20 Jan 1984 - (Diner and Wollman, 1980), a PSII LHC (Burke et al., 1978), a cytochrome b-f complex (Hurtand Hauska, 1981), anal- ogous to theb-c1 ...
The EMBO Journal vol.3 no.4 pp.701-706, 1984
Partial characterization of the biosynthesis and integration of the Photosystem II reaction centers in the thylakoid membrane of Chiamydomonas reinhardtii
Philippe Delepelaire Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, 75005 Paris, France Communicated by J. -D. Rochaix
Pulse-labeling of wild-type and a Photosystem II mutant strain of Chlamydomonas reinhardtii was carried out in the presence or absence of inhibitors of either cytoplasmic or chloroplast ribosomes, and their thylakoid membrane polypeptides were analyzed by polyacrylamide gel electrophoresis. A pulse-chase study was also done on the wild-type strain in the presence of anisomycin, an inhibitor of protein synthesis on cytoplasmic ribosomes. The following results were obtained: the Photosystem II reaction center is mainly composed of integral membrane proteins synthesized within the chloroplast. Several of the proteins of the Photosystem II reaction center are post-translationally modified, after they have been inserted in the thylakoid membrane. Key words: Chlamydomonas/protein function/protein synthesis/thylakoid membrane/Photosystem II Introduction In plant cells, the chloroplast is a semi-autonomous organelle whose development is dependent upon both the nucleus and the chloroplast itself (Hoober, 1976). The chloroplast contains supercoiled circular DNA and its own transcription and translation apparatus, with bacterial-like ribosomes (Herrmann and Possingham, 1980; Wollgiehn and Parthier, 1980). Chloroplast proteins are coded for either by the chloroplast DNA or the nuclear DNA and synthesized either on chloroplast (70S) ribosomes or cytoplasmic (80S) ribosomes; those chloroplast proteins synthesized in the cytoplasm are subsequently transported into the chloroplast. The thylakoid membranes of the chloroplast carry out the electron transfer reactions from H20 to NADP + catalyzed by light energy. They contain several complexes which can be extracted by detergent treatment in a functional state: a Photosystem I (PSI) reaction center complex (Mullet et al., 1980), a PSI light-harvesting complex (LHC, Mullet et al., 1981), a Photosystem II (PSII) reaction center complex (Diner and Wollman, 1980), a PSII LHC (Burke et al., 1978), a cytochrome b-f complex (Hurt and Hauska, 1981), analogous to the b-c1 mitochondrial complex and an ATPase complex containing both CFo and CF1 (Pick and Racker, 1979). Each of these complexes contains several proteins whose function and origin is known in several instances from direct biochemical studies, the use of mutants defective in one of the photosynthetic functions (Bennoun et al., 1981), antibiotics studies (Chua and Gillham, 1977) and, more recently, the isolation of structural genes coding for individual proteins (Herrmann et al., 1983). For some of these proteins the events occurring during their biosynthesis and integration are partly characterized (Grebanier et al., 1978). © IRL Press Limited, Oxford, England.
Since, in Chlamydomonas reinhardtii, many proteins have been functionally characterized, it is a particularly suitable organism in which to study the biosynthesis and integration of membrane proteins into functional complexes. We have chosen the PSII reaction center complex as a model system, since it is easy to purify and there are mutants deficient in PSII activity. Results We have used inhibitors, following the approach developed by Chua and Gillham (1977), to study thylakoid membrane polypeptides: whole cells are pulse-labeled with [14C]acetate either with no inhibitor or in the presence of chloramphenicol (CAP, Conde et al., 1975), an inhibitor of protein synthesis on 70S ribosomes, or anisomycin (ANI, Lizardi and Luck, 1972), an inhibitor of protein synthesis on 80S ribosomes. The pitfalls of this method have been reviewed by Gillham (1978), the main one being secondary effects due to long-term action of the inhibitors. In our case, since there is a perfect complementarity between the three samples for all but one of the proteins (see Figure 1: 0 sample = ANI sample + CAP sample), the sites of synthesis which we have determined for the thylakoid membrane polypeptides should be correct. This first approach was substantiated using a nuclear mutant, F34, devoid of PSII reaction centers. Whenever a protein, not detected by the stain and missing in the pulse-label of the mutant in the presence of CAP, is present in the wild-type (WT) pulse-label, it is of cytoplasmic origin; whenever a protein, missing in the pulse-label of the mutant in the presence of ANI, is present in the WT pulse-label, it is of chloroplast
origin. We have also isolated active PSII particles from the WT strain after pulse-labeling either with no inhibitor or with CAP or ANI. This approach is complementary to, and less ambiguous than, that using mutants and it has been used to study the PSI reaction center and CF1-CFO complex (Nechustai and Nelson, 1981). Finally, we have carried out a pulse-chase experiment to study the integration of several of these proteins into the thylakoid membrane. Figure 1 shows the analysis of the thylakoid membrane polypeptides of the wild-type strain of C. reinhardtii on a 12- 18O%o polyacrylamide gradient gel containing 8 M urea. The stained gel (lane 1) displays 40 prominent bands numbered from 1 to 37 in order of decreasing apparent mol. wt. We used a published nomenclature (Piccioni et al., 1981), which, for bands 1-24, arises from a comparison of the patterns observed on a urea gel and on a 7.5- 150/o polyacrylamide gradient gel (Delepelaire, 1983). Some of these bands arise from co-migration of several polypeptides (Piccioni et al., 1982), shown by differential solubility in organic solvents or in 6 M guanidine-HCl. Lanes 2, 3 and 4 show the autoradiograms of the corresponding gel for WT cells pulselabeled either with no inhibitor (lane 2), with CAP (lane 3) or with ANI (lane 4). Lane 4 also presents the nomenclature we -
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7.5 - 15 o polyacrylamide gradient gel without urea in the second). The following polypeptides made inside the chloroplast are either missing or partly deficient in F34: polypeptides 5 and 6 (respectively the apoproteins of the chlorophyllprotein complexes III and IV), D2.1, L5, L6, L7 and L8. It should be noted that, relative to the other bands, the label of L3 is greater in the F34 mutant than in WT. Table I summarizes the results obtained with F34. Because of possible pleiotropic effects of the mutation (although difficult to distinguish since many independent mutants give rise to the same phenotype), we have used isolated active PSII particles which, a priori, should give a clearer picture of the sites of synthesis of their constituent polypeptides, provided they are not contaminated by other membrane fractions. These particles were isolated from the WT strain of C.
reinhardtii following a protocol primarily designed to isolate PSI particles (Mullet et al., 1980). It occurred to us that, after Triton X-100 solubilization of the membranes and sucrose gradient centrifugation, a band on the gradient corresponded to PSII particles. These particles are quite similar to the PSII particles isolated from a double mutant of C. reinhardtii devoid of both ATPase and PSI reaction centers (Diner and Wollman, 1980). The main difference is that they do not contain polypeptides 12, 19, and 24 (respectively 30, 20 and 17 kd) which are the equivalent of the polypeptides seen by others on the donor side of PSII (Murata et al., 1983). They also contain somewhat more antenna chlorophyll associated with the LHC polypeptides (in the region 25-30 kd). The sites of synthesis of all these polypeptides have been determined by pulse-labeling experiments which are shown in lanes 2, 3 and 4 of Figure 4. The proteins synthesized in the cytoplasm (lane 3) are mainly those of the LHC and some of the low mol. wt. polypeptides. The following proteins, synthesized in the chloroplast, are constituents of the PSII particle: polypeptides 5, 6, 6', D2.1, D2.2, Dl, L4, L5, L6, L7 and L8. Except for L4, the stoichiometry of these labeled bands is roughly the same as in the whole membrane. In our opinion this is fairly good evidence that there is not an artifactual association of one of these proteins with the PSII particle. On the basis of their extractibility from the thylakoid membrane by 6 M guanidine-HCI (Matlin, 1979), the following PSII proteins are considered to be intrinsic membrane proteins (i.e., tightly bound to the lipid bilayer): 5, 6, 6', DI, D2.1, D2.2, L5, L6, L7 and L8. Most of these proteins present a fragment exposed to the outside of the thylakoid membrane, as they are partly digested with trypsin (Delepelaire, 1983). It is now known that DI is the equivalent of the socalled '32 kd protein' of higher plants and that it is able to bind herbicides (Erickson et al., 1984). It should be noted that, except for 34 (and in our preparation 12, 19 and 24), there is an almost perfect complementarity between the polypeptides co-purifying with the PSII particle and those 703
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missing in the PSII mutant, strengthening the idea that the PSII mutant lacks one membrane complex. To provide information on the biosynthetic pathway followed by these proteins, we carried out a pulse-chase experiment. The pulse, in the presence of ANI, was for 5 min; it was followed by a chase of 1 h in the presence of both ANI and CAP. Under these conditions there is no protein synthesis at all during the chase period, since both 80S and 70S ribosomes are blocked by the inhibitors. During the pulse period there is protein synthesis in the chloroplast (and the mitochondria) but not in the cytoplasm. Both purified thylakoid membranes and whole cell proteins were analyzed on a 12-18% polyacrylamide gradient gel containing 8 M urea.
Figure 5 shows the autoradiograms of the thylakoid membrane polypeptides from cells pulsed for 5 min (lane 1) and then chased for 30 and 60 min (lanes 2 and 3). Several modifi704
Fig. 5. Analysis of thylakoid membrane polypeptides and of whole cell proteins from C. reinhardtii cells pulse-labeled in the presence of ANI for 5 min and then chased for 30 and 60 min in the presence of ANI plus CAP. The analysis was by a 12-18% polyacrylamide gradient gel containing 8 M urea. Lanes I-3: autoradiograms of the thylakoid membrane polypeptides from cells labeled 5 min (lane 1) and then chased 30 and 60 min (respectively, lanes 2 and 3). Lanes 4-6: autoradiograms of whole cell proteins from cells labeled 5 min (lane 4) and then chased for 30 and 60 min (respectively, lanes 5 and 6). LS corresponds to the large subunit of RubP carboxylase.
cations appear in the labeling pattern as the chase proceeds. After 5 min of pulse (lane 1) there is no label in the bands 6', D2. 1, L5 and L6 and there is a relatively more intense labeling in bands L3 and D2.2. All the other bands detected after a 45 min pulse are already present (see Figure 1, lane 4). As the chase proceeds, (lanes 2 and 3) the band 6' appears as well as D2. 1, L5 and L6. During the same period, the relative label in D2.2 decreases and L3 completely disappears. During the chase, the shape of D1 changes, becoming slightly less diffuse. It should be noted that during the chase there is no net change of the overall radioactivity incorporated during the pulse. A priori, there were three possible explanations for these observations. (i) There is still some protein synthesis occurring during the chase: although very hard to rule out completely, this seems very unlikely since the chase was in the presence of saturating concentrations of both CAP and ANI. Besides, it does not account for the disappearance of L3 and
Thylakoid protein synthesis and function in C. reinhardtii
the relative decrease of D2.2 during the chase. Finally, as mentioned earlier, there is no overall increase of the label during the chase period. This implies that pools of precursors, if they exist, should be very small. (ii) The polypeptides which appear during the chase period in the thylakoid membrane fraction originate from another compartment of the chloroplast (stroma or envelope) and there is a lag for their integration in the membrane. (iii) The modifications seen during the chase occur in situ, once the polypeptides have been inserted into the thylakoid membrane. To choose between these possibilities, we carried out the same analysis on total cell proteins, reasoning that any protein whose biosynthesis takes place in another compartment before its integration in the thylakoid membrane should be found at the beginning of the chase in whole cells. This analysis was made possible by the relatively high percentage of the thylakoid membrane in the total cell proteins. The analysis of total cell proteins for the pulse-chase experiment is shown in Figure 5, lanes 4, 5 and 6 (respectively 5 min pulse, 30 and 60 min chase). With the main exception of the large subunit of the RubP carboxylase (LS), and a few other minor bands (either stromal proteins or mitochondrial proteins), the pattern of radioactivity is exactly the same for the thylakoid membrane fraction and the total cell proteins (compare lanes 1 and 4, 2 and 5, 3 and 6). This is good evidence that all the modifications we see during the chase period occur directly on the thylakoid membrane, as a result of posttranslational events, once the polypeptide has been inserted into the thylakoid membrane. Additional results were also obtained on those thylakoid membrane polypeptides synthesized inside the chloroplast: (i) pulse-labeling of whole cells in the presence of [32P]orthophosphate shows that, in addition to the LHC polypeptides, polypeptides 6', D2.1, L5 and L6 are phosphorylated, whereas polypeptides 6, D2.2 and L3 are not (Delepelaire and Wollman, unpublished); (ii) Cleveland digests of D2.1 and D2.2 with papain (over a range of concentrations from 0.001 to 0.1 jig) give rise to identical patterns (results not shown); (iii) the appearance of D2.1 is at least partly dependent upon the redox state of the plastoquinone pool which functions as an intermediate electron carrier between PSII and PSI: the appearance of D2.1 is favoured when the plastoquinone pool is oxidized (not shown); (iv) digestion of purified thylakoid membrane with trypsin shows that D2. 1, D2.2 as well as DI are exposed to the outside of the thylakoid membrane (Delepelaire, unpublished observation).
Discussion Here we have shown that the use of a photosynthesis mutant and photoactive PSII particles coupled with pulse-labeling studies allows a tentative identification of the function of the thylakoid membrane polypeptides synthesized inside the chloroplast. The pulse-chase study has also provided information on the biosynthetic pathway of some of these proteins. The PSII reaction center is mainly made of integral membrane proteins synthesized inside the chloroplast: polypeptides 5, 6, 6', D2.1, D2.2, DI, L3, L5, L6, L7 and L8. Among these bands, 5, 6 and L8 clearly correspond to stained polypeptides. A close examination of the 2-dimensional gels also shows that D2.1 and D2.2 correspond to stained polypeptides. Although they are quite heavily labeled, Dl, L5, L6 and L7 do not correspond to stained polypeptides: either they
do not bind the stain or they turn over rapidly. Our results indicate that, in the 32-kd range in Chlamydomonas, there are three distinct polypeptides synthesized inside the chloroplast, two of them, D2.1 and D2.2, correspond to stained bands, the other, DI, to a non-stained band. This region includes the polypeptides responsible for the herbicide binding (Edelman and Reisfeld, 1980; Grebanier et al., 1978; Steinback et al., 1981; Pfister et al., 1981; Matoo et al., 1981; Arntzen et al., 1982). The precursor-product relationship between D2.2 and D2.1 is substantiated by both the concomitant increase of D2.1 and decrease of D2.2, and the identity of the Cleveland digest patterns for both proteins. This post-translational modification occurs only after D2.2 has been inserted into the thylakoid membrane. It is not a glycosylation of the protein (tunicamycin has no effect on it) but a phosphorylation. The reason for this phosphorylation is unknown but, since it does not exist in the F34 mutant lacking PSII reaction centers (see Figures 2 and 3), it could be involved in the integration of the PSII center into the thylakoid membrane. Both D2.1 and D2.2 correspond to intrinsic membrane polypeptides and are found in PSII particles. Since there is no change between the 30 min and 60 min chase points, we propose that only a part of D2.2 is converted to D2.1 under our conditions, or that there is an equilibrium between D2.2 and D2.1. Polypeptide 6' might be a product of 6. The best evidence for this is the absence of both 6 and 6' in the PSII mutant. Both 6 and 6' correspond to intrinsic membrane polypeptides; 6' is phosphorylated whereas 6 is not. Again only a part of 6 would be modified. Concerning L3, L5 and L6, it is tempting to propose that L3 is a precursor of L5 and L6. There are two observations favouring this: (i) the kinetics of disappearance of L3 and of appearance of L5 and L6 are parallel; (ii) in the PSII mutant in which L5 and L6 are missing there is a relative accumulation of L3 as compared with the other proteins (Figure 2, lane 4). We should mention that whereas L3 is an extrinsic membrane, L5 and L6 are intrinsic. In any case, even though the precursor-product relationship is not directly established in the above experiments, unique transitory proteins or the successive appearance of proteins (Miller and Price, 1982) cannot explain our results since these modifications occur in the absence of protein synthesis and the experiments are done with stationary cultures. It is worth mentioning that all these modifications concern PSII-associated proteins and correspond to phosphorylations. The PSII mutant we have used was of nuclear origin, and although it does not synthesize and/or integrate most of the polypeptides of the PSII reaction center, polypeptides 5, D2.2, Dl, L3, L7 and L8 are still labeled during a pulse in this mutant. This means there are several steps which control the synthesis and assembly of such a complex system as the PSII reaction center. Materials and methods Two strains of C. reinhardtii have been used throughout this work: a wildtype strain WT + and a PSII mutant strain, F34, of nuclear origin (Chua and Bennoun, 1975). The strains were grown in Tris-acetate-phosphate medium (TAP) at 25°C under a light intensity of 250 lux. The pulse-labeling was carried out as previously described (Delepelaire, 1983). Pulse-chase: 300 ml of a cell suspension (2 x 106 cells/ml) were spun down at 600 gm. for 10 min and washed once in minimal medium. They were then resuspended at the same cell density and illuminated at 250 lux for 1 h on
705
P.Delepelaire a rotary shaker at 25°C. ANI was then added to a final concentration of 2.5 x 10-4 M and the cells incubated in its presence for 10 min before the beginning of the labeling. The labeling was with [14C]acetate (sp.act. 50 mCi/mmol) and was allowed to proceed for 5 min at a concentration of lzCi/ml. At the end of the labeling period, CAP was added (100 ttg/ml) and the cell suspension (300 ml) was immediately chilled with 300 ml of pre-cooled TAP medium containing both CAP (100ytg/ml) and ANI (2.5 x 10-4 M). This cell suspension was then divided into three aliquots of 200 ml each. One of them was directly processed for the isolation of purified thylakoid membranes. The two others were spun down at 600 gm. for 10 min and the cell pellet resuspended in 100 ml TAP medium containing both CAP (100ytg/ml) and ANI (2.5 x 10-4 M) in order to follow the chase of radioactivity in the absence of protein synthesis. One of these samples was processed for the isolation of the thylakoid membranes after 30 min and the other after 60 min of chase. For each time point (0, 30 and 60 min) an aliquot of the cell suspension was spun down and the cell pellet immediately dissolved in boiling SDS. The isolation of purified thylakoid membranes was carried out according to Chua and Bennoun (1975). PSII particles were prepared according to Girard et al. (1980). It appeared that during this preparation a band on the sucrose gradient consisted of PSII particles quite similar to the PSII particles isolated by Diner and Wollman (1980); (Delepelaire, unpublished observation). Gel electrophoresis was carried out as previously described (Delepelaire, 1983).
Acknowledgements The author wishes to express his thanks to Dr. Pierre Bennoun for helpful comments and the use of the mutant strain F34, and to Dr. Francis-Andre Wollman for fruitful discussions. This work was supported by an EEC grant, no. ESD-017-F.
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Acad. Sci. USA, 78, 1572-1576. Miller,M.E. and Price,C.A. (1982) FEBS Lett., 147, 156-160. Mullet,J.E., Burke,J.J. and Arntzen,C.J. (1980) Plant Physiol., 65, 814-822. Mullet,J.E., Leto,K. and Arntzen,C.J. (1981) in Akoyunoglou,G. (ed.), Proceedings of the Fifth International Congress on Photosynthesis, Vol. 5, Balaban International Services, Philadelphia, pp. 557-568. Murata,N., Miyao,M. and Kuwabata,T. (1983) in Sixth International Congress on Photosynthesis, abstract 411-19. Nechustai,R. and Nelson,N. (1981) J. Biol. Chem., 256, 11624-11628. Piccioni,R.G., Bennoun,P. and Chua,N.H. (1981) Eur. J. Biochem., 117, 93-102. Piccioni,R.G., Bellemare,G. and Chua,N.H. (1982) in Edelman,M. et al. (eds.), Methods in Chloroplast Molecular Biology, Elsevier Biomedical, Amsterdam, pp. 985-1014. Pick,U. and Racker,E. (1979) J. Biol. Chem., 254, 2793-2799. Pfister,K., Steinback,K.E., Gardner,G. and Arntzen,C.J. (1981) Proc. Nat!. Acad. Sci. USA, 78, 981-985. Steinback,K.E., Mclntosh,L., Bogorad,L. and Arntzen,C.J. (1981) Proc. Natl. Acad. Sci. USA, 78, 7463-7467. Wollgiehn,R. and Parthier,B. (1980) in Reinert,J. (ed.), Chloroplasts, Springer Verlag, Berlin, pp. 97-145. Received on 24 August 1983; revised on 20 January 1984
