THE JOURNAL OF BIOLOGICAL CHEMWIW

Vol. 251, No. 9, Issue of May 10, pp. 2848-2853, 1976

Printed in U.S.A.

Protein Synthesis in Plant Leaf Tissue

THE SITES OF SYNTHESIS OF THE MAJOR PROTEINS

(Received for publication, March 24, 1975)

ANTHONY R. CASHMORE
From the Applied Biochemistry Division, D.S.I.R., Palmerston North, New Zealand

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

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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.

One of the first requirements in studies concerned with the cytoplasmic ribosomes in the synthesis of chloroplast proteins
biosynthesis of plant proteins is to determine their respective has been confirmed. In addition, it is shown that cytoplasmic
sites of synthesis. A popular approach to this problem has been protein synthesis is also required for the integration into the
through the use of inhibitors like cycloheximide and chloram- chloroplast lamellae of at least one of the chloroplast-synthe-
phenicol (l-5), which selectively inhibit synthesis either on the sized proteins.
cytoplasmic ribosomes, or on the prokaryotic-like ribosomes
EXPERIMENTAL PROCEDURE
found within the plant organelles. Such studies have shown, for
Chlamydomonas reinhardi (I, 2) and Vicia faba (3), that MateriaZs_Pea (Pisum satiuum var. Massey) and spinach
(5pinacia oleracea, hybrid 102) seeds were obtained from Yates.
ribosomes both in the cytoplasm and the chloroplast are Radiochemicals were obtained from The Radiochemical Centre, Amer-
required for the synthesis of chloroplast lamellar proteins. A sham/Searle. Tris (Sigma), glycine (Ajax Chemicals Ltd., Sydney),
similar sharing of function seems to exist for the synthesis of and acrylamide (Eastman) solutions were all filtered through a 0.8-p
ribulose-1,5-diphosphate carboxylase. Isolated pea chloro- Millipore filter prior to use in gel electrophoresis. Phenylmethylsul-
fonylfluoride and Coomassie brilliant blue R were obtained from
plasts have been shown to synthesize the large subunit of this
Sigma, sodium diethyldithiocarbamate and sodium dodecyl sulfate
protein (6) and inhibitor studies with barley (7) show the from BDH Chemicals Ltd., Nonidet P-40 from Shell and Miracloth
cytoplasm to be the likely site of synthesis for the small from Calbiochem. Kodak royal blue and Kodirex films were used for
subunit. Recently, the partial in oitro synthesis of the small autoradiography, and development was with Kodak liquid X-ray devel-
subunit on polysomes derived from the cytoplasm of Phaseolus oper (type 2) and Kodak liquid fixer.
Labeling and Extraction of Pea Seedlings-Pea seedlings, grown in
uulgaris has been reported (8). Thus, at least in the case of pumice at 20 with a 12.hour light period, were harvested 9 days after
higher plants, the synthesis of the two subunits of ribulose-1,5- planting by slicing the epicotyl approximately 2.5 cm below the leaves.
diphosphate carboxylase seems to occur at distinct sites, They were labeled by standing the excised seedling in a small volume
although the inhibitor studies concerned with the site of of amino acid mixture containing [SsS]methionine. During labeling,
the seedlings were irradiated with a 250.watt projector lamp, focused,
synthesis of the small subunit are far from unequivocal. For the
and cooled through a spherical flask containing water. After labeling,
green algae, the situation is not clear, the possibility existing the total leaf tissue, including the meristematic tissue, was homoge-
that both subunits are synthesized within the chloroplast (1, nized in a conical glass tissue grinder (Kontes) with approximately 10
9-12). volumes of extraction buffer (10 rn~ Tris; 10 rnM glycine; 1 rn~
As a prelude to studies concerning the regulation of protein phenylmethylsulfonylfluoride, and 1% 2.mercaptoethanol, pH 8.9).
After centrifugation through Miracloth, the filtrate was fractionated
synthesis in the leaf tissue of pea seedlings, it was of interest to
into soluble and particulate components by centrifugation for 30 min
characterize the major proteins being synthesized and their at 27,500 x g. The supernatant was treated with x0 volume of 10%
sites of synthesis. In the course of these studies, the role of sodium dodecyl sulfate and immediately incubated for 2 min at 100.

2848
 Protein Synthesis in Plant Leaf Tissue
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.
Sodium Dodecyl Sulfate-Acrylamide Gel Electrophoresis-The slab
gel apparatus described by Reid and Bieleski (13) was used. Acrylam-
ide gels (15% acrylamide, 0.085% bisacrylamide; 14 x 11 x 0.15 cm)
were prepared in 0.1 M Tris, 0.1 M glycine, and 0.1% sodium dodecyl
sulfate (pH 8.9). The gels were pre-electrophoresed for 1 hour at 150
volts using an electrode buffer of 0.1 M Tris, 0.1 M glycine, and 0.1%
sodium dodecyl sulfate (pH 8.9). The samples (5 to 20 ~1; previously
made 10% in glycerol) were loaded (15 min, 20 volts) and then electro-
phoresis was for 3 hours at 150 volts. After staining (3 hours; 0.25%
Coomassie blue in acetic acid/methanol/water, l/5/5), gels were de-
stained by shaking for several days in acetic acid/methanol/water,
i
l/4/15. The gels were dehydrated onto Whatman 3MM paper accord- 9
ing to the procedure described by Maize1 (14), and autoradiographs
were prepared.
Preparation of Chloroplast Lamellar Proteins-Fifteen grams of
tissue (12-day pea leaves or the young leaves from 5-week glass-house-
grown 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

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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) I I I I
I
2 4 6 8 10
was recovered from the gradient and treated with sodium dodecyl MOBILITY (cm)
sulfate prior to electrophoresis.
Purification of Fraction I Protein-Leaves from a-week pea seedlings FIG. 1. Sodium dodecyl sulfate-acrylamide gel electrophoresis of
were extracted in a pressure cell (15) with 3 volumes of 50 mM soluble and particulate proteins from pea seedling leaves. The molecu-
phosphate buffer (pH 7.5) containing 5 rnM 2.mercaptoethanol and 10 lar weights of the major soluble (a) and particulate (b) proteins are
mM sodium diethyldithiocarbamate. A 33 to 50% saturated ammonium indicated in kilodaltons. They have been calculated by co-electro-
sulfate cut was obtained, and this was fractionated by sequential gel phoresis with the following standards of indicated molecular weights:
filtration on Sepharose 6B and Bio-Gel A-(0.5m) in 25 mM Tris-H,SO, bovine serum albumin (BSA, 68,000); pyruvate kinase (PyK, 57,000);
(pH 7.5), 5 mM 2-mercaptoethanol and 5 rnM EDTA. Pure Fraction I lactate dehydrogenase (LDH, 36,000); carbonic anhydrase (CA,
protein (AZBO:AzBO = 1.90) was obtained as indicated by a single band 29,000); lysozyme (L, 14,300); and cytochrome c (C, 12,400). The
on gel electrophoresis in the absence of sodium dodecyl sulfate, and diagram on the left illustrates that in this gel system, a linear
two bands (molecular weights equal to 14,000 and 53,000) on gel relationship is obtained between log (molecular weight) and mobility.
electrophoresis in the presence of sodium dodecyl sulfate. The samples for electrophoresis contained 58 /lg of soluble protein and
Preparation of Antiserum and Immunoprecipitation-Fraction I 40 fig of particulate protein.
protein (3.6 mg in 0.5 ml of the above Tris-H,SO, buffer) was
emulsified with an equal volume of Freunds complete adjuvant and standing in water prior to the 2-hour labeling with [35S]methio-
injected intramuscularly into a New Zealand white rabbit. The
injections were repeated twice, without the adjuvant, at 2.weekly nine.
intervals, and after a further 2 weeks, the serum was prepared, It was of interest to characterize the major peptides depicted
fractionated by 40% saturated ammonium sulfate precipitation, and in Fig. 2. The,large and small subunits of Fraction I protein (17,
assayed by standard procedures. An excess of antiserum was added to 18) (ribulose-1,5-diphosphate carboxylase) were expected to
an [SSS]methionine-labeled, unfractionated, seedling extract (in 0.1 M
KCl, 50 rnM Tris-HCl (pH 8.0), 0.5% deoxycholate, and 0.5% Nonidet form major components of the soluble proteins, and this was
P-40). The mixture was incubated for 30 min at 30 and then 20 hours confirmed by comparison with purified labeled Fraction I
at 4. The precipitate was collected, washed three times in the protein prepared by immunoprecipitation (Fig. 2). The inter-
detergent-containing buffer, and then dissolved in sodium dodecyl nal lamellar structure of the chloroplast contains a significant
sulfate-containing.buffer for gel electrophoresis.
portion of plant protein, and consequently, it seemed likely
that many of the particulate proteins would be derived from
RESULTS AND DISCUSSION the chloroplast lamellae. This comparison is made in Fig. 2, c
Characterization of the Major Proteins-A fractionation by and d. Quite clearly, the majority of the particulate proteins
sodium dodecyl sulfate-acrylamide gel electrophoresis of the are seen to correspond to chloroplast lamellar proteins. Not
peptides derived from the total proteins present in the leaves of surprisingly then, the predominant protein synthesis in the
g-day light-grown pea seedlings is shown in Fig. 1. By leaves of young light-grown pea seedlings is associated with the
co-electrophoresis with appropriate standards (16), the molec- development of the stromal and lamellar systems of the
ular weights of the major peptides have been calculated. chloroplast.
In order to determine which proteins were being actively An attempt was made to characterize further the lamellar
synthesized, excised pea seedlings were light-irradiated for 2 proteins PI and PI1 depicted in the autoradiograph of Fig. 2. In
hours while being labeled with [YS]methionine. The proteins Fig. 3 is shown a stained sodium dodecyl sulfate-acrylamide gel
were again fractionated by electrophoresis, and in Fig. 2, the fractionation of the proteins derived from both pea and spinach
autoradiograph shows which proteins are being synthesized in lamellae, and by comparative electrophoresis, the labeled PI
the leaves of g-day pea seedlings. An essentially identical and PI1 proteins have been shown to correspond to the major
profile is obtained if the labeling is done with a C-amino-acid lamellar proteins in this diagram. An unusual feature of some
mix (Amersham CFB.25), and similarly, an identical profile is chloroplast lamellar proteins is that they run as undissociated
obtained if the seedling is light irradiated for 2 hours while protein-chlorophyll complexes on electrophoresis in sodium
2850 Protein Synthesis in Plant Leaf Tissue

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FIG. 2 (left). Sodium dodecyl sulfate-acrylamide gel electrophore- ing to the method described under Experimental Procedure. The
sis of [3sS]methionine-labeled proteins from pea seedling leaves. green chloroplast lamellar bands (250 ~1; 1.16 g/ml) were recovered
Seedlings were labeled by standing in a 60-J solution of amino acids from the gradient and made 1% in sodium dodecyl sulfate and
including [%]methionine (33.9 Ci/mmol, 25 pM; 19 other amino acids, 2-mercaptoethanol. One-half of each sample was kept on ice and the
166 FM). For the preparation of lamellar proteins, 0.5 ml of leaf extract rest incubated for 2 min at 100. Twenty-microliter samples were
was layered directly onto a linear sucrose gradient and sedimented as fractionated by acrylamide gel electrophoresis as described under
described under Experimental Procedure. The green lamellar band Experimental Procedure except that electrophoresis was for 6 hours
was recovered (200 ~1) and treated with sodium dodecyl sulfate. The at 4. After electrophoresis, the chlorophyll bands were marked (PSI,
autoradiograph shows a, Fraction I immunoprecipitate (2,704 cpm); b, PSIIa, PSII, and Chl) prior to staining in Coomassie blue. a, pea
soluble proteins (53,825 cpm); c, particulate proteins (50,466 cpm) and lamellar proteins, not heated in sodium dodecyl sulfate; b, pea
d, chloroplast lamellar proteins (72,350 cpm). lamellar proteins, heated in sodium dodecyl sulfate; c, spinach
FIG. 3 (right). Sodium dodecyl sulfate-acrylamide gel electropho- lamellar proteins, not heated in sodium dodecyl sulfate; d, spinach
resis of lamellar proteins from pea and spinach chloroplasts. Chloro- lamellar proteins, heated in sodium dodecyl sulfate.
plast lamellae were purified by sedimentation through sucrose accord-

dodecyl sulfate (3). In the present investigation, four chloro- the pea chloroplast contains two major proteins, PI and PII, of
phyll-containing bands (PSI, PSIIa, PSII, and Chl) were respective molecular weights 58 and 29 kilodaltons (obtained
observed if lamellar preparations were fractionated by electro- according to Fig. 1). PI1 appears to be strongly associated with
phoresis at 4 and the prior dissociation in hot sodium dodecyl chlorophyll within the chloroplast, and from the work of others
sulfate was omitted. However, if the lamellar preparations (19-22), it forms the major protein of photosystem II. In
were heated at 100 prior to electrophoresis, which was contrast, the author finds no evidence that PI forms a
standard procedure throughout this investigation, then only protein.chlorophyll complex, although it does appear to form
one chlorophyll band was found (presumably free chloro- the major photosystem I protein (19-22). The nature of the
phylls). In the heated sample, in contrast to previous claims protein moieties associated with the complexes PSI and PSIIa,
(19), no protein band was found with electrophoretic properties of apparent molecular weights 96 and 70 kilodaltons, has not
similar to the photosystem I protein-chlorophyll complex been clarified.
(PSI). When the lamellae are labeled with [YS]methionine and Sites of Synthesis of Major Proteins-The sites of protein
treated according to the procedure in Fig. 3, then, by autoradi- synthesis have been examined with the aid of the inhibitors
ography, the complexes PSI, PSIIa, and PSI1 are shown to be cycloheximide and chloramphenicol. In Table I, the effect of
labeled, indicating that they are indeed protein complexes. In these inhibitors on the incorporation of [35S]methionine is
the case of the PSI1 protein .chlorophyll complex, it seems shown. Cycloheximide is seen to decrease total protein synthe-
likely that dissociation here gives rise to protein PII, of sis significantly, and this effect is most pronounced in the case
molecular weight 29 kilodaltons (19-22). Often two proteins are of the soluble proteins. The effect of cycloheximide and
observed in this region (29 and 31 kilodaltons). It has been chloramphenicol on the synthesis of specific proteins is illus-
shown that PSI contains mainly chlorophyll a, whereas PSI1 trated by the autoradiographs in Figs. 4 and 5. It is observed
contains both chlorophyll a and b (3). The nature of the that the synthesis of the large subunit of Fraction I protein is
chlorophyll associated with the minor PSIIa complex has not unique among the major soluble proteins in that it is sensitive
been determined, although from the color of the band, both to chloramphenicol and relatively insensitive to cycloheximide.
before and after staining with Coomassie blue, it is similar to The small subunit of Fraction I protein, along with all of the
PSI1 and distinct from PSI. other major soluble proteins, is synthesized on ribosomes
From Fig. 3 then, it is concluded that the lamellar system of sensitive to cycloheximide. These observations are consistent
 Protein Synthesis in Plant Leaf Tissue 2851

TABLE I
Effect of chloramphenicol and cycloherimide on protein synthesis in
pea seedling leaves
Pea seedlings were light irradiated for 15 min while standing in
water (50 Al), chloramphenicol (200 fig/ml), or cycloheximide (20
&ml). To each sample were then added 10 pl of [%]methionine (34.2
Ci/mmol, 0.15 mM; 19 other amino acids, 1.0 mht) and the irradiation
continued for a further 2 hours. The leaves were then homogenized in 1
ml of extraction buffer, fractionated into soluble and particulate
components, and treated with sodium dodecyl sulfate as described
under Experimental Procedure. Protein synthesis was determined
by trichloroacetic acid precipitation, collection of precipitates on
Whatman GFC filters, and liquid scintillation counting.
[3?3]Methionine
incorporated/ 9-c 5%
seedling distribution of control
(cpm x 10-Y

Soluble ;;tE- Soluble +

Particulate

Control 16.02 9.25 63.4 36.6

Cycloheximide 3.37 5.62 37.5 62.5 35.6
Chloramphenicol 14.11 7.53 65.2 34.8 85.6 I

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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 cyto-
plasm of bean leaves (P. vulgar-is) (8). It is noted that whereas
both inhibitors used show some selectivity, this is not com-
plete, particularly in the case of cycloheximide. This presum-
ably 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 chlor-
amphenicol 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 synthe-
sis 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
FIG. 4. Effect of chloramphenicol (CAM) and cycloheximide (CH)
Lamellar Assembly-A further interesting piece of information on the synthesis of specific proteins in pea seedling leaves. [s5S]-
is depicted in Fig. 4. This concerns protein PI, which is quite Methionine-labeled proteins were prepared according to Table I. The
clearly a chloroplast lamellar protein (Fig. 2), and conse- samples (10 ~1) were fractionated by electrophoresis, an autoradio-
quently, in Fig. 4, it occurs in the particulate fraction. graph was prepared, and this was scanned with a densitometer. a,
However, in the presence of cycloheximide, an inhibitor which control, soluble proteins (112,622 cpm); b, chloramphenicol, soluble
proteins (73,622 cpm); c, cycloheximide, soluble proteins (27,438 cpm);
does not appear to influence its synthesis, approximately 50% d, control, particulate proteins (101,358 cpm); e, chloramphenicol,
of what appears to be PI is found in the soluble fraction. particulate proteins (60,120 cpm); f, cycloheximide, particulate pro-
Confirmation of the relationship between this soluble protein teins (81,808 cpm).
 2852 Protein Synthesis in Plant Leaf Tissue

25%

CAM

7%
1_-1___

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FIG. 5. Effect of chloramphenicol
sis of chloroplast
[Slmethionine
lamellar
according
proteins were extracted
proteins.
and cycloheximide on the synthe-
Pea seedlings were labeled with
to the procedure
with 1 ml of extraction
described in Table I. Leaf
buffer per seedling and
:
0.5 ml of filtered extract was layered directly onto a linear sucrose FIG. 6. Immunoprecipitation of Fraction I protein synthesized in
gradient. After centrifugation, the lamellar band was recovered, the presence of chloramphenicol (CAM) or- cycloheximide (CH).
treated with sodium dodecyl sulfate, and fractionated by electrophore- IYSlMethionine-labeled proteins were prepared according to Table I.
sis. The autoradiograph shows proteins synthesized in a, control To &l-~l aliquots of the soluble fractions were added 500 il of Fraction
(28,876 cpm); b, chloramphenicol (21,448 cpm); and c, cycloheximide I antiserum. The immunoprecipitates were prepared as described
(17,986 cpm). under Experimental Procedure, solubilized in 200 ~1 of sodium
dodecyl sulfate-containing buffer, and then fractionated by sodium
dodecyl sulfate-acrylamide gel electrophoresis. The percentage figures
and PI protein has been attained by comparative isoelectric shown on the densitometer tracings of the autoradiograph refer to the
amount of [*%]methionine present in the immunoprecipitates as a
focusing. It appears that a continuous supply of a cytoplasmi- percentage of the control. In the control sample a, an aliquot of 5 ~1 was
tally synthesized component is required for the incorporation used for electrophoresis, whereas in b (chloramphenicol), and c
of PI into the chloroplast lamellae. A possible identity of such d (cvcloheximide), 20-pl aliquots were used.
component is PI1 protein, as it represents the major lamellar
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Protein synthesis in plant leaf tissue. The sites of synthesis of the major proteins.
A R Cashmore
J. Biol. Chem. 1976, 251:2848-2853.

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