Protein Synthesis in Plant Leaf Tissue THE SITES OF SYNTHESIS OF THE MAJOR PROTEINS

Protein synthesis in the leaves of green pea seedlings (Pisum satiuum) is examined by short term labeling with [35S]methionine and autoradiography of the labeled proteins after fractionation by sodium dodecyl sulfate-acrylamide gel electrophoresis. The two subunits of ribulose-1,5-diphosphate carboxylase and the chloroplast lamellar proteins are identified as the major proteins being synthesized. Three protein.chlorophyll complexes are characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis; all three complexes are disrupted by heating to 100” in sodium dodecyl sulfate solution. Studies with inhibitors of protein synthesis indicate that the large subunit of ribulose-1,5-diphosphate carboxylase is synthesized in the chloroplast, in contrast to the majority of the soluble proteins, including the small subunit of ribulose-l,&diphosphate carboxylase, which is synthesized in the cytoplasm. PI1 protein, the major lamellar protein associated with photosystem II, is also synthesized on cytoplasmic ribosomes. However, many of the lamellar proteins are synthesized within the chloroplast. Integration into the lamellar system of at least one of the chloroplast-synthesized proteins is shown to be dependent on cytoplasmic protein synthesis.

Integration into the lamellar system of at least one of the chloroplast-synthesized proteins is shown to be dependent on cytoplasmic protein synthesis.
One of the first requirements in studies concerned with the biosynthesis of plant proteins is to determine their respective sites of synthesis.
A popular approach to this problem has been through the use of inhibitors like cycloheximide and chloramphenicol (l-5), which selectively inhibit synthesis either on the cytoplasmic ribosomes, or on the prokaryotic-like ribosomes found within the plant organelles. Such studies have shown, for Chlamydomonas reinhardi (I, 2) and Vicia faba (3) was treated with x0 volume of 10% sodium dodecyl sulfate and immediately incubated for 2 min at 100".
The pellet was resuspended in one-half the original volume of extraction buffer, heated with sodium dodecyl sulfate as above, and the solubilized proteins clarified by centrifugation for 30 min at 27,500 x g.
Preparation of Chloroplast Lamellar Proteins-Fifteen grams of tissue (12-day pea leaves or the young leaves from 5-week glass-housegrown spinach) were homogenized with a Lourdes homogenizer in 50 ml of 0.1 M Tris-HCl (pH 8.0) and 14 rnM 2-mercaptoethanol. After filtration through Miracloth the extract was centrifuged for 15 min at 106,000 x g. The pellets were resuspended in the extraction buffer (2 ml for pea and 1 ml for spinach), homogenized, and 250 ~1 layered onto a linear sucrose gradient (20 to 50% sucrose in 0.1 M Tris-HCl, pH 8.0, and 14 mM P-mercaptoethanol) and sedimented for 1 hour at 35,000 rpm in a Beckman SW 39 rotor. The green lamellar band (1.16 g/ml) was recovered from the gradient and treated with sodium dodecyl sulfate prior to electrophoresis. Purification of Fraction I Protein-Leaves from a-week pea seedlings were extracted in a pressure cell (15) with 3 volumes of 50 mM phosphate buffer (pH 7.5) containing 5 rnM 2.mercaptoethanol and 10 mM sodium diethyldithiocarbamate.
A 33 to 50% saturated ammonium sulfate cut was obtained, and this was fractionated by sequential gel filtration on Sepharose 6B and Bio-Gel A-(0.5m) in 25 mM Tris-H,SO, (pH 7.5), 5 mM 2-mercaptoethanol and 5 rnM EDTA. Pure Fraction I protein (AZBO:AzBO = 1.90) was obtained as indicated by a single band on gel electrophoresis in the absence of sodium dodecyl sulfate, and two bands (molecular weights equal to 14,000 and 53,000) on gel electrophoresis in the presence of sodium dodecyl sulfate. sodium dodecyl sulfate-acrylamide gel electrophoresis of the peptides derived from the total proteins present in the leaves of g-day light-grown pea seedlings is shown in Fig. 1. By co-electrophoresis with appropriate standards (16), the molecular weights of the major peptides have been calculated.
In order to determine which proteins were being actively synthesized, excised pea seedlings were light-irradiated for 2 hours while being labeled with [YS]methionine.
The proteins were again fractionated by electrophoresis, and in Fig. 2, the autoradiograph shows which proteins are being synthesized in the leaves of g-day pea seedlings. An essentially identical profile is obtained if the labeling is done with a 'C-amino-acid mix (Amersham CFB.25), and similarly, an identical profile is obtained if the seedling is light irradiated for 2 hours while standing in water prior to the 2-hour labeling with [35S]methionine. It was of interest to characterize the major peptides depicted in Fig. 2. The,large and small subunits of Fraction I protein (17, 18) (ribulose-1,5-diphosphate carboxylase) were expected to form major components of the soluble proteins, and this was confirmed by comparison with purified labeled Fraction I protein prepared by immunoprecipitation (Fig. 2). The internal lamellar structure of the chloroplast contains a significant portion of plant protein, and consequently, it seemed likely that many of the particulate proteins would be derived from the chloroplast lamellae. This comparison is made in Fig. 2, c and d. Quite clearly, the majority of the particulate proteins are seen to correspond to chloroplast lamellar proteins. Not surprisingly then, the predominant protein synthesis in the leaves of young light-grown pea seedlings is associated with the development of the stromal and lamellar systems of the chloroplast.
An attempt was made to characterize further the lamellar proteins PI and PI1 depicted in the autoradiograph of Fig. 2. In Fig. 3 is shown a stained sodium dodecyl 'sulfate-acrylamide gel fractionation of the proteins derived from both pea and spinach lamellae, and by comparative electrophoresis, the labeled PI and PI1 proteins have been shown to correspond to the major lamellar proteins in this diagram. An unusual feature of some chloroplast lamellar proteins is that they run as undissociated protein-chlorophyll complexes on electrophoresis in sodium observed if lamellar preparations were fractionated by electrophoresis at 4" and the prior dissociation in hot sodium dodecyl sulfate was omitted. However, if the lamellar preparations were heated at 100" prior to electrophoresis, which was standard procedure throughout this investigation, then only one chlorophyll band was found (presumably free chlorophylls). In the heated sample, in contrast to previous claims (19), no protein band was found with electrophoretic properties similar to the photosystem I protein-chlorophyll complex (PSI). When the lamellae are labeled with [YS]methionine and treated according to the procedure in Fig. 3, then, by autoradiography, the complexes PSI, PSIIa, and PSI1 are shown to be labeled, indicating that they are indeed protein complexes. In the case of the PSI1 protein .chlorophyll complex, it seems likely that dissociation here gives rise to protein PII, of molecular weight 29 kilodaltons (19)(20)(21)(22). Often two proteins are observed in this region (29 and 31 kilodaltons).
It has been shown that PSI contains mainly chlorophyll a, whereas PSI1 contains both chlorophyll a and b (3). The nature of the chlorophyll associated with the minor PSIIa complex has not been determined, although from the color of the band, both before and after staining with Coomassie blue, it is similar to PSI1 and distinct from PSI. From Fig. 3  the pea chloroplast contains two major proteins, PI and PII, of respective molecular weights 58 and 29 kilodaltons (obtained according to Fig. 1). PI1 appears to be strongly associated with chlorophyll within the chloroplast, and from the work of others (19)(20)(21)(22), it forms the major protein of photosystem II. In contrast, the author finds no evidence that PI forms a protein.chlorophyll complex, although it does appear to form the major photosystem I protein (19)(20)(21)(22). The nature of the protein moieties associated with the complexes PSI and PSIIa, of apparent molecular weights 96 and 70 kilodaltons, has not been clarified.

Sites of Synthesis of Major
Proteins-The sites of protein synthesis have been examined with the aid of the inhibitors cycloheximide and chloramphenicol. In Table I, the effect of these inhibitors on the incorporation of [35S]methionine is shown. Cycloheximide is seen to decrease total protein synthesis significantly, and this effect is most pronounced in the case of the soluble proteins. The effect of cycloheximide and chloramphenicol on the synthesis of specific proteins is illustrated by the autoradiographs in Figs. 4 and 5. It is observed that the synthesis of the large subunit of Fraction I protein is unique among the major soluble proteins in that it is sensitive to chloramphenicol and relatively insensitive to cycloheximide. The small subunit of Fraction I protein, along with all of the other major soluble proteins, is synthesized on ribosomes sensitive to cycloheximide.
These observations are consistent with the finding that the large subunit of Fraction I protein is synthesized in isolated pea chloroplasts (6), and the small subunit is synthesized on polysomes derived from the cytoplasm of bean leaves (P. vulgar-is) (8). It is noted that whereas both inhibitors used show some selectivity, this is not complete, particularly in the case of cycloheximide. This presumably reflects a rather tight coupling between protein synthesis in the cytoplasm and the chloroplast. These inhibitor studies are extended by the results shown in Fig. 6. Here, Fraction I protein has been purified with the aid of specific antiserum. Clearly, the synthesis of the small subunit of Fraction I protein continues to some extent in the absence of large subunit synthesis. However, in the presence of cycloheximide, where the synthesis of the small subunit is severely restricted, so also is the synthesis of the large subunit, or at least its incorporation into the native molecule. Similar results to those depicted in Fig. 6 have been obtained by purifying Fraction I protein by sucrose gradient centrifugation (1).
The particulate proteins present a very different picture (Figs. 4 and 5), as in this case, the synthesis of many of the proteins is sensitive to chloramphenicol.
In the case of the chloroplast lamellar proteins (Fig. 5), this sensitivity to chloramphenicol is almost certainly due to their synthesis on chloroplast ribosomes. PI1 protein is exceptional in that its synthesis is sensitive to cycloheximide and insensitive to chloramphenicol.
Similar results regarding the sites of synthesis of bean (V. fuba) chloroplast lamellar proteins have been reported (3).
All of the results obtained with chloramphenicol ( Fig. 4) have been duplicated using lincomycin.
Cytoplasmic Protein Synthesis Is Required +or Chloroplast Lamellar Assembly-A further interesting piece of information is depicted in Fig. 4. This concerns protein PI, which is quite clearly a chloroplast lamellar protein (Fig. 2), and consequently, in Fig. 4 Pea seedlings were labeled with ["Slmethionine according to the procedure described in Table I. Leaf proteins were extracted with 1 ml of extraction buffer per seedling and 0.5 ml of filtered extract was layered directly onto a linear sucrose gradient.
After centrifugation, the lamellar band was recovered, treated with sodium dodecyl sulfate, and fractionated by electrophoresis. The autoradiograph shows proteins synthesized in a, control (28,876 cpm); b, chloramphenicol (21,448 cpm); and c, cycloheximide (17,986 cpm). and PI protein has been attained by comparative isoelectric focusing.' It appears that a continuous supply of a cytoplasmitally synthesized component is required for the incorporation of PI into the chloroplast lamellae. A possible identity of such d component is PI1 protein, as it represents the major lamellar protein and its synthesis is sensitive to cycloheximide. Similar results pertaining to PI have been obtained with anisomycin, indicating that the phenomenon is indeed a reflection of the inhibition of cytoplasmic protein synthesis.
In this investigation, it has been shown in pea seedlings that the small subunit of Fraction I protein is apparently synthesized within the cytoplasm and this finding is in agreement with the work of others (7,B). In Nicotians, the small subunit of Fraction I protein is coded by the nuclear genome (23), and this presumably holds for the pea Fraction I subunit. It has also been shown that the cytoplasm is the likely site of synthesis for the major protein of photosystem II, and this protein is also coded for by the nucleus (24). The cytoplasmic origin of the major photosystem II protein is also found in bean (V. fuba) (3) and in an unicellular green alga (C. reinhardii) (25). These conclusions regarding the site of synthesis of the small subunit of Fraction I protein and PI1 protein have recently been confirmed by in vitro studies.z