462

Biochimica et Biophysics Acts, 503 ( 1 9 7 8 ) 4 6 2 - - 4 7 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 47547

INSIDE-OUT MEMBRANE VESICLES ISOLATED FROM SPINACH THYLAKOIDS

BERTIL A N D E R S S O N

and HANS-ERIK ~ k K E R L U N D

Dept. of Biochemistry 1, University of Lund, Chemical Center, P.O. Box 740, S-220 07 Lund (Sweden) (Received March 20th, 1978)

Summary Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: {1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; {4} concave EF and convex PF freeze-fracture faces.

Introduction The asymmetry of biological membranes is an important feature of membrane organization and function [ 1 ]. This has been studied extensively in membrane systems where isolation of inside-out vesicles has been possible, for example the red blood cell [2] and the inner mitochondrial membrane [3]. For the chloroplast thylakoid membrane system the lack of a preparation method of inside~aut vesicles has made a study of the inner thylakoid surface difficult. Conclusions on the asymmetric arrangement of components across this membrane have therefore been drawn mainly from effects of antibodies and other non-penetrating reagents on the outer surface of the membrane [4]. In previous studies [5--7], partition in an aqueous dextran/polyethylene glycol two-phase system has been used for separation of press>? I-I 1 MIN

Fig. 1. T w o - p h a s e f r a c t i o n a t i o n o f t h e l o w salt d i s i n t e g r a t e d g r a n a e n r i c h e d f r a c t i o n ( 4 0 Kls). T h e t w o m a i n f r a c t i o n s , T 2 a n d B3, w e r e c o l l e c t e d and c h a r a c t e r i z e d w i t h r e s p e c t to m e m b r a n e s i d e d n e s s . Fig. 2. L i g h t - i n d u c e d p r o t o n t r a n s p o r t a s s o c i a t e d w i t h p h e n y l - p - b e n z o q u i n o n e reduction., m e a s u r e d w i t h a ~ e l e c t r o d e . T h e a ~ a y m e d i u m w a s c o m p o s e d of: 4 0 m M KCI, 0 . 3 7 m M p h e n y l - p - b e n z o q u i n o n e a n d c h l o r o p l a s t m a t e r i a l c o r r e s p o n d i n g t o 1 0 0 /~g c h l o r o p h y l l / m L Initial p H in t h e m e d i u m was 6 . 5 a n d t h e e x t e n t o f p r o t o n u p t a k e o r e x t r u s i o n w a s d e t e r m i n e d a f t e r e a c h e x p e r i m e n t b y a d d i t i o n o f 50 n m o l NaOH.

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~S Fig. 3. p H c h a n g e s a f t e r a s i n g l e - t u r n o v e r f l a s h m e s , u r e d as t h e a b s o r b a n c e c h a n s e o f b r o m c r e s o l p u r p l e in t h e T 2 a n d B3 m e m b r a n e f r a c t i o n s . T h e a s s a y r e e d / u r n w a s c o m p o s e d o f 4 0 m M KCI, 0 . 3 m M p h e n y l p - b e n z o q u i n o n e , 2 5 # M b r o m c r e s o l p u r p l e . Inlt/al p H w a s set t o 6 . 5 . T h e s i g n a l - t o - n o i s e r a t i o w a s i n c r e a s e d b y a v e r a g i n g f o r N s u c c e s s i v e flashes. Fig. 4. p H c h a n g e s i n t h e i n t e r n a l p h a z e o f t h e T 2 a n d B3 m e m b r a n e v e ~ c l e s w e r e m e a s u r e d as t h e Lightinduced absorbance chanse of Neutral Red (NR) in the presence of the external buffer bovine serum s l b u m i n . A s s a y m e d i u m w a s c o m p o s e d o f : 3 7 m M KCI, 6 . 5 m S b o v i n e s e r u m a ] b u m i n / m l ( p H 6.S), 1 2 . 5 ~M N e u t r a l R e d , 0 . 3 m M p h e n y l - p - b e n z o q u i n o n e a n d c h l o r o p l a s t m a t e r i a l c o r r e s p o n d l n ~ t o 4 0 ~ g c h l o r o p h y l l / m l . T o i n c r e a s e t h e =~_~n-]-to-~oise r a t i o t h e signal w a s a v e r a g e d f o r 1 6 r e p e t e t i v e f l l u m i n a tions.

thylakoid v e s i c l e s which are turned inside-out. A criticaltest of this susgestion would be provided by an investigation of whether the internal p H of the B3 material increases upon illumination, as would be expected for an inside-

TABLE I EFFECT OF NH4CI, DBMIB AND DCMU ON STEADY STATE LEVEL OF LIGHT-INDUCED PROTON TRANSPORT AND OXYGEN EVOLUTION ASSOCIATED WITH PHENYL-P-BENZOQUINONE REDUCTION

IN T H E 4 0 KIs, T 2 A N D B 3 M E M B R A N E F R A C T I O N S

E x p e r i m e n t a l c o n d i t i o n s as in Fig. 2 a n d w h e n i n d i c a t e d N H 4 C L D B M I B a n d D C M U w a s a d d e d t o y i e l d c o n c e n t r a t i o n s o f 1 0 m M , l . S # M a n d I 0 # M , r e s p e c t i v e l y . S t e a d y s t a t e level o f p r o t o n t r a n s p o r t ( ~ H * ) a n d r a t e o f o x y g e n e v o l u t i o n ( 0 2 ) a r e e x p r e s s e d as n m o l H ÷ / m g c h l o r o p h y l l a n d # m o l O 2 / m s c h l o r o p h y l l per h, respectively. Fract/on

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467 o u t vesicle. Such a study was performed using the pH indicator Neutral Red and buffering the external medium with bovine serum albumin (Fig. 4}. The internal compartments of the T2 material showed an acidification upon illumination as is normally seen for thylakoid membranes. In contrast a rise in the internal pH of the B3 vesicles was registered, supporting the conclusions drawn from the measurements of the external pH changes. The absorbance change without addition of Neutral Red was probably due to light scattering changes.

Trypsin treatment The effects on electron transport activities after tryptic digestion of chloroplast lamellae have been studied by several workers [13--15]. It is assumed that the resulting effects are caused by specific proteolysis of groups on the outer thylakoid surface, since trypsin should not be able to penetrate the lipid bilayer. Therefore, provided the T2 and B3 material are of opposite sidedness, different c o m p o n e n t s should be digested by trypsin, leading subsequently to different inhibitory effects. Fig. 5 shows the effect of trypsin treatment on Photosystem I electron transport activity, in the T2 and B3 fractions. The

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Fig. 5. I n h i b i t i o n o f t h e P h o t o s y s t e m l r e d u c t i o n o f m e t h y l v i o l o g e n a f t e r t r y p s i n i n c u b a t i o n o f t h e T 2 a n d B3 m e m b r a n e f r a c t i o n s . A t r y p s i n / c h l o r o p h y l l r a t i o o f 0 . 6 w a s u s e d . O x y g e n c o n s u m p t i o n w a s m e a s t ~ e d w i t h a C l a r k t y p e o x y g e n e l e c t r o d e in 2.5 m l o f a m e d i u m c o m p o s e d o f : 40 m M s o d i u m p h o s p h a t e b u f f e r ( p H 7.4), 1 m M NaC1, 10 m M s u c r o s e , 0 . 6 m M NAN3, 0 . 1 2 m M m e t h y l viologen0 0 . 2 8 8 m M 2 , 6 - d i c h l o r o p h e n o l - i n d o p h e n o l , 3 2 m M s o d i u m a s c o r b a t e , 10 ~M D C M U a n d c h l o r o p l a s t m a t e r i a l c o r r e s p o n d i n g t o 1 0 0 ~g o f c h l o r o p h y l l . T h e 1 0 0 % r a t e s w e r e 1 1 7 a n d 54 ~ m o l / m g c h l o r o p h y l l p e r h f o r t h e T 2 a n d B3 m a t e r i a l , r e s p e c t i v e l y . Fig. 6. I n h i b i t i o n o f t h e r e a c t i o n H 2 0 ~ P h o t o s y s t e m II ~ p h e n y l - p - b e n z o q u i n o n e a f t e r t r y p s i n i n c u b a t i o n o f t h e T 2 a n d B3 m e m b r a n e f r a c t i o n s . A t r y p s i n / c h l o r o p h y l l r a t i o o f 0.1 w a s u s e d . T h e o x y g e n e v o l u t i o n w a s m e a s u r e d w i t h a C l a r k t y p e e l e c t r o d e in 2 . 5 m l o f a m e d i u m c o m p o s e d o f : 28 m M s o d i u m p h o s p h a t e b u f f e r ( p H 6 . 5 ) , 4 . 8 m M NaCI, 0 . 2 4 m M phenyl-p-benzoquinone a n d c h l o r o p l a s t m a t e r i a l c o r r e s p o n d i n g t o 50 ~g c h l o r o p h y l l . T h e 1 0 0 % r a t e s w e r e 82 a n d 1 1 6 ~ m o l O 2 / m g c h l o r o p h y l l p e r h f o r t h e T 2 a n d B3 m a t e r i a l r e s p e c t i v e l y .

100

468

F i g . 7. F r e e z e - f r a c t u r e a p p e a r a n c e oe t h e T 2 a n d B3 m e m b r a n e

f r a c t i o n s . Bar r e p r e s e n t s 0 . 5 Urn.

activity o f the B3 fraction was inhibited 40--45% while under identical conditions the activity of the T2 fraction was inhibited by only approx. 15%. It is well established that this particular reaction is not affected by trypsin treatment o f u n b r o k e n chloroplast lamellae [13,14]. T herefore the most likely

469 explanation for the relatively high inhibition of the B3 fraction is that it contains membranes exposing the inner thylakoid surface. Correspondingly the low inhibition of the T2 material confirms that it is a right-side-out fraction. From Fig. 6 it is evident that the Photosystem II activity was also affected differently in the T2 and B3 fractions by trypsin treatment. The slope of the inhibition curve showed a biphasic appearance. First, a very rapid phase, resulting in about 40% decrease in activity of both fractions within 5 min of incubation. This rapid phase was followed by a slow phase in which there was a pronounced difference in activity decrease between the T2 and B3 materials. Between 5 and 90 min incubation the Photosystem II activity of the T2 fraction declined from 60% to below 10%. After the same incubation period, over 30% of the activity still remained in the B3 fraction. The results obtained from the trypsin inhibition experiments thus support the interpretation that the T2 and B3 material are of opposite sidedness, with the latter containing mainly inside-out thylakoids.

Freeze-fracture studies Freeze-fracturing of thylakoid membranes results in the visualization of faces with characteristic topographies [16]. The fracture face next to the intrathylakoid space (EF) shows relatively few, large distinct particles while the fracture face next to the stroma {PF) consists of mainly closely packed small particles. Simple vesicles derived from thylakoid membranes would show convex EF and concave PF faces provided they have retained their original sidedness (for nomenclature, see ref. 17). Consequently, inside-out thylakoid vesicles should ha-e concave EF and convex PF faces. In fig. 7 are shown electron micrographs of representative replicas from the T2 and B3 fractions. A predominance of convex PF and concave EF faces was found for the B3 material as expected for inside-out thylako~d vesicles, while mainly rightside-out vesicles were observed for the T2 fraction. Approx. 160 vesicular faces in each fraction were examined to estimate the relative contents of right-side-out and inside-out vesicles. Residual complex membrane structures were omitted from the calculations since a subdivision into concave and convex faces is not applicable for these. The results revealed that the B3 fraction contained 74% inside-out and 26% right-side-out vesicles while the T2 fraction contained 89% right-side-out and only 11% inside-out vesicles. Discussion

Independently of each other, the different experiments presented in the previous section all provide evidence that phase partition of disintegrated chloroplast lamellae yields two subpopulations of chloroplast fragments with opposite sidedness. Initially, the existence of inside-out thylakoid vesicles in the bottom-phase was suggested from observations on light-induced reversible proton extrusion associated with phenyl-p-benzoquinone reduction [5]. This interpretation is, however, correct only if the mechanism for proton translocation by the B3

470 material is the same as that normally observed for thylakoid membranes. In view of the present results this prerequisite is fulfilled since: {a) addition of the inhibitors d i b r o m o t h y m o q u i n o n e and DCMU affected both proton translocation and electron transport in a similar way {Tabel I}; {b} approximately the same degree of uncoupling by NH4Cl was observed for both T2 and B3 material {Table I); (c) the external proton release by the B3 vesicles was associated with a pH rise in the internal space of the vesicles while the opposite was observed for the T2 vesicles {Fig. 4). Other investigators have reported light-induced proton release by chloroplasts where part of the coupling factors has been removed by EDTA or NaBr treatment [18,19]. It has been suggested that these types of proton release are the results of conformational changes [19]. Moreover, these types of proton release are insensitive to uncouplers but can be converted into proton uptake by treatment of the coupling factor depleted thylakoids with dicyclohexylcarbodiimide. The proton release by the B3 vesicles, however, has earlier been shown to be dicyclohexylcarbodiimide insensitive [5]. Obviously, the proton extrusion by the B3 vesicles does not resemble the proton extrusion observed for coupling factor depleted thylakoids. The values for proton translocation in Table I are corrected for the weak "acidification"-response of the pH-electrode to light in the presence of phenylp-benzoquinone. In a previous report [5] we assumed that a minor uncoupler and DCMU insensitive part of the proton release by the b o t t o m phase material might be due to some kind of conformational change. This residual proton release was, however, overestimated since no correction was made for the response of the electrode to light. The proton gradient observed for thylakoids of normal sidedness, such as the T2 material, in the presence of phenyl-p-benzoquinone is suggested to be a result of internal release of protons from the water oxidation reaction and uptake of external protons by reduction of phenyl-p-benzoquinone. For the B3 material the only difference would be that the protons from the water oxidation reaction are released to the external medium and the protons for the reduction of phenyl-p-benzoquinone are taken from the internal space. At the present stage it is not known if the plastoquinone loop is involved in phenyl-pbenzoquinone reduction. The electron transport in intact thylakoids using phenyl-p-benzoquinone as acceptor was, however, insensitive to uncouplers. This observation corresponds to the results obtained for reduction of dibromot h y m o q u i n o n e and other class III acceptors [20], indicating that only coupling site II is involved in phenyl-p-benzoquinone reduction. The two fractions T2 and B3 showed different resistance to tryptic digestion as revealed from electron transport inhibition studies. These observations can most easily be explained by assuming a different sidedness for the two subpopulations, thereby exposing different components sensitive for the membrane impermeable proteolytic enzyme. The conclusions drawn from these trypsin treatment experiments demands that the electron acceptor used can penetrate the thylakoid membrane sufficiently. Methyl viologen is known to penetrate the chloroplast envelope freely [21] and is also likely to penetrate the thylakoid membrane in the small catalytic amounts needed for the Mehler reaction. For the highly hydrophobic

471 phenyl-p-benzoquinone no accessibilityrestrictionwould be expected. At the present stage the effects of tryptic digestion on the T2 and B3 material should not be taken as evidence for the assymmetric localization of certain reaction sequences or components across the membrane. Such interpretations need extensive investigations, including comparative studies on inhibition of several partial electron transport reactions of the two different membrane populations. Such studies are in progress. The replicas from freeze-fracturing of the B3 material demonstrated that this material was dominated by inside-out vesicles, since mainly concave E F and convex PF faces were observed. The replicas of the T2 and B3 fractions also gave a rough estimation of the relativeamount of right-side-outand insideout vesicles in each fraction. To avoid errors in the interpretation caused by residual unbroken thylakoids, only true vesicular shaped faces without surrounding crosssections were considered. A more extended study on freezefracture and freeze-etch appearance of the fractions will be published elsewhere [221. Obviously the Yeda press treatment under the present conditions disintegrates the thylakoid membranes in a way which allows some membrane fragments to reseal into vesicles with the intra thylakoid surface turned outside and others to reseal into vesicles of original sidedness. Thus, the 40 Kls fraction contains a mixture of inside-out and right-side-out vesicles. Due to asymmetric arrangement of the thylakoid membrane [4,231 the surface properties of the inside-out vesicles would differ from those of the right-side-out vesicles. This is the basis for their separation by phase partition, since this technique utilizes differences in membrane surface properties, such as charge and hydrophobicity, for separation [9]. Probably, varying amounts of inside-out vesicles are usually formed during different disintegration procedures described for the chloroplast lamellae. The reason why insideout thylakoid vesicles have not been isolated previously would be that the separation methods used earlier depend predominantly on size and density differences of particles. The phase partition method has also been used successfully for isolation of inside-out erythrocyte ghosts [24]. The B3 fraction is apparently still contaminated with right-side-out material as judged by freeze-fracturing. Attempts are therefore in progress to improve the purity of the inside-out fraction using extensive centrifugation and phase partition procedures. Despite this contamination, the B3 material should be extremely suitable for exploring the inner thylakoid surface, to improve our knowledge of the asymmetric arrangement of components across this membrane.

Acknowledgements W e are grateful to Professor Per-.~ke Albertsson for stimulating discussions and encouragement throughout the cource of this work. W e are also indebted to Miss Agneta Persson for skilful technical assistance. The experiments with bromcresol purple was carried out at the Max-Volmer-Institute fiir Physikalische Chemie und Molekularbiologie, Berlin, in collaboration with Dipl.Phys. R. Tiemann and Dr. P. Gr~iber. Freeze-fracturing was performed by Dr.

472

D.J. Simpson, Department of Physiology, Carlsberg Laboratory, Copenhagen. Dibromothymoquinone was a generous gift from professor A. Trebst. This work was supported by the Swedish Natural Science Research Council and the Carl Trygger foundation.

References 1 R o t h m a n , J.E. and Lenard, I. (1977) Science 195, 743--753 2 Steck. T.L. and Kant, J.A. (1974) Methods Enzymol. (Fleischer, S. and Packer, L.0 eds.)0 Vol. 31A. pp. 172--180. Academic Pre~. New York 3 DePier~e, J.W. and E~nster, L. (1977) Ann. Rev. Biochem. 46° 201--262 4 Trebst, A. (1974) Ann. Rev. Plant. Physiol. 25. 4 2 3 - - 4 5 8 5 A n d e r s ~ n . B., Aker~und, H.-E. and A l b c r t ~ o n , P.-A. (1977) FEBS Lett. 77, 141--145 6 Akeriund, H.-E., A n d e r s o n , B. and Albert~on~ P.-A. (1976) Biochim. Biophys. Acta 449, 525-.535 7 A n d e r ~ o n , B., AkeHu~d, H.-E~ and A l b e r t ~ o n , P.-A. (1976) Biochim. Biophys. Acta 423. 122--132 8 Alherts~on. P.-A. (1971) Partition of Cell Particles and Mac~omolecules, 2rid edn~ Almq~ist and Wiksell, S t o c k h o l m 9 A l b e r t ~ o n , P.-A. (1974) Methods Enzymol. (Fleischer, S. and Packer, L.0 eds.). Vol. 31A, pp. 761--789, Academic Pre~, New York 10 Shneyor, A. and Av~on0 M. (1970) FEBS Lett. 8, 164--166 11 ~ob~n~qon, G., Ha~tman0 A. and A l b e r t ~ o n , P.-A. (1973) Eur. J. Biochem. 33, 379--386 12 Ausl~nder, W. and Junge~ W. (1975) FEBS Lett. 59, 3 1 0 ~ 3 1 5 13 Selman, B.R. and Bannister, T.T. (1971) Biochim. Biophys. Acta 253, 428--436 14 Regitz. G. and Ohad, I. (1976) J. Biol. Chem. 2 5 1 , 2 4 7 - - 2 5 2 15 Renger, G., Eflxon, K , D6ring~ G. and Wolf. Ch. (1976) Biocl~im. Biophys. Acta 440,278-..286 16 Staehelin0 A. (1976) J. Cell Biol. 71. 136--158 17 Branton, D., Bul]ivant, S., Gflula. N.B., Karnovsky, M.J., Moor, ~., Mtthlethaler. K., Northcote, D.H., Packer. L., Satir, B , Satir, P., Speth, V., Staehlin, L . A , Steere, R.L. and Weinstein0 R.S. (1975) Science 190. 54--56 ~8 Uribe, E. (1972) Biochemistry 1 1, 4228-4235 t 9 Kamienietzky, A. and Nelson. N. (1975) Plant Physiol. 55, 282--287 20 Gould, I.M. and lzawa, S. (1974) Biochirn. Biophys. Acta 333, 509--524 21 Robinson, S.P. and Wl~ldch, J.T. (1976) Biochim. Biophys. Acta 440, 131--146 22 Anderson, B.. Simpson, D.J. and H~yer-Har~en, G. (1978) Car]sberg Re$. Commt~. 48, 7 7 - 8 9 23 Anderson, J.M. (1975) Biochim. Biophys. Acta 416, 191--235 24 Steck, T,L. (1974) in Methods in Membrane Biology (Ko m , E.O., ed.), Vol. 2, pp, 245--281, Plenum Press, N e w York