Photosynthesis Research 6, 193-199 ~1985 Martinus Nijhoff/Dr W. Junk Publishers,

Dordrecht, Printed in the Netherlands.

193

The use of polyclonal antibodies to identify peptides exposed on the

stroma side of the spinach thylakoid

STAN IVEY and STEVEN P. BERG

Department of Biological Sciences, University of Denver, Denver, CO 80208, USA

Kay Words: Thylakoids, antibodies, photosynthesis, photosystem II, membrane topography.

Abstract : We have raised polyclonal antibodies against an oxygen-evolving photosystem II preparatio~ Western Blot analysis of the whole serum reveals antibodies specific for at least 15 Coomassie visible bands ranging from 59 to 11 kD~ These antibodies are specific for proteins located on both sides of the membran~ Included are antibodies specific for Tris-removable peptides (33, 25 and 18 kda], which are thought to be exposed on the lumen surface of the PS II comple~ Since the whole serum agglutinates thylakoids, antibodies specific for the stroma side of the PS II complex are also present. A sub-popula~on of antibodies can be isolated by allowing the antibodies in whole serum to bind to EDTA- treated thylakoid membrane~ The antibodies which specificatly bind are cross-reactive with peptides with Mr of 59, 57, 34, 28, 27, 2B, end 23 kO~ Our data indicate that these peptides have antigenic determinants exposed on the stroma side of the thylakoid membrane

I n t r o d u c t i o n

The proteins which participate in photosynthetic electron transport and

photophosphorylation are now known to exist in discrete transmembrane

protein complexes. A remaining research goal is to understand the

topography of these various complexes in detail sufficient to explain the

structure/function relationships which exist among these protelns.

Efforts to understand the topography of the Photosystem II (PS II)

protein complex have resulted in a number of widely disparate models

[4,5,12,15]. We describe here an experimental protocol for the isolation

of antibodies specific for the stroma side of the thylakoid membrane.

These antibodies have been used to identify PS II peptides which are

exposed to the stroma side of the PS II complex. This technique has

allowed us to identify seven bands which seem to have stromal exposure.

The three extrinsic proteins associated with water oxidation, which are

proposed to be located on the lumenal side of the thylakoid [I]° do not

react significantly with antibodies specific for proteins exposed to the

stroma side of the membrane.

M a t e r i a l s a n d N e t h o d s

Highly purified PS II complexes were isolated from spinach by the

protocol of Kuwabara and Murata [i0], as we have described elsewhere [7].

194

The preparation had no Coomassie detectable PSI or cytochrome f/b6

complex. A 1.6 ml PS II preparation (2.7 mg chl/ml and 23 mg protein/ml)

was split into eight 200 ~I aliquots each of which was injected into a

different subcutaneous site on a rabbit's back. No adjuvant was employed

because the aggregated PS II complexes were already particulate.

Identical secondary injections of the same material were given after

eight weeks. The rabbits were bled and the whole serum was frozen at

-8~C until needed. Western Blotting was done as described in [17].

Prior to electrophoresis, samples were dissolved in a solution containing

35% glycerol, 6% lithium dodecylsulfate (LDS), 5% mercaptoethanol and 0.5

mM TRIS, pH 6.8. LDS-PAGE (4.5 mA/cm 2) was done on 10 to 15% gradient

gels containing 2% lithium dodecylsulfate at 4°C. Other conditions were

hke those described elsewhere [6]. Antibodies specific for the stroma

side of the thylakoid membrane were isolated by exposing whole PS II

anti-serum to unstacked thylakoids which had been prepared by washlng

stacked thylakoids three times in 200 mM sucrose containing 1 mM EDTA (pH

7.5) at 20°C. The resulting unappressed thylakoids were resuspended to

0.3 mg chl/ml in 200 mM sucrose containing I0 mM NaCI, i0 mM TES (pH 7.5)

and 3% gelatin (BioRad, Richmond, CA). The gelatin was found to

significantly reduce non-specific antibody binding to thylakoids. After

30 min the gelatin-coated thylakoids were collected by centrifugation and

the pellet was resuspended to 1 mg chl/ml in 200 mM sucrose containing i0

mM NaCI, i0 mM TES (pH 7.5), and 1% gelatin. Three ml of whole serum

were brought to 200 mM sucrose before the serum was added dropwise to 65

ml of the thylakoid suspension. After a 14 h incubation at 20°C, the

thylakoids were collected by centrifugation at 40,000Xg. The thylakoids

with bound antibody were washed three times with 200 mM sucrose

containing 20 mM Tris (pH 7.5) and 500 mM NaCI. The pellets were

stripped of specifically bound antibody by resuspension in 40 ml of 200

mM sucrose containing I00 mM glycine (pH 2.3) at 0°C. After stirring

slowly tot 25 minutes at 0°C, the stripped thylakoids were removed by

centrifugation. The antibody-containing supernatant was adjusted to I%

gelatin, the pH was adjusted to 7.5 with 1M Tris (pH 9.2) and NaCI was

added to a final concentration of 0.i M. This solution contains the

putative stroma-side antibodies.

R e s u l t s and D i s c u s s i o n

The process of Western Blotting begins with the electrophoretlc

separation of peptides in polyacrylamide gels. The peptides are then

transferred electrophoretically to nitrocellulose paper, where they bind

tightly. When the polyacrylamide gels were stained with Coomassie after

the electrophoretic transfer (data not shown), no Coomassie visible bands

remained, implying that peptide transfer from the gel was complete.

195

Figure I: Polypeptide profiles on nitrocellulose paper stained with Buffalo Black [Lanes 1-5) or labeled by Western Blotting [17] (Lanes 6-I0]. Lane 11 is s polyacrylsmide gel stained with Coomassie. Lanes 1 and 6, membrane peptides of Streptococcus san~uis 34 (SS- 34]. Lanes 2 and 7, molecular weight standard~ Lanes 3 and 8, peptides present in PS II preparatio~ Lanes 4 and g, thylakoid peptide~ Lanes 5 and 10, peptides remaining after washing the PS II preparation with NaC1/Tri~ Lane 11, Coomassie visible PS II peptides present after LDS-PAG~

Figure 1 (Lanes 1-5) shows Buffalo Black stained peptides on

nitrocellulose and the peptides (Lanes 6-10) identified by antibodies

raised against the PS II preparation. Lane 6 shows that a Triton extract

of Streptococcus sanguis 34 (SS-34) has no antlgenic determlnants which

cross-react with antibodies raised against the Triton extracted PS II

preparation. The detergent extract of bacterial membranes contains

residual stabilizing Triton, but this nonionic detergent is presumably

exchanged with dodecylsulfate during LDS-PAGE so that virtually no Triton

remains associated with the separated peptides on the nitrocellulose.

Antibodies specific for Triton which are probably produced by the

rabbits, find no Triton determinants after the LDS-PAGE. The data of

Lane 6 obviate our concern that Western Blottzng is merely identifying

residual Triton associated with any peptide present.

Lane 8 of Figure 1 shows results obtained when the separated

peptides of PS II are challenged with the antibodies raised against the

PS II preparatzon. A comparison of the Buffalo Black stazned

196

nitrocellulose (Lane 3) and the antibody stained profile of Lane 8,

indicates that antibodies have been raised against most of the Buffalo

Black visible peptides of the PS II preparation. When the separated

peptides from thylakoids are challenged with the same antibodies (Lane 9)

the peptides identified are very similar to those of the isolated PS II

complex. The most notable difference between the thylakoid profile (Lane

9) and the PS II profile (Lane 8) is the identification of a 50 kDa

peptide in the thylakoid profile. This band is never seen with

thylakoids which have been washed with EDTA at low ionic strength, or

with isolated PS II preparations. Thus, we are certain that this 50 kDa

peptide is neither a component of PSI or PS II. Together these results

indicate that antibodies raised against PS II are not cross reactive with

other thylakoid peptides (except the 50 kDa), including those associated

with PS I.

Lanes 5 and 10 of Figure 1 show the separated peptides of a PS II

preparation which has been washed with 2 M NaCI and 1M Tris. Tris

treatment is known to remove at least three peptides from PS II

preparations [19], and these three peptides are absent in the Buffalo

Black (Lane 5) and the antibody labeled (Lane i0) profiles. After the

NaCl/Tris treatment a diffuse doublet remains at 34 and 33 kDa. The

diffuse 33 kDa band appears to comigrate with the Tris-removable 33 kDa

peptide. These diffuse bands might be any one of several proteins known

to migrate between 30-35 kDa [18,11,14,16J. Since we can make no

definitive assignments for any of these bands, we can only conclude that

we might have antibodies specific for any of HBP32 [18], D2 [14,16J or

the 34 kDa intrinsic protein [11]. Further experiments are underway to

allow us to make these band assignments. When Lane 8 is compared with

Lane 10, we conclude that the whole serum contains antibodies speclfic

for the lumen side of the PS II complex. This conclusion is based on the

observation (Lane 8) of antigenically identifiable bands at 18, 25 and 33

kDa which can be removed from the PS II preparation by treatment with

NaCI and Tris [19,20]. These peptides have been shown to be extrinsic

proteins on the lumen side of the thylakoid membrane [1,20]. We have

also observed that our whole serum strongly agglutinates EDTA-treated

thylakoids suggesting that antlbodies are present whlch are specific for

the stroma side of the PS II complex as well.

Once the agglutinated EDTA-treated thylakoids mentloned above are

washed to remove nonspecifically bound antibody, the specifically bound

antibody can be removed with a low pH wash [8,13]. The thylakoids are

removed by centrifugation and the pH of the supernatant containing the

released antibodies can be titrated back to pH 7.5, thus reactivating the

antibodies. These reactivated antibodies should be those specific for

the stroma side of the PS II complex. If none of the EDTA-treated

thylakoids are turned inside-out during their isolation, there should be

197

Figure 2: Pslypeptide profiles on nitrocellulose paper stained by Western Blotting. Whole serum antibodies were used for the primary identification in Lanes 4-6, Antibodies which bound tightly to the stroma side of thylaksid membranes were used for the primary identification of peptides in Lanes I-3. Lanes I and 4 are the peptide profiles from the PS II preparation Lanes 2 and 5 are the peptide profiles after the PS II preparation was washed with 2 M NaC1. Lanes 3 and 6 are the peptide profiles obtained after the salt washed PS II preparation was treated with 1M Tri~

no contamination with antibodies specific for antigenic determlnants

exposed on the lumen side of the thylakoid membrane.

Figure 2 shows the Western Blots of peptide profiles challenged wlth

whole serum (Lanes 4-6) or with the antibodies which bound specifically

to the EDTA/gelatin-treated thylakoids (Lanes 1-3). Clearly present in

Lane 4 are prominent bands at 33, 25 and 18 kDa. Treatment with 2 M NaCI

(Lane 5) results in the apparent loss of the 25 kDa band and a

substantial reduction in the 18 kDa band. Loss of these bands produced a

reduction in the rate of electron transport activlty by 58%. A

subsequent wash with 1 M Tris completely inhibited electron transport,

reduced the 18 kDa band to near invisibility, and also removed the

prominent band at 33 kDa (Lane 6). A faint band remained at 33 and 34

kDa. A second Tris wash did not further reduce the intensity of this

band, suggesting that the band does not represent the 33 kDa extrinsic

protein. Lanes 1-3 show peptide profiles which have been challenged with

antibodies which have specifically bound to EDTA/gelatin-treated

thylakoids. The faint doublet at 33 and 34 kDa is clearly present (Lanes

198

I-2) except in Lane 3 where only the 34 kDa band is visible. Very faint

bands at 25 and 18 kDa appear to be present, suggesting that there may be

very alight whole serum contamination. The faint band at 34 kDa which

appears to comigrate with the 33 kDa extrinsic protein [11] could be

HBP32 [18J, the 34 kDa intrinsic protein [11] or D2 [14,16J. If we

ignore any faint bands which might be due to contamination with whole

serum antibodies, we would conclude that the major bands identlfied in

Lanes 1-3 are proteins which have antigenic determinants exposed on the

stroma side of the PS II complex. These bands include peptides with Mr

of 59, 57, 34, 28, 27, 26 and 23 kDa.

Stromal exposure of PS II peptides with similar molecular weights

have been suggested by others. For instance, Andersson et al. [3] raised

antlbodies against purified light-harvesting complex. They found

antibodies specific for peptides of 28, 27, 25, and 23.5 kDa. They also

found that the antiserum would agglutinate unstacked right-side-out and

inside-out thylakoids, allowing them to conclude that the light-

harvestlng complex was transmembraneous. However, they did not

definitively show which, if any, of the peptides actually had antigenic

determinants on both sides of the membrane. Our data identlfied peptides

which are exposed on the stromal surface of the thylakoid membrane, but

at this time we are unable to definitely assign any of these visible

bands or to correlate them with the observations ot Andersson et al.

However, work is underway with "green'" gels, urea gels, and secondary

LDS-PAGE to make these assignments. Since there is evidence that the

herbicide binding protein has a stromal exposure [21] and since Vermaas

et al. L18] has shown that HBP32 comigrates with the Tris removable

LRP32, we would tentatively identify the 34 kDa band as HBP32.

Figure i (Lane 11) shows the Coomassie visible peptides in a

polyacrylamide gel after LDS-PAGE. Clearly visible are peptides at 40

and 44 kDa which are probably CP43 and CP47 respectively [5]. Western

Blot analysis (Lanes 8-10) shows that these bands are visible when the

peptides are challenged with whole serum, indicating that these peptides

have been transferred to the nitrocellulose and that antlbodies to these

peptides are present. In spite of their presence, the 40 and 44 kDa

peptldes are not stained with Buffalo Black (Figure i, Lanes 3 and 5),

and these peptides are not identified with stroma-side antibodies (Figure

2, Lanes 1-3). This suggests that there is not significant direct

exposure of the PS II reaction center to the aqueous stroma.

With our PS II antibodies, we routinely observe an antlgenically

identified doublet at about 59 and 57 kDa. As with the peptides

discussed above we can not yet offer a clear assignment for these bands,

but we believe that they are probably two oligomeric species of LHCPI or

LHCP2 which have been described by Anderson [2] in her re-electrophoresis

studies. Andersson et al. found a similar high molecular weight band

199

when their nitrocellulose was challenged with antibodies specific for LHC

[3]. In summary, we report here that immunization of rabbits wzth

purified PS II complexes results in the production of antibodies specific

for many of the peptides present in the complex. From these antlbodies,

it is possible to select those which are specific for antigenic

determinants exposed on the stromal surface of the thylakoid membrane.

By coupling the technique of Western Blotting to the selection of stroma-

side-specific antibodies, we can identify peptides which are exposed on

the stroma surface of the PS II complex. Further work is underway to

identzfy the peptides exposed on the lumen surface of the thylakoid, and

to unambiguously assign the identity of the antibody-identified peptides

exposed on both surfaces.

Acknowledgement

This work was supported by a grant (82-CRCR-I-1125) from the Competitive

Grants Office of the USDA to SPB. The authors are grateful to Dr. Victor

Reusch for generously supplying the SS-34 membrane preparation used in

this study.

~ f e ~ n ~ 8

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