficult

70 to distinguish by SDS-gel electrophoresis because of the similarity in their apparent molecular weights. Of these polypeptides, two are found in the core complex. These are the so-called Qb-binding protein (also called the 33 kDa herbicide-binding protein [23], or DI, and the polypeptide called D2 [10]. D2 is chloroplast encoded and is believed to be part of the PS II reaction centre core [31] serving perhaps to carry Qb [28]. Metz and co-workers have identified a mutant of Scenedesrnus which is impaired in water-splitting activity and has an altered 34 kDa protein [25]. This protein, which is found in PS II core preparations has recently been identified as D 1 [25, 26]. Thus a polypeptide of the so-called core appears to support some of the functions of water splitting as well as electron transport. The extrinsic water splitting complex is generally considered to consist of a 33kDa polypeptide and two smaller ones (24 and 16kDa) [13, 22]. These polypeptides have been defined to be extrinsic by phase separation into Triton X-114 [4]. The fact that the polypeptides are surface exposed [32] tends to support the idea of their intrinsic nature. Recently, there have been a number of reports of PS II cores with a limited number of polypeptides which are capable of carrying out both core functions and water splitting [14, 17, 18, 33]. These core preparations do, however, have more than 5 polypeptides and include several prominant ones in the 3 ~ 3 4 kDa region on SDS-electrophoresis gels. Tang and Satoh [33] report that their PS II core preparation contains the 33 kDa extrinsic polypeptide (33 kDa EP) from the water splitting complex. Since it appears that the function of 33 kDa EP is to preserve manganese in the photosynthetic oxygen-evolving complex [27], its presence might reasonably be expected in a preparation which evolved oxygen. While conjectures have been made about the nature of the association of the 33 kDa EP with the membrane [20, 21], the putative presence of the 33 kDa EP in a core preparation suggests that this apparently extrinsic protein shows a tenacious attachment to core proteins in particular. Most previous workers have used Coomassie blue staining of gels to assay for the presence or absence of 33 kDa EP in fractions. In the present work, we use the much more sensitive technique of Western blotting with antibody to the 33 kDa EP to evaluate various techniques for washing Triton-prepared PS II particles. We confirm and extend the earlier work of Kuwabara and Murata [20, 21] by showing that a significant fraction of the 33 kDa EP remains attached to the membrane even after extensive washing. It is our contention that the PS I! core and water splitting complex are closely associated in the thylakoid membrane, and that this reflects the association of their functions.

Methods

Washing procedures Photosynthetic oxygen evolving particles (PS II particles) were prepared from grocery store spinach by the method of [1] as modified by [11]. A single Triton

71 extraction was used at a detergent/chl ratio of 25/1. The particles were stored frozen at - 8 0 ° in 15mM NaC1, 5raM MgC12, 20raM HEPES pH 7.5 and 400mM sucrose. (This is the B4 medium of [11].) These particles were used to compare different published methods for removal of 33 kDa EP. Unless otherwise noted, all operations were carried out in dim light.

1.1 M CaCl2/cholate. The method of Ono and Inoue [30] as modified in [19] was followed. Frozen PS II particles were washed three times in 200 mM sucrose, 20mM HEPES pH 7.5. A HEPES buffer was substituted for the MOPS of Imaoka et al. [19] because the particles had been frozen in a HEPES buffer. The particles were suspended in 1 M CaC12, 300 mM sorbitol, 10 mM NaC1, 40 mM MES pH 6.5 at a final concentration of 1 mg/ml and gently shaken on ice for 30rain. The material was centrifuged for 30rain at 35,000 x g. The pellet was resuspended in 200raM sucrose, 20raM MES pH 6.5, 20raM Na cholate at 2 mg chl/ml. This was gently shaken on ice in the dark for 15 rain, then centrifuged 15rain at 35,000 x g. The pellet was washed once in 200raM sucrose, 20raM MES pH 6.5, and then resuspended in the B4 medium of [11] at a final concentration of 1 mg/ml. 2. NaCl/urea. PS II particles were washed three times in 300 mM sucrose, 10 mM NaC1, 25 mM MES pH 6.5, after the method of Miyao and Murata [27]. The particles were resuspended to 500/~g chl/ml in 1 M NaC1, 300raM sucrose, 25 mM MES pH 6.5. This was allowed to stand for 30 rain under room light, on ice. The particles were centrifuged for 30rain at 39,000 x g. The pellet was resuspended in 2.3M urea, 10raM NaC1, 25raM MES pH 6.5 to a final concentration of 0.5 mg chl/ml. The material was gently shaken for 1 hr on ice in room light. The particles were pelleted at 35,000 x g for 15rain, and were resuspended in B4 medium. 3. Tris wash. PS II particles were resuspended in 16raM Tris-HC1 pH 8.3, 10 mM NaC1 at a final concentration of 0.25 mg/ml, and were shaken gently on ice in the dark for 15rain [21]. The particles were pelleted at 35,000 x g for 15 rain, then were resuspended as before. After a second incubation and centrifugation, the particles were resuspended in B4 medium. 4. Detergent wash. PS II particles were prepared by the method of Kuwabara and Murata [22] from 6 day old barley plants (var. Bonanza) using a detergent/ chl ratio of 15/1. They were stored at - 80 °C in 300raM sucrose, 10raM NaC1 and 25 mM MES pH 6.5. An aliquot was thawed, centrifuged and washed once with 16 mM Tris buffer as in item 3 above. The washed pellet was resuspended in 30 mM octyl glucoside in 20 mM MES pH 6.8 to a final detergent/chl ratio of 10/l. After a centrifugation of 30rain at 110,000 x g at 4°C in a Beckman 50 Ti rotor, the pellet was resuspended in 30 mM octyl glucoside in 20 mM tricine, pH 8.0, to a final detergent/chl ratio of 10/1. After centrifugation at 110,000 x g, the pellet was taken up in 65 mM Tris buffer pH 6.8, 10% glycerol.

72 An aliquot of 0.1ml of this pellet was washed with 10ml of 0.8M Tris (unbuffered). Another aliquot was used for electrophoresis with the addition of 0.4% SDS. In addition, an aliquot of the original PS II particles was washed with 5 ml of 0.8 M Tris (unbuffered).

Electrophoresis of samples All samples were electrophoresed on 10% acrylamide gels using the method previously described [5]. Samples in octyl glucoside were loaded directly on gels (with the addition of 10% glycerol or ethylene glycol). Other samples were made to 65 mM Tris, pH 6.8, 10% glycerol, 12 mM dithiothreitol and 2% SDS. These were incubated at room temperature for 20 minutes or at 70 °C for 10 min.

Preparation of antibodies Antibodies monospecific to the 33 kDa polypeptide were raised in rabbits by intramuscular and intradermal inoculation of the purified protein (very generously supplied to us by Dr Michael Seibert, Solar Research Institute, Golden, CO) in an emulsion with Freund's complete adjuvant. A total of ca 100 #g of protein per rabbit was used as the primary immunogen. Booster immunizations were given i.m. at 3 and 8 weeks subsequent to the primary immunization, using 100#g of protein in Freund's incomplete adjuvant. The IgG fraction was isolated from whole serum by precipitation with 40% saturated ammonium sulfate [15], with subsequent separation from other immunoglobulin classes by DEAE-cellulose chromatography in 10 mM sodium phosphate, pH 8. The IgG fraction, concentrated by ultrafiltration, was titrated by dot-blot assay against 10ng spots of antigen and found to be > 1/65000.

Western blotting Electrophoresis gels were blotted overnight onto Bio-Rad nitrocellulose in 50 mM acetate buffer, pH 7 at a current of 220 mA. The blots were reacted with the rabbit antibody against the 33 kDa EP (IgG fraction) at a dilution of 1:200. Antigen-antibody complexes were visualized using goat-anti rabbit linked to alkaline phosphatase (Kirkegaard and Perry Labs) [34]. The relative amount of 33 kDa EP was determined by densitometery. Blots were scanned on a Helena R&D densitometer, using a green filter. Since electrophoresis of larger amounts of proteins resulted in wider bands, each determination of intensity was multiplied by a factor representing the width of the band. This resulted in linear relationships between volume of material applied to a gel and the corrected intensity of the resulting coloured spot. Correlation coefficients of the points about the line going through the origin were ~>0.98.

73

Fig. 1. SDS-gels and immunoblots of Triton-prepared PS II particles following various washing procedures. Cont: Control PS II particles; lanes A C: particles washed with A: 1M CaCI2; B: 1M NaC1 followed by 2.3M urea; C; 16raM Tris buffer, pH 8.3. Left side of figure: Coomassie blue-stained gel; right side: Western blot of duplicate gel reacted with anti-33 kDa EP. Each lane was loaded with 10 pg chl in 2% SDS. Samples were not heat denatured. The faint band toward the bottom of the Western blot is chl. Photosystem H assay Water-splitting was m e a s u r e d by the ability of particles to use water as a d o n o r for D C P I P reduction. The reaction was carried out in saturating light, in a m e d i u m consisting of 0 . 0 2 5 r a M D C P I P , 1 0 r a M M E S p H 6, 15raM NaC1 a n d 5 m M MgC12. The electron d o n o r D P C was used to a c o n c e n t r a t i o n of 0.5 m M to assay core activity.

Results

Tenacity of binding of 33 kDa EP to PS H membrane Figure 1 (left) shows Coomassie blue-stained lanes of control a n d washed PS II m e m b r a n e s . The heavily stained b a n d labelled 33 k D a runs with an a p p a r e n t molecular weight of 31 k D a in the present system. It was identified as the 33 k D a

74 Table 1. Water-splitting and core activities of frozen spinach PS II particles after washing treatments to remove 33 kDa EP.

PS II activity (/~moles/mg chl-hr)

Treatment

H20--~ Control CaC12/cholate NaCI/urea Dilute tris

DCP

DCPIP

36.4 + 2.2 12.3 ,+ 0.9 0 0

~ DCPIP

88.1 _+ 1.8 88.5 _+ 24.4 75.2 _+ 10.7 89.4

values from 3 determinations _+ s.d.

EP by immunoblotting (Fig. 1, right). It is clear that the three washing treatments vary in their ability to remove the 33 kDa EP from membranes. The 16 mM Tris wash (pH 8.3) (Fig. 1, lane C) is least effective in protein removal although it completely eliminated the ability of these particles to use water as an electron donor (Table 1). The CaC12/cholate wash is somewhat more effective in removing the protein, although almost 33% of the original water splitting activity remains. (The relatively low control activity is probably due to the winter-grown spinach used to prepare the membranes, which we find yields less active preparations than spring-grown plants). The CaC12 wash is frequently cited as providing complete removal of the 33 kDa EP (e.g. [9, 19]); however, in our hands a faint but definite trace of the protein is seen even with Coomassie staining. Our data shows that the most complete removal of the 33 kDa EP resulted from a two-step procedure; a NaC1 wash to remove the 24 and 16 kDa polypeptides, followed by a urea wash to remove the 33 kDa EP (Fig. 1, lane B). The protein could not be detected by Coomassie staining, although immunoblotting revealed that a small amount remained bound. This procedure leaves most of the PS II core activity but results in complete depletion of water splitting activity. This is in contrast to a report [27] in which 25% of water-splitting activity (as measured by DCPIP reduction) remained after the two-step wash. An attempt was made to quantify the 33 kDa EP remaining after NaC1/urea treatment by using densitometric scans of the immunoblots. A range of aliquot sizes from each of the treatments was electrophoresed and immunoblotted. In Table 2. Quantification by immunoblotting of 33 kDa EP remainingin spinach PS II particles after various washing procedures

Relative units of 33 kDa EP Control membranes

Ca + + / cholate

Na +/urea

Dilute tris

1643 868 495

675 (41.1%) 440 (50.7%) 240 (48.5%)

140 (8.5%) 77 (8.9%) 60 (12.1%)

1300 (79.1%) 630 (72.6%) 480 (97.0%)

/~g chl loaded 2.5 1.25 0.625

Average remaining

46.8 _+ 5.0%

9.8 ,+ 2.0%

82.2 + 17.9%

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Fig. 2. Western blot showing presence of 33 kDa EP in barley Triton-prepared PS II particles after sequential washes with Tris and octyl glucoside. Lane 1: control particles, 2.5#g chl; lane 2: supernatant from first wash (16mM Tris buffer) 50#1, containing no chl; lane 3: supernatant from second wash (octyl glucoside, pH 6.8), 50 #1 containing 0.37 #g chl; lane 4: supernatant from third wash (octyl glucoside pH 8.0) 50 #1 containing 1.0 #g chl; lane 5: pellet from third wash (10 #1); lane 6: final pellet after additional wash with 0.8 M Tris (10#1). The volumes in which pellets were resuspended were adjusted so that all three pellets represent the same amount of starting material (i.e. 2.5 #g chl). The arrow shows the position of a faint line in lane 6.

each case, w h e n the w i d t h - c o r r e c t e d i n t e n s i t y (see M e t h o d s ) was p l o t t e d a g a i n s t the v o l u m e l o a d e d , a l i n e a r r e l a t i o n s h i p was o b t a i n e d ( c o r r e l a t i o n coefficients e q u a l to or greater t h a n 0.98) ( T a b l e 2). E v e n in the case o f t h e m o s t effective w a s h (NaC1/urea), in excess o f 10% o f the o r i g i n a l 33 k D a E P r e m a i n s ; h o w e v e r t h a t a m o u n t is c o n s i s t e n t l y b e l o w the level o f d i s c r i m i n a t i o n by C o o m a s s i e staining.

76

Effect of oetyl glucoside In addition to the salt and urea washes in the above experiment, we also looked at the effect of repeated washes with the non-ionic detergent octyl glucoside (see Detergent Wash, in Methods). Figure 2 shows an experiment in which PS II particles were washed twice with 30 mM octyl glucoside (once at pH 6.8 and once at pH 8.0) and finally with 0.8 M Tris (unbuffered). It was expected that since octyl glucoside extraction at relatively low pH values extracts a minimum of chl-protein complexes [6] this treatment would result in loss of the 33 kDa EP without concomitant loss of chl. The subsequent treatment at pH 8.0 removes some of the chl-protein complexes and we assumed that any remaining extrinsic proteins would be extracted. The results of this experiment are shown in Fig. 2. Significant quantities of 33 kDa EP are removed by each of the detergent washes, but a detectable amount remains membrane bound, as shown by the blot of the washed pellet in Fig. 2, lane 5. A final wash of the remaining pellet in 0.8 M Tris results in almost complete depletion of 33 kDa protein from the pellet. On the other hand, the use of 0.8 M Tris as an initial wash step does not result in complete removal (data not shown). The Tris treatment results in a more effective removal of the 33 kDa EP than treatment with detergent, but maximum depletion seems to require treatment with both the detergent and the ionic solution.

Role of dithiothreitol In Fig. 2, lane 5, there are two bands which react with anti-33 kDa EP. This effect was not visible except when small amounts of antigen were loaded on gels. Since the amino acid sequence of the protein from spinach [29] indicates one pair of cysteines, it appears most likely that differences in the apparent molecular weights could reflect the presence of a disulfide bridge in incompletely reduced samples. Earlier workers [21] had noted that the presence of reducing agent changed the apparent molecular weight of the 33 kDa EP, and also attributed it to the formation of disulfide bridges. We examined the concentration range over which this phenomenon occurred. PS II particles and extracted 33 kDa EP from spinach were suspended in buffer containing 2% SDS with various D T T concentrations, but were not heated prior to electrophoresis. The results of this experiment are shown in Fig. 3. In the case of PS II particles suspended in buffer with no exogenous reducing agent, 33 kDa EP runs as a single band of apparent MW 31 kDa. With 15 mM DTT, a second band with an apparent MW of 31.5 kDa is present. In 50mM D T T only the higher MW band is present. This behaviour is consistent with reduction of a disulfide, bridge to give a more extended form of the polypeptide which therefore has a lower electrophoretic mobility. In the case of the extracted 33 kDa EP, most of it is converted to the more slowly running form by 5 mM DTT. It is quite reasonable to expect that the

77

Fig. 3. The effecft of dithiothreitol concentration on the electrophoretic behaviour of the 33 kDa EP on spinach membranes and after extraction. A: Silver-stained gel of PS II particles. Each lane contains 2~g chl of Triton-prepared particles prepared without heating in 2% SDS, 65mM Tris-HC1 pH 6.8, 10% ethylene glycol and the indicated amount of DTT. B: Silver-stained gel of extracted 33 kDa EP. Each lane contains the CaCl2-extract from the equivalent of 5#g of PS II particles. The extract was dialyzed overnight against 50 mM Tris-HC1 pH 7.6, before the addition of sample buffer (see above) containing the amount of DTT indicated in the figure.

33 k D a p o l y p e p t i d e is in different e n v i r o n m e n t s when on the m e m b r a n e a n d after extraction. H o w e v e r , it is unlikely t h a t the b i n d i n g o f 33 k D a E P to the m e m b r a n e is directly affected by the degree o f r e d u c t i o n o f this p a i r o f cysteines, since PS II particles can be i n c u b a t e d in buffer c o n t a i n i n g up to 100 m M D T T with no c h a n g e in the a m o u n t o f 33 k D a E P released into the soluble supern a t a n t ( d a t a n o t shown).

78 Discussion

It is clear from our experiments involving different methods of washing that it is extremely difficult to effect complete removal of the 33 kDa EP. Of the methods tested, the most efficient was the two step NaC1/urea method [27] which removed ca. 90% of the 33 kDa EP. In contrast, we did not find that CaC12extraction removed all the 33 kDa EP, although this method has been cited by some workers as giving complete removal (e.g. [9, 19]). Other workers have similarly demonstrated less than complete removal (see figures in [24]). Therefore, the CaC12-wash method should be viewed only as a convenient method for significant depletion, but not necessarily for perfect removal of the 33 kDa EP. As summarized in Table 1, all four washing procedures are effective for the reduction of water-splitting activity. However, some activity remains after CaC12/cholate treatment, consistent with the results of [30]. Among these four treatments there does not appear to be any correlation between residual PS II activity and the amount of 33 kDa EP visible on Coomassie-stained or immunoblotted gels. Treatment with non-ionic detergent octyl glucoside does not completely remove the 33 kDa EP from the membrane. In addition, when the solubilized material is centrifuged on a sucrose gradient in the presence of octyl glucoside [7], 33 kDa EP is found not only at the top of the gradient but also migrates into the gradient and is found in the same fractions as D1 and CP a-1 (CP 47) [8]. That this apparently extrinsic protein is attached very firmly to the PS II core particle was first demonstrated by Tang and Satoh [33] who identified it in their oxygen-evolving cores on the basis of Coomassie blue staining. In addition, Hinz [16] concluded that the 32 kDa polypeptide in PS II cores from barley was the 33 kDa EP, since it contained lysine, in contrast to D1 which does not (at least in the case of spinach and a tobacco) [35]. Kuwabara and Murata [20, 21] commented on the firm attachment of the 33kDa EP to the thylakoid membrane, and suggested that there might be different domains on the surface of the 33 kDa EP. Recent data suggest that there might be more than one way in which the 33 kDa EP interacts with the chloroplast membrane. Bowlby and Frasch [3] showed high-affinity and a low-affinity binding sites for the 33 kDa EP on CaC12-washed PS II particles. The high-affinity site is filled at low concentrations of the 33 kDa EP, whereas the low-affinity sites becomes filled at higher concentrations. The different binding modes of 33 kDa EP to the membrane suggest different kinds of interaction with the membrane. It is possible that the 33kDa EP possesses a hydrophobic region which is associated either with the hydrophobic intrinsic proteins of the PS II core or lipids which are firmly associated with them. The primary structure of this protein shows no extended hydrophobic regions [29] and the protein as a whole is clearly hydrophilic [4]. However, a hydrophobic domain could be generated by the secondary and tertiary structure of the protein. In fact, a hydropathy plot [12] of the 33 kDa EP indicates two

79 s o m e w h a t h y d r o p h o b i c d o m a i n s t o w a r e d the C - t e r m i n a l e n d n e a r r e s i d u e s 109-136 a n d 192 219 ( d a t a n o t s h o w n ) . T h e r e is c o n s i d e r a b l e e v i d e n c e for h e t e r o g e n e i t y in P S II r e a c t i o n c e n t r e s [2] a n d it is c o m p l e t e l y p o s s i b l e t h a t the w a y the 33 k D a b i n d s to the m e m b r a n e d e p e n d s u p o n the t y p e o f r e a c t i o n c e n t r e w i t h w h i c h it is a s s o c i a t e d .

Acknowledgements T h i s w o r k w a s s u p p o r t e d by N S E R C g r a n t 67-4688 to B R G a n d by N I H g r a n t G M 2 2 9 1 2 to L A S .

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