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THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 247, No. 1, Issue of January 10, pp. 122-129, 1972
Printed in U.S.A.
Biogenesis of Chloroplast Membranes
VIII. MODULATION OF CHLOROPLAST LAMELLAE COMPOSITION AND FUNCTION INDUCED BY
DISCONTINUOUS ILLUMINATION AND INHIBITION OF RIBONUCLEIC ACID AND PROTEIN
SYNTHESIS DURING GREENING OF CHLAMYDOMONAS REINHARDI y-l MUTANT CELLS
(Received for publication, June 21, 1971)
GERA EYTAN AND ITZHAK
OHAD
From the Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel
SUMMARY
Modulation of the synthesis of membrane proteins and
chlorophyll during the light-dependent formation of the
chloroplast membranes in
Chlamydomonas reinhardi
y-l was
achieved through use of inhibitors of protein and RNA syn-
thesis and alternating exposure to the light or dark.
The relative content of membrane proteins of cytoplasmic
origin (L protein), of chloroplast origin (activation proteins),
and of chlorophyll in the growing membrane can be changed
so as to obtain membranes of differing composition. Mem-
branes formed in a normal greening process have a relatively
constant proportion of L protein, activation proteins, chloro-
phyll, and lipids. Synthesis of activation proteins can be in-
hibited by chloramphenicol, resulting in the formation of
photosynthetically inactive membranes relatively enriched in
L protein and chlorophyll. Transfer of greening cells to the
dark immediately blocks the synthesis of chlorophyll while
allowing synthesis of L protein and activation proteins to
continue for a short period. Transfer of cells containing
membranes enriched in L protein, but relatively deficient in
chlorophyll or activation proteins, to conditions which permit
the synthesis of these latter components results in the inte-
gration of these components into the membranes which then
regain a normal photosynthetic activity.
If excess of L protein is present, activation proteins can
be synthesized in the dark for long periods of time. Inhibi-
tion of RNA synthesis by actinomycin D was used to show
that the synthesis of L protein is associated with the presence
of an unstable RNA the synthesis of which depends on con-
comitant conversion of protochlorophyll to chlorophyll.
The regulation by light of the greening process is explained
as a control by the light-dependent conversion of protochloro-
phyll to chlorophyll on the synthesis of L protein at the trans-
cription level.
Dark grown
Chlamydomonas
reinhardi y-l cells contain a
partially differentiated plastid lacking the photosynthetic lamel-
lar apparatus (1). Upon exposure to the light, chlorophyll (2),
lipids (3), and specific membrane proteins (4-6) are synthesized
and assembled into normal photosynthetic membranes (2, 7-9).
It has been shown previously that two major types of proteins
participate in the formation of the chloroplast membranes: pro-
teins produced by the cytoplasmic ribosome (L protein (6))
which can be formed and assembled together with chlorophyll
and lipids to form a nonactive membrane and proteins produced
by the chloroplast ribosomes whose addition to the above mem-
brane is essential in order to acquire photosynthetic activity
(44).
In a previous work, it was suggested that the presence of an
excess of L protein is a prerequisite for the synthesis of both
chlorophyll and activation proteins (6). Thus, one can consider
the possibility that a frame or ‘Wr” membrane (a term utilized
by Siekevitz et al. (10)) consisting of L protein and lipids is an
obligatory intermediate step in the formation of the photosyn-
thetic membranes. The secondary addition of certain compo-
nents to a pre-existing membrane indicates that growth might
occur stepwise and thus membrane composition and function
can be modulated during the growth process.
In the present work the sequential synthesis and integration
of cytoplasmic and chloroplast proteins as well as that of chloro-
phyll was modulated by use of protein and RNA synthesis in-
hibitors and alternate exposure of greening cells to the light and
dark. Measurements of rates of synthesis of L protein, chloro-
phyll, activation proteins, and photosynthetic activity under a
variety of conditions support the following conclusion. Con-
version of photochlorophyll to chlorophyll controls the synthesis
of L protein at the transcriptional level, and an excess of L pro-
tein is required for the synthesis of chlorophyll and activation
proteins. However, the synthesis of both L protein and ac-
tivation proteins is not a light-dependent process.
MATERIALS AND METHODS
Growth, harvesting, and greening of C.
reinhardi y-l cells
were
carried out as previously described (2, 6). Pulse labeling of cells
was done by use of [3H]acetate (10 &?i per pmole) added to cells
greening in normal growth medium supplemented with KHzPOl
(2 pmoles per ml) and containing only 1 mM acetate (6). The
pulse was ended by add&ion of unlabeled acetate to a final con-
centration of 100 mM and three successive washings in fresh
growth medium. The cells were then further processed for
analysis as described below. In experiments in which labeling
of L protein was measured in the presence of actinomycin D,
122
This is an Open Access article under the CC BY license.
Issue of January lo,1972
G. Eytan and I. Ohad
123
P
W
0
7
c500
z
v
the specific radioactivity of the acetate was 100 $Zi per pmole,
A
at a final concentration of 1
mM
and the labeled cells were proc-
essed together with a lo-fold amount of unlabeled cells to serve
as a carrier for the electrophoretic analysis of the lamellar pro-
teins. In all experiments, the pulse was continued for 15 min.
In chase experiments, following pulse labeling with [aH]acetate,
the washed cells were resuspended in fresh growth medium and
further incubated for an additional hour.
Fractionation of Cells and Acrylamide
Electrophoresis of
Lamel-
lar Proteins-A
fraction enriched in chloroplast membranes was
prepared by centrifugation of a cell homogenate on a linear
Urografin density gradient as described before (6). The mem-
brane fraction was collected, washed by centrifugation, and freed
from lipids before electrophoresis was carried out in an acidic
medium following a modification of the method of Takayama
et al.
(11) as described by Eytan and Ohad (6). For estimation
of radioactivity in the different peaks, the cells were sliced,
dissolved, and counted in a scintillation spectrometer as de-
scribed (6).
Measurements of Photosynthetic
Activity-Photophosphoryla-
tion was measured with an “open cell” preparation as described
by Wallach
et
al. (8) ; see also Reference 6.
System II-dependent photophosphorylation was measured
with ferricyanide (2.10e6
M)
; System II- and I-dependent phot,o-
phosphorylation were measured with diquat (l , l’-ethylene-2,2’-
dipyridylium dibromide, 9.10+
M).
Protein, chlorophyll, and cell count were done as described
before (1). Chloramphenicol was obtained from Abic Labora-
tories, Tel-Aviv, Israel. Cycloheximide was purchased from
Calbiochem. Urografin was obtained from Schering Corpora-
tion, West Germany, and 13H]acetate was obtained from The
Radiochemical Centre, Amersham, England. Actinomycin D
was purchased from Merck, Sharp and Dohme. All other re-
agents used in this work were of analytical grade.
RESULTS
L
D
Synthesis of
Lamellar
Proteins
during Discontinuous Greeningi-
j+&q&&df It was shown before that when greening Chlamydomonas
y-l
i..,.-c
cells are transferred to the dark chlorophyll synthesis stops im-
mediately but resumes upon re-exposure to the light at the same
initial rate as that attained at the time light was shut off (2, 12).
The cessation of chlorophyll synthesis in the dark is due to the
fact that the mutant cells have lost the ability to enzymatically
convert protochlorophyll to chlorophyll. Since this conversion
can be attained through a nonenzymatic photochemical reduc-
tion, chlorophyll can be synthesized by the mutant only in the
light. The question arises whether cessation of chlorophyll
synthesis will cause also an immediate arrest of the lamellar
I I I proteins synthesis and their assembly into the photosynthetic
20
30 40
5o membranes. In order to answer this question greening cells
SLICE No.
were pulse labeled just before and at different times after trans-
fer to the dark and the radioactivity incorporated into the differ-
FIG. 1. Incorporation of [aH]acetate into lamellar proteins after
^ ” .* 1,. -3
11 ent lamellar proteins was measured using the gel electrophoresis
transter ot greening cells to tne aark. Hark-grown ceils were
washed, resuspended in fresh growth medium at a final concentra-
tion of 107 cells per ml and either incubated in the dark or illumi-
nated for 5 hours and transferred back to dark. Just before
transferring the cells to the dark (A) and 5 min (B) , 30 min (C),
90 min (D), and 180 min (E) after transfer the cells were pulse-
labeled for 15 min, with [aH]acetate. The cells incubated con-
tinuously in the dark (F) were pulse-labeled 6 hours after the start
of the experiment. Labeled cells were fractionated in a linear
Urografin gradient and the proteins of the membrane fractions
technique as described under “Materials and Methods.” The
results, shown in Fig. 1, indicate that following transfer to the
1 Experiments in which greening cells were exposed to alter-
nating light and dark incubation will be referred to as discon-
tinuous greening.
run on acrylamide gels as described under “Materials and Meth-
ods.” The gels were sliced and counted for radioactivity. The
L protein peak corresponded to Slice 28.
75001/40 F
.^ 12 ‘f
!G . .L
.
E 2500 ‘,
t“‘
2
5 ‘-._ ---o -“b 20 g
-. J
---_
i
c
--------w’ -‘a 2
I I I I I I I I I I
I&II05
2 4 G 8 10
0 1 2 3” 120 TIME ( hours)
TIME (hours)
FIG. 3. Rate of synthesis of L protein and chlorophyll during a
FIG. 2. Rate of decay of L,protein synthesis after transferring discontinuous greening. Dark-grown cells were incubated in the
cells to the dark. Based on data of the experiment described in light for 6 hours (control) or for 4 hours followed by 3 hours of
Fig. 1. the rate of svnthesis of L protein after transfer to the dark incubation in the dark and an additional 2 hours in the lieht. At
is plotted as counts per min found in L protein after labeling the different times, samples were taken for measurements ofu chloro-
cells at different times upon transfer back to dark (---). The phyll content and optical density of the Amido black-stained L
same results are plotted as percentage of the total radioactivity protein peak on acrylamide gels as described under “Materials
in the gels found in the L protein peak. and Methods.” The amount of L protein synthesized in the
light could be estimated from the increase in the optical density
dark a reduction in the incorporation of radioactive acetate into
of the Amido black-stained gels (6). The data are expressed as
cumulative relative units, (AO.D.tr - to) + (AO.D.t, - tr) + . . . .
the L protein peak was detected after 5 min (20 min if one takes
etc., when tr, tz, represents the time of sampling in the light. After
into account the duration of the pulse). Following further in-
transfer of the cells to the dark, the increase in the ontical density
cubation in the dark an additional reduction in the rate of label-
of the L protein peak become too small to be measurkd accurately
ing of the L protein was observed as well as a reduction in the
due to reduction in its rate of synthesis and use of shorter sam-
pling time intervals. Therefore. the followine method was used.
incorporation of radioactivity in all the other protein peaks
The-cells were pulse-labeled with [3H]acetateuas described under
(Fig.
1).
The decay rate of L protein synthesis expressed as the
“Materials and Methods” and the amount of radioactivity incor-
apparent half-life of the maximal rate of synthesis varied in the
porated into the L protein peak was measured before and after
different experiments between 20 min to about 1 hour (Fig. 2). transfer of the cells to the dark. The rate of labeling in the light
In contrast to chlorophyll synthesis, the rate of L protein syn-
was found to parallel the rate of increase in optical density, i.e.
the specific activity (counts per min per A0.D.t’ - t) was constant.
thesis upon re-exposure to the light is not equal to that attained
The incorporation rate became progressivelv lower after transfer
before the transfer to the dark (Fig. 3). A lag period can be
of the cells to the dark. Assuming that the specific activity of the
observed which seems to last for about
1
hour before the maxi-
L protein synthesized in the dark is similar to that obtained in
mal rate of synthesis is resumed.
the light, the increase in L protein in the dark was calculated from
the rate of labeling of the L protein peak in t.he dark and the con-
The changes in the rate of L protein synthesis are not a reflec-
stant specific activity value measured before transfer of the cells
tion of a total nonspecific reduction of acetate incorporation to the dark (counts per min pulse dark/specific activity light =
into dark-incubated cells. This can be seen from Fig.
1 where A0.D. dark). The data for L protein synthesis were taken from
it is
clear that the reduction in the labeling of L protein precedes
several experiments.
Squares,
L protein; circles, chlorophyll;
the fall in the labeling of the other membrane proteins. The
dark jigures, incubation in the dark;
open
figures, incubation in
the light.
possibility exists that the labeling of L protein is at least par-
tially due to turnover which might assume different rates under thesis of L protein lagged behind (Fig. 3). However, as the lag
different conditions. However, the contribution of label due to ended and L protein synthesis was resumed at the maximal rate,
turnover is expected to be extremely low in view of the stability
the relative constant proportions between L protein and chloro-
of the chloroplast membranes in this organism (1) and the long phyll were obtained again. This seems to have been achieved
half-life time of its proteins (13). through a compensation of the surplus of L protein synthesized
The results of several experiments in which the rate of L pro-
in the dark by the chlorophyll synthesized during the lag period
tein synthesis was measured at different times of a discontinuous of L protein synthesis.
greening are exemplified in Fig. 3. The data are shown as cumu-
In all the above experiments, radioactivity was measured only
lative amounts of L protein synthesized and were calculated on in the lamellar proteins found in the membrane fraction. How-
the basis of measurements of optical density in the L protein ever, it is possible that lamellar proteins continue to be synthe-
peak and radioactivity following short pulse labeling in the peak sized in the dark but are not incorporated into the membranes.
as measured by the polyacrylamide gel electrophoresis method.
In order to test this possibility, the cells were pulse-labeled with
It is evident that during the incubation in the light, the mem- [*HIacetate 90 min after transfer to the dark when labeling of L
branes contained a constant proportion of L protein and chloro- protein was reduced by 80%. The cells were then washed free
phyll. However, after the dark incubation period a significant of radioactive [*HIacetate and further incubated either in the
amount of L protein accumulated in excess to the chlorophyll dark or light for an additional hour. At the end of this chase
content of the membranes. Transfer of the dark-incubated cells period the membranes were analyzed again by gel electrophoresis
to the light initiated a rapid chlorophyll synthesis while the syn-
for radioactivity in the different lamellar proteins. The results
124 Biogenesis of Chloroplast Membranes. VIII Vol. 247, No. 1
Issue of January 10, 1972 G. Eytan and I. Ohad 125
shown in Fig. 4 demonstrate that no additional radioactivity
appeared in any of the membrane proteins either in the cells
chased in the dark nor in those chased in the light. This indi-
cates that labeled proteins did not accumulate exterior to the
membranes in such a way as to be reutilized during incubation
in light when synthesis of L protein(s) and their assembly into
membranes occurs. These results seem also to exclude the possi-
bility that lamellar proteins were indeed synthesized in the dark
but were immediately degraded since under the experimental
conditions used even proteins with a half-life of about 10 min
should have been detected if one assumes a similar rate of syn-
thesis during the pulse in the dark to that obtained in the light.
Changes in sensitivity of y-l cells to Protein Synthesis Inhibitors
during Discontinuous Greening-In the experiments described
above it was shown that L protein can be synthesized and accu-
mulated in excess of chlorophyll in the membranes following
transfer of greening cells to the dark. The question arises
whether under these conditions the cells will be able to synthe-
size also activation proteins and utilize them both upon addition
of chlorophyll during a second illumination period. It was men-
tioned previously that chloramphenicol blocks specifically the
synthesis of activation proteins and the development of photo-
synthetic activity while cycloheximide inhibits the synthesis of
L protein resulting in complete inhibition of chlorophyll and
membrane synthesis (6).
If both L protein and activation proteins are indeed accumu-
lated in excess to chlorophyll in the dark, one would expect that
addition of each of the above inhibitors after a dark incubation
will have an initially reduced effect on chlorophyll synthesis and
on the increase in photosynthetic activity as compared with that
obtained if the inhibitors were added before transfer to the dark.
The results of an experiment in which this possibility was
tested are in agreement with the above expectations. Wh.en
chlorophyll synthesis during a discontinuous greening was meas-
ured before and after a period of incubation in the dark a relative
resistance to both inhibitors was observed as compared to that
exhibited if the inhibitors were added to the greening cells before
transfer to the dark (Fig. 5).
Similar results were observed when the relative fluorescence of
whole cells was measured as an indication for the degree of
photosynthetic activity of the newly formed membranes (14).
Incubation of cells greening continuously in the light, in the
presence of chloramphenicol, induced a steep rise in the relative
fluorescence indicating formation of faulty photosynthe%ic mem-
branes. However, incubation of greening cells with chloram-
phenicol after a dark incubation period had no effect on the
relative fluorescence (Fig. 6).
These results were confirmed by measurements of photophos-
phorylation activities of both photosystems (Table I). Again,
the development of both activities was more resistant to chlor-
amphenicol and cycloheximide if the cells were incubated in
presence of inhibitors after a dark incubation period.
The apparent discrepancy between the effect of cycloheximide
which has no effect on fluorescence (Fig. 6) as compared with a
marked inhibition of photophosphorylation (Table I) can be
explained by the fact that cycloheximide added to cells which
have not accumulated excess L protein blocks completely all
membrane protein synthesis (6) and thus prevents formation of
faulty fluorescent membranes. One should mention that accu-
mulation of L protein and activation proteins during incubation
in the dark does not increase the specific photosynthetic activity
500
250
500
250
10 20 30 40 50
SLICE No
FIG.
4. Incorporation patterns of [aH]acetate into the lamellar
proteins following pulse-labeling in the dark and after chase in
the light or dark. Dark-grown cells were incubated in
the light
for five hours and then transferred to the dark. After 90-min
incpbation in the dark the cells were pulse-labeled for 15 min and
then washed and resuspended in fresh growth medium and further
incubated for 1 hour in the light or dark. Samples were taken
immediately after the pulse (A), after the chase in the dark (B),
and after the chase in the light (C) and processed for analysis of
lamellar proteins labeling as described under “Materials and
Methods.”
a
* G
5
”
1
-I
1 I I I I I I I I I I
2 4 G
a 10
TIME (hours)
FIG.
5. Effect of protein synthesis inhibitors on chlorophyll
synthesis during a discontinuous greening experiment. Dark-
grown cells were washed, resuspended in fresh growth medium, and
exposed to light
(0).
After 4% hours as indicated by the arrow
( l ) the cell suspension was divided into four parts. One part was
transferred to the dark (
l
) while the rest of the suspensions were
incubated in the light without any additions (0) or with addition
of chloramphenicol
(A)
or cycloheximide (a). The cell sus-
pension incubated in the dark
was
divided in three parts and re-
exposed to light at the time indicated by the arrow (1) without
(0) or with addition of chloramphenicol
(A)
or cycloheximide
(Lx.
of the membranes unless chlorophyll is added upon subsequent
illumination (9).
Regulation of L Prokin Xynthesis-Since L protein can be
synthesized for short periods after transfer of greening cells to
126
Biogenesis of Chloroplast Membranes. VIII
Vol. 247, No. 1
the dark (Figs. 1 to 3) the question arises why its synthesis does
not continue for longer periods of dark incubations. Several
factors can be considered to control the synthesis of L protein.
It is known that the conversion of protochlorophyll to chloro-
phyll stops upon transfer of greening cells to the dark. Thus,
it is possible that either lack of chlorophyll or accumulation of
protochlorophyll or some precursor of its synthesis might affect,
the synthesis of L protein. It seems most likely that this effect
occurs at the transcription or translation levels and not the as-
sembly level, as the results of Fig. 4 tend to eliminate this pos-
sibility.
Greening cells were transferred to the dark after 5 hours of
incubation in the light. After an additional incubation of 3
hours in the dark the cells were washed in fresh growth medium
and then part of the cells were illuminated continuously, another
part illuminated for 5 min and then returned to the dark and
0.8
L
2 4 10
TIME(hours)
FIQ. 6. Effect of protein synthesis inhibitors on the relative
fluorescence of
Chlamydomonas
reinhardi cells during discon-
tinuous greening experiments. Same conditions as in Fig. 5.
Fluorescence was measured as described under “Materials and
Methods.” 0 and
l
, control cells in the light (open pgures) or
in the dark (j&Z figures) ; A, cells incubated in the presence of
cycloheximide; 0, cells incubated in t.he presence of chloram-
phenicol.
the rest of the cells were kept in the dark without illumination.
L protein synthesis was measured by pulse labeling and gel
electrophoresis 15 min after the transfer of the greening cells to
the light or after washing the cells which were kept continuously
in the dark; no effect of the short (5 min) illumination was noted
on the synthesis of L protein (Fig. 7). These results indicate
that release of repression of the synthesis of protochlorophyll
precursors and transformation of the accumulated protochloro-
phyll to chlorophyll which should have occurred during the 5
min of illumination had no triggering effect on the synthesis of
L protein.
A short, illumination period was insufficient to re-establish the
maximal rate of L protein synthesis in the dark as shown in the
previous experiment. Also a lag of about. 1 hour was observed
in the synthesis of L protein upon the re-exposure of greening
cells to continuous illumination following a dark incubation
period (Fig. 3). These results suggest that although incorpora-
tion of amino acids into L protein can occur in the dark, the
reinitiation of L protein synthesis after its cessation following
incubation of the cells in the dark is a slow process which seems
to be light-dependent. Such a situation might arise if one as-
sumes that the synthesis of L protein is regulated by rapidly
turhing over RNA whose synthesis is light-dependent. In order
to test this possibility, the effect of actinomycin D (which was
shown to block RNA synthesis in y-l cells) on chlorophyll and
L protein synthesis was measured in a discontinuous greening
experiment. The drug was added under three different con-
ditions: (a) before transfer to the dark, (a) after transfer to the
dark, and (c) after transfer to the light following a 2-hour in-
cubation in the dark.
The degree of inhibition of L protein and chlorophyll synthesis
differed according to the time of actinomycin D addition (Table
II). Thus, both L protein and chlorophyll synthesis were
strongly inhibited before transfer to the dark while L protein
synthesis was significantly less inhibited after transfer in the
dark. However, when actinomycin D was added at the onset,
of the second illumination period the reinitiation of L protein
synthesis was blocked completely whereas synthesis of chloro-
phyll was only slightly affected (Table II).
These results suggest that, L protein synthesis depends on the
TABLE
I
Effect of protein synthesis inhibitors on development of photophosphorylation activity during discontinuous greening
Similar experimental conditions as Fig. 5 with the difference that the incubation in the dark lasted 3 hours. Photophosphorylation
activities were measured 2 hours after the addition of the inhibitors during the second incubation using ferricyanide or diquat as co-
factors.
Treatment
First incubation I Second incubation
5 hours of greening
5 hours of greening and then
3 hours of dark incubation
Light control
Light + cycloheximide
Light + chloramphenicol
Light control 18.3
Light + cycloheximide 18.3
Light + chloramphenicol 16.8
Photosystem II
Total
activity Inhibition
%
29
59
I
0
3
Specific
activity
/.l?deS
A TP/mg
chloro~hyll/hr
383
540
182
390
I
520
370
-
Photosystem II + I
Total
activity Inhibition Specific
activity
Jwh!s
I I /moles
ATP/108 % ATP/mg
cells/hr chloro$hyll,‘hr
18.3 390
12.3 33 513
9.6 48 234
18.7 398
16.3 12 465
13.3 29 296
Issue of January 10, 1072
G. Eytan
and I.
Ohad 127
FIQ. 7. Effect of a short illumination on L protein synthesis in
a discontinuous greening experiment. Dark-grown cells were
illuminated for 5 hours and then transferred to the dark. After
s-hours incubation in the dark, the cells were washed and trans-
ferred to fresh growth medium and then part of them left in the
dark (C), part illuminated for 5 min and returned to the dark (B),
and the rest exposed to continuous illumination (A). Twenty
minutes after transfer to the fresh medium the cells were labeled
and then analyzed for radioactivity distribution in the lamellar
protein by using the gel electrophoresis technique as described
under “Materials and Methods.” Slice 28 corresponds to the L
protein peak.
I I I I 1
B
I,
10 20 30 40 50
SLICE No.
TABLE II
Effect of RNA synthesis
inhibition
on
chlorophyll and
L protein synthesis during discontinuous greening
Dark grown cells were incubated in fresh growth medium at a ml). The cells were then labeled for 15 min with [*HIacetate as
final concentration of 10’ cells per ml. Incubation was continued described under “Materials and Methods.” At the end of the
for 5 hours in light or 5 hours in light with an additional incubation pulse the cells were processed for membrane isolation and gel elec-
in the dark for 2 hours. The cells were then washed, resuspended trophoresis of the lamellar proteins. The radioactivity in the
in fresh growth medium, and further incubated in the light or dark different peaks was measured. The chlorophyll content wss
for 30 min without or with addition of actinomycin D (150 pg per determined at the end of the pulse.
Treatment
Second incubation
First incubation
0 hours light
5 hours light and 2 hours dark
Light control
Light + actinomycin D
Dark control
Dark + actinomycin D
15,400
5,075
1,505
790
%
0 0
67 75
0
100
48
%
48
44
33
25
Light control
2,800 0 0 38
Light + actinomycin D
660 77
110
26
Dark control
870
100
27
-
Inhibition
0 Percentage of inhibition of radioactivity in L protein relative
to the radioactivity in the uninhibited sample (light or dark con-
trol).
b Percentage of inhibition of radioactivity in L protein relative
to the light minus the dark control.
presence of an unstable RNA which is not available after a 2-
hour incubation of the cells in the dark.
DISCUSSION
synthetic electron transfer and energy coupling activity. Sec-
ondly, the control mechanism by which light regulates the
synthesis of membranes seems to reside at the transcription level
of the cytoplasmic L protein.
The results presented in this work emphasize two major fea- Modulation of Membrane Composition during Their Develop-
tures of the system responsible for the chloroplast membrane ment-The concept that membrane composition can be modu-
biogenesis in C. reinhardi y-l cells. Firstly, the membranes lated in such a way as to obtain membranes enriched in one of
grow as a flexible system in terms of composition and function the different classes of components is based on the following find-
by an orderly sequential addition of three different classes of ings. L protein can be accumulated in excess to chlorophyll,
components. Proteins of cytoplasmic origin (L protein) form activation proteins, or both. Excess of L protein to activation
together with lipids a frame or “Ur membrane” which serves as proteins and chlorophyll can be achieved when greening is car-
an acceptor for chloroplast made proteins and chlorophyll the ried out in presence of chloramphenicol (5, 6). Excess of L
addition of which confers to the complete membrane its photo- protein and activation proteins over chlorophyll can be obtained
Chlorophylld Inhibition
/dlO~ cells
1.5
0.6
0.0
0.0
1.0
0.8
0.0
%
0
66
0
20
c Percentage of radioactivity in L protein relative to the total
radioactivity found in the corresponding gels.
d Amount of chlorophyll synthesized during the second incuba-
tion period (45 min).
128
Biogenesis of Chloroplast Membranes. VIII
Vol. 247, No. 1
when cells are transferred to the dark in discontinuous greening
experiments. The missing components, chlorophyll, and chloro-
plast proteins can be added together to the membranes enriched
in L protein following greening in the presence of chlorampheni-
co1 as shown when repair of nonphotosynthetic membranes is
carried out in the presence of cycloheximide in the light thus
permitting synthesis of both chlorophyll and chloroplast made
proteins. Addition of activation proteins can occur also alone
if the repair process is carried out in the dark and chlorophyll is
not synthesized.
The modulation of membrane composition as shown above
demonstrates that the addition of the different membrane com-
ponents can occur in a sequential manner either in the normal
greening process (discontinuous greening) or in greening experi-
ments in which protein synthesis has been specifically inhibited.
However, one condition seems to be critical for this modulation.
It appears that an excess of L protein to the other components
in the growing membranes is a prerequisite for the chloroplast
made proteins or chlorophyll to be synthesized and integrated.
Thus, L protein appears to be a limiting factor for the growth
of the photosynthetic membranes.
The alternation of membrane composition results in changes
of photosynthetic activity (5, 8, 9). Recent measurements of
the illumination intensity required for saturation of photosyn-
thetic activity seems to confirm that different parts of the photo-
synthetic reaction chain can become limiting factors for photo-
phosphorylation by membranes whose composition was altered
by use of chloramphenicol or discontinued greening as described
in this work (9). Photosynthetic membranes in C.
reinhad
y-l
cells grow by intussusception of newly formed components in a
DNA
Protochlorophyll
- synthesising e
system
1
I [Protochlorophylll
FIQ. 8. Schematic representation of the proposed control pat-
tern involved in the formation of chloroplast lamellae in Chlamy-
domonas reinhardi y-l mutant cells. Heavy arrows indicate syn-
thesis of building blocks (circles); light U~TCWS designate control by
the presence of one component on the synthesis of other compo-
nents; crossed arrows indicate site of inhibitors action. For ex-
planation see text. CHZ, cycloheximide; CAP,
chloramphenicol.
random way as demonstrated by autoradiographic technique
(13). Recently, it was shown that the growing membranes
preserve their homogeneity throughout the process whether all
the components are added at once or sequentially (14).
Regulation of
Synthesis and Assembly
of
Membrane Components
-Recent work has shown that the enzymatic system responsible
for the synthesis of protochlorophyll in the y-l mutant includes
one or several unstable enzymes whose synthesis is coded by a
rapidly turning over RNA (12). However, this RNA and all
the enzymes necessary for protochlorophyll synthesis can be
formed in the absence of light (12). Since chloramphenicol in-
hibits only partially the synthesis of chlorophyll at saturative
concentrations (5, 6) and rifampicin was reported to have no
effect on its synthesis (15), one can tentatively conclude that
both RNA and the enzymes involved in the synthesis of chloro-
phyll are of cytoplasmic origins.
Although L protein can be synthesized for short periods of
time in the dark as shown in this work, its long term synthesis
is light-dependent. The control of L protein by light seems to
be mediated by chlorophyll or chlorophyll biosynthesis inter-
mediates. This conclusion is based on the fact that the wild
C.
reinhurdi
can synthesize chlorophyll and normal chloroplast
membranes in the dark while the y-l mutant which has lost the
ability to convert protochlorophyll to chlorophyll enzymatically
(1) requires continuous illumination also for sustained synthesis
of L protein.
In this work, it was demonstrated that actinomycin D effects
a remarkably rapid inhibition of L protein synthesis when added
during a continuous greening process and even a more marked
effect if added after a dark incubation in discontinuous greening
experiments. However, chlorophyll synthesis is not inhibited by
actinomycin D after the dark incubation period during the 1st
hour of illumination. This can be readily explained by the fact
that while RNA synthesis required for the formation of chloro-
phyll-synthesizing enzymes can occur in the dark (12)) the RNA
necessary for the formation of L protein cannot be synthesized
in the absence of illumination. At the end of a dark incubation
period the cells will be depleted of the latter RNA but not of the
former. This will enhance the inhibition of L protein synthesis
by actinomycin D but will have initially little effect on the syn-
thesis of chlorophyll. This conclusion is further supported by
the finding that cycloheximide has a very similar effect on both
chlorophyll and L protein synthesis during a discontinuous
greening as shown in this work (cf. Reference 6).
One should mention that the inhibitory effect on the synthesis
of L
protein is greater if the cells are transferred to the dark than
if they are treated with actinomycin D in the light (Table II).
This might suggest that the RNA coding for L protein synthesis
is less stable in the dark. However, it is possible that other
factors might contribute to the control of L protein synthesis
the nature of which is not yet known.
The fact that addition of actinomycin D after transferring the
cells to the dark has an additive inhibitory effect over that of the
dark control might indicate that the dependence of the RNA
synthesis coding for L protein on simultaneous conversion of
protochlorophyll to chlorophyll is not stringent.
The formation of chloroplast membranes in C. reinhardi y-l
cells can withstand a wide range of experimentally induced
modulations. However, under normal greening conditions it
seems that an accurate balance is kept in the assembly of the
different membrane components. This balance is due to a
regulatory mechanism apparently operating at the transcrip-
tional level of the RNA coding for the synthesis of L protein. for his valuable suggestions and for reading the manuscript, and
to Dr. I. Goldberg for many useful discussions.
It seems that L protein controls the synthesis of chlorophyll and
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129
