THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 245, No. 17, Issue of September 10, pp. 4297-4307, 1970

Printed in U.S.A.

Biogenesis of Chloroplast Membranes

VI. COOPERATION BETWEEN CYTOPLASMIC AND CHLOROPLAST RIBOSOMES IN THE SYNTHESIS OF PHOTOSYNTHETIC LAMELLAR PROTEINS DURING THE GREENING PROCESS IN A MUTANT OF CHLAMYDOMONAS REINHARDI y-l *

(Received for publication, March 3, 1970)

GERA EYTAN AND ITZHAK OHAD

From the Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

SUMMARY

Proteins from a membrane fraction enriched in chloroplast material obtained by density gradient centrifugation were analyzed by electrophoresis on acrylamide gels. About 15 to

20 bands could be resolved. The electrophoretic pattern of

the proteins obtained from dark-grown cells, lacking the chloroplast lamellae, differed from that of light-grown photosynthetic cells. When dark-grown cells were exposed to light, a main protein peak designated as L protein ap- peared parallel to the increase in the chlorophyll content. Continuous labeling and pulse labeling of the proteins during the greening process, in the presence or absence of cycloheximide and chloramphenicol, indicated that the L protein fraction is synthesized de novo; its synthesis is inhibited by cycloheximide but not by the latter compound.

Membranes synthesized in the presence of chlorampheni- co1 have a very low photosynthetic activity although a normal amount of L protein is present in the cells. Removal of the

inhibitor allows synthesis of new proteins and activation of the defective membranes. This process does not require synthesis of additional L protein or chlorophyll since it is not inhibited by cycloheximide and it can occur in the dark. Pulse labeling of proteins after transfer of greening cells from chloramphenicol to cycloheximide indicates progressive reduction in the labeling of the L protein peak and appearance of radioactivity in other protein peaks which are not labeled in the presence of chloramphenicol. Cycloheximide com- pletely blocks the synthesis of chlorophyll and protein when added at any time during the greening process in a normal noninhibited system. However, prior incubation of the greening cells with chloramphenicol leading to accumulation of L protein protects the system against the complete inhibi- tion exerted by cycloheximide.

Based on these data it is suggested that in the process of synthesis and assembly of the photosynthetic membranes, proteins of cytoplasmic and chloroplast origin concur to form a functional membrane. The synthesis of these two types of protein is regulated by a feedback-like mechanism; its features include control of the level of the lame&u proteins of cytoplasmic origin by the level of chlorophyll and control of the level of proteins of chloroplast origin by that of the

* For Part V of this series see Reference 6.

cytoplasmic proteins. In addition, the light-dependen chlorophyll synthesis is controlled by the level of the lamellar proteins of chloroplast and cytoplasmic origin.

Mutants of Chlamydomonas reinhardi have been extensively used as a suitable tool for the investigation of the mechanism of the photosynthetic electron transfer system (1) and more recently for the study of the biogenesis of the photosynthetic lamellae (2-7). In previous work (2, 3) it has been shown that the Chlamydomonas y-l mutant cells grown on acetate in the dark contain a partially different’iated chloroplast lacking the photo- synthetic lamellar system. When exposed to light the dark- grown yellow cells synthesize photosynthetic pigments and lipids as well as proteins (3, 5, 7), and the content of photosynthetically active lamellae increases parallel to the increase in the chloro- phyll content (3, 7, 8). It was shown that at least some of the lamellar proteins, such as the photosynthetic cytochrome f, are present in the dark-grown cells in large amounts (2, 4, 9) in an inactive form and become photosynthetically active during the greening (3,4). Therefore, it was reasonable to assume that the formation of the new membranes consists of a process of assembly of pre-existing and newly synthesized components into normally active membranes. The aim of the present work was to detect, identify, and measure the formation of chloroplast membrane specific proteins during the greening process and to try to correlate these data with the appearance of the membrane struc- ture and photosynthetic function. It was hoped that from such a study it would be possible to obtain more information about the mechanism of the assembly of the photosynthetic membranes.

MATERIALS AND METHODS

Preparation of Cells and Labeling during Greening Experiments -C. reinhardi y-l cells were grown on a mineral medium con- taining acetate (7 mM) as a carbon source as described by Ohad, Siekevitz, and Palade (2, 3) with addition of 50 pg per ml of penicillin. Cells were harvested after five to six generations of growth in the dark (5 to 6 days), when still in the logarithmic phase (cell concentration about 1.5 x lo6 cells per ml), by centrifugation at 5000 x g for 10 min at 4”. For greening, the

4297

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4298 Biogenesis of Chloroplast Membranes. VI Vol. 245, iSo. 17

cells were resuspended in fresh growth medium with the addition of 2 mM KH2P04 at a final concentration of 5 to 10 x lo6 cells per ml and incubated under white fluorescent light (700 foot- candles) (3, 7). At different times, 5-ml samples were taken for analysis of chlorophyll, protein, and radioactivity content; 100- ml samples were taken for the preparation of membrane fractions (see below). For continuous labeling, 3H-acetate was added (5 PCi per pmole, 7 rn~) at onset of illumination. For pulse labeling, samples were taken at different times, and the cells were pelleted by centrifugation at room temperature, resuspended in growth medium without acetate, and incubated further in light or dark for 10 min. 3H-Acetate was then added (20 PC1 per ~molc, 0.5 IIIM), and incubation was continued for 20 min. The pulse was ended by addition of 100 mM unlabeled acetate and three successive washings by centrifugation in growth medium in the cold. The cells were then either processed immediately or after storage in the pellet in ice overnight.

Preparation of Menlbrane Fractions-Pelleted cells were resusl)ended at a final concentration of 2.5 x 108 cells per ml in ice-cold Tris buffer (30 IIIM, pH 7.4) containing 8 mM P-mercapto- ethanol. The suspension was passed through a French press operated between 3,000 and 5,000 psi., increasing pressure being used for cells containing increasing amounts of chlorophyll. Three milliliters of the disrupted cell suspension were layered on top of 25 ml of urografin linear gradient in the same buffer (specific density from 1 .OO to 1.31). The tubes were centrifuged in a Spinco L4 SW25 rotor at 24,000 rpm for 120 min at 4”. Fractions were collected with a Pasteur pipette. Fractions 3 and 5 (see Fig. 1) containing most of the chlorophyll, were centrifuged at 140,000 x g for 30 min at 4’. The resulting pellet was resuspended by homogenization with a Teflon pestle and washed once in Tris buffer by centrifugation as above. Samples were taken for determination of protein, chlorophyll, and radio- activity content, and the pellet was dissolved and analyzed by electrophoresis on acrylamide gels as described below.

Preparation of Samples and Gels for ElectrophoresisFrac- tions for analysis were dissolved in a mixture of phenol-acetic acid-water (4 :2: 1, w/v/v) containing 1 y0 of the nonionic detergent Nonidet P40 and 0.5% /3-mercaptoethanol, Solvent A. The protein content was adjusted to 1.5 to 3.0 mg of protein per ml and the solution was kept at room temperature overnight. The solubilized protein did not sediment when centrifuged at 100,000 x g for 1 hour. To separate the lipids from the proteins present in the solution, 10 volumes of ether were added and the samples were kept in ice for 20 min. The white protein precipi- tate which appeared was sedimented by centrifugation at 1,000 X g for 5 min and washed once in ether. The precipitate was easily formed when the initial concentration of protein in Solvent A was above 1.0 mg per ml. Extraction of lipids with 80% acetone prior to solubilization resulted in incomplete solubilization of proteins. Treatrnent with ether removed 99% of the lipids, as tested by use of radioactive lipids admixed with nonlabeled membranes.

The lipid-free protein was redissolved in Solvent A and used for electrophoresis immediately or after storage at -2O”, with no apparent change in the electrophoretic pattern.

Acrylarnide gels were prepared by a modification of the method described by Takayama et al. (10) and Razin and Rotem (11). The gels were prepared by polymerization of a mixture of 7.5%

acrylamide, 35% acetic acid, 5 M urea, 0.2% Nonidet P40, and 0.227, ethylene diacrylate (12). For polymerization, 0.06%

tetramethylethylenediamine and 45 mg of ammonium persulfate were added to 12 ml of the above mixture, and polymerization was carried out at 37” for 60 min. Gels were prepared in glass tubes with an internal diameter of 5 mm and a length of 100 mm. Samples containing 100 to 300 pg of protein in 0.15 ml of solvent were mixed with half a volume of 40% sucrose in 35% acetic acid, layered on top of the gels, and overlayed with 0.1 ml of 75% acetic acid. Electrophoresis was at room temperature. The samples were first run for I5 min at 0.5 ma per tube and then for 2 to 3 hours at 3 ma per tube. A solution of 10% acetic acid was used for the upper and lower buffer. The lower electrode served as the cathode. The gels were stained with Amido black as described by Razin and Rotem (11). For analysis of the distribution of the radioactivity in the gels, 0.75-mm slices were cut using a tissue slicer (H. Mickle, Gomshall, Surrey, England). During slicing, the gels were held in place by a piece of wet filter paper. The slices were transferred to glass scintillation vials containing 0.5 ml of ammonia and were dissolved by heat,- ing at 80” for 30 min. The vials were then cooled to -lo, and 10 ml of Bray solution were added for counting (13).

AssaysProtein was det,ermincd as described by Lowry et al. (14). The samples were dissolved in 0.1 N NnOII voutaining 0.2% sodium lauryl sulfate. Chlorophyll leas determined nc- cording to the method of Arnon (15). Cell conrentration was measured by counting in a hemocytometer. Radioactivity of samples was measured using a Packard model 3320 s:cintillat,ion counter. For determination of protein radioactivity in samples from gradients or whole cells, the samples were extracted twice with 80% acetone, washed twice with 100/o trichloracetic acid, resuspended in 57, of the same medium, and heated at 90” for 20 min. The samples were then centrifuged (2000 x g, 15 min) and the pellets dissolved for counting as described above.

Densitometer tracings of the stained gels were obtained with a Gilford-Beckman spectrophotometer, model 2000, equipped wit,h a linear transport scanner and O.l-mm slit.

Succinic dehydrogenase activity was measured as described 1)~ King (16). For measurements of photosynthetic proton ~)ump activity, the cells were washed twice by centrifugation (5000 x g, 5 min) in a medium containing 0.5 M mannitol, 20 mM KC’1 and NaCl, 2 mM MgC12, 1 rnM MnClz, 0.1 mg per ml of bovine serum albumin, 5y0 dextran T40, and 2.50y0 Ficoll. The washed cells were resuspended in the same medium, and were pressurized through a French press operated at 700 p.s.i.* Proton uptake was measured on the pressured cells as described by Schuldiner and Ohad (4) with addition of 0.1 M KC1 and 50 PM pyocyanine.

Acrylamide, tetramethylethylenediamine, and amrnonium persulfate were purchased from Matheson, Coleman and Bell. Ethylene diacrylate was ljurchased from K and K Laboratories, Plainview, New York. hrnido black 1OB Michrome Brand was obtained from George Gurr, Ltd., London, England. Chlor- amphenicol (D-three isomer, 99% pure) was a gift from Abic Laboratories, Tel Aviv, Israel. Cycloheximide was obtained from three different sources: Nutritional Biochemicals, Parke- Davis, and Sigma. Dextran T40 and Ficoll were purchased from Pharmacia. Pyocyanine perchlorate was obtained from 12ann and bovine serum albumin, grade A, from Calbiochem. Pcni- cillin was obtained from Rafa Inc., Jerusalem, Israel. Nonidet P40, a nonionic detergent, was obtained from The British Drug Houses, Ltd. Urografin was obtained from Schering

1 D. Wallach, M.Sc. thesis, The Hebrew University, manuscript in preparation.

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Issue of September 10, 1970 G. Eytan and I. Ohad 4299

FIG. 1. Schematic representation of the distribution of the bands on OMOGENATE LAYE urografin gradients used for the

-------

fractionation of C. reinhardi cells. ORANGE (TURBID) - ---,---

YELLOW (CLEAR) - ---z----

Same experiment as in Table I. GREEN (TURBID) - ----3---

Homogenates obtained from dark- grown cells before (I) and after il- CLEAR -

lumination for 5 hours (II) were analyzed. Fractions 3 and 5 con-

GREEN (TURBID) -c

tained most of the chlorophyll ini- CLEAR - ---e---

tially present in the homogenate layered on the gradient. The sedi-

WHITE (AGGREGATES) -

merit contained unbroken cells (- CLEAR -

l%), starch granules, and other cellular debris.

w---------w SEDIMENT

II I

Corporation, West Germany, and 3H-acetate was obtained from The Hadiochemical Centre, Amersham, England. All other reagents used throughout this work were of analytical grade.

RESULTS

Synthesis of Protein during Greening ProcessWhen dark- grown cells are incubated in the light, the kinetics of total protein synthesis parallels that of the chlorophyll synthesis. The net increase in total protein amounts to about 40 to 50% over the initial protein content of 200 pg/lO cells. Protein synthesis occurs also in dark-incubated cells but only at a third of the rate of synthesis in the light (cj. Reference 3). In order to test whether specific proteins associated with the photosynthetic apparatus are synthesized during the greening the following experiments were performed. Dark-grown cells were incubated in the presence of 3H-acetate. At different times samples were taken and the cells were fractionated as described under “hla- terials and Methods.” The pattern of fractions obtained by density gradient centrifugation is shown in Fig. 1. The bands were analyzed for chlorophyll, protein, and radioactivity of protein and for succinate dehydrogenase as a mitochondrial marker. The results are shown in Table I. Most of the chloro- phyll was found in Fractions 3 and 5. Fraction 5 contained about 55% of the chlorophyll and 35% of the total succinic dehgdrogenase activity. When the incorporation of 3H-acetate into insoluble proteins of the different fractions was measured during the greening process it was found that the specific activity (counts per min per mg of protein) in Fraction 5 was the highest and paralleled the increase in chlorophyll. No significant difference was found between the specific activity of the different fractions in the dark-incubated cells. Therefore, further analysis of the electrophoretic pattern of the insoluble proteins (mem- brane proteins) was carried out on Fraction 5 of the gradient.

Acrylamide Gel Electrophoresis-The electrophoretic pattern of membrane proteins from light-incubated cells is shown in Figs. 2 and 4. About 15 distinct bands can be discerned (see also Fig. 5) including several major bands and a heavy band located about the middle of the gel. The intensity of the stain in the different bands was proportional to the protein concentration (Fig. 2). As shown in Fig. 3, the intensity of two arbitrarily selected bands was proportional to the total amount of protein layered on the gel and the ratio of their optical density was con- stant between 50 pg and 250 pg per gel. About 70% of the total protein layered on the gel penetrated the gel during electro- phoresis as tested by measurements of radioactivity distribution of proteins from cells evenly labeled through growth on 3H- acetate in the light for two generations or from cells labeled for

ORANGE (TURBID) GREEN (CLEAR) DARK GREEN (TURBID)

CLEAR

DARK GREEN (TURBID )

CLEAR

GREEN (AGGREGATES) CLEAR

TABLE I

Distribution of protein, chlorophyll, and succinic dehydrogenaxe activity in different fractions of broken cells separated by

centrifugation on urograjin gradients

Dark-grown cells (1.2 X 108) were harvested before and after incubation for 5 hours in the light. The cells were homogenized and fractionated on urografin gradients as described under “Ma- terials and Methods.” The parallel fractions obtained from the gradients were combined and analyzed for insoluble protein con- tent (sedimented at 100,000 X g for 30 min), succinic dehydro- genase activity (micromole per min), and chlorophyll content. Fraction I consisted of homogenate layer and Bands 1 and 2 of the gradient; Fraction II consisted of Bands 3 and 4 of the gradi- ent; Fraction III represented Band 5 of the gradient and Fraction IV Bands 6, 8, and sediment (see Fig. 1). The 100% value was 171 pg of chlorophyll, 770 pmoles of succinate oxidized per min, and 2.6 mg of protein for the dark-grown cells. For the light- incubated cells it was 220 fig of chlorophyll, 670 &moles of suc- cinate oxidized per min, and 3.5 mg of prot,ein. The recovery from the gradiedts was &thin 95%.

Fractior

Dark I.... II III.. IV....

Light I...... II..... III.. IV.

IS

--

Succinic Chloro-

PW dehydro- Insoluble

gepc+ protein activity

0.0 0.0 37.8 57.0 50.7 38.1 10.3 15.3

0.0 0.0 46.8 64.6 53.2 35.4 0.0 0.0

% total

3.0 54.0 31.0 12.0

2.8 56.7 40.5

A

L

mount 0 chlo-

rophyll

pebr”g protein

46.4

106.5 52 .5

52.5 217 0.72 84.5 169 1.52

fP

1

P

unaunt 0

succinic dehydre

pegez;r protein

318 382 37ti

-

Ratio of chloro-

phyll to succinic

dehydro- genase

%

0.(X

1 .23

o.ti7

7 hours in the dark. The electrophoretic pattern was similar when analysis was carried out prior to, or after lipid extraction, by ether precipitation of the proteins. However, since radio- active 3H-acetate was incorporated also in the lipid and pigments fraction (5), lipids were routinely extracted in order to prevent their interference with the determination of the radioactivity of the proteins and the densitometry tracings of Amido black- stained gels.

Changes in Electrophoretic Pattern of Membrane Proteins during Greening-The electrophoretic pattern of membrane proteins of dark-grown cells differed from that of light-incubated cells.

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4300 Biogenesis of Chloroplmt Membranes. VI Vol. 245, No. 17

The heavy band present at the middle of the gel of proteins of samples from light-incubated cells (L protein) was replaced by three distinct separate bands in samples from dark-grown cells. The remaining bands were similar in location and intensity in membrane proteins obtained from both light- and dark-incubated cells. When proteins from Fraction 3 of gradient were analyzed, about 50 bands could be resolved showing a pattern different from that of the proteins from Fraction 5. When the electro- phoretic patterns of proteins from Fraction 3 obtained from light- and dark-incubated cells were compared it was again found that

0 50 100 150 200 250 300

)1g PROTEIN /GEL

FIG. 2. Staining intensity of the different bands in acrylamide gels as a function of the amount of protein load. Dark-grown cells were incubated in the light for 6 hours. The cells were homogenized and fractionated on urografin gradients. Frac- ion 5 of the gradient was processed for electrophoresis as de- scribed under “Materials and Methods.” The chlorophyll con- tent was 16.5 fig/lo’ cells. Peaks 7 and L are identified according to the densitometer tracing shown in Fig. 5.

PEA! i

A’

ZO- J o--~--oo--~

/ PEAK 7

0 A

3.0

2.0 o i= B ii d

1.0

)ig PROTEIN / GEL

FIG. 3. Increase in the optical density of Peaks 7 (A-A) and L (A-A) and the ootical densitv ratio of Peak L to Peak 7 (O---O) as a function of the amouni of protein loaded on the gel. Same experimental conditions as in Fig. 2.

the main difference was the presence of the L protein band in the light sample and its absence in the dark one. The same results were obtained when proteins, which could be sedimented from the total homogenate at 100,000 X g for 30 min, were analyzed, and it was found that the gels contained about a hundred bands. Thus it was tentatively concluded that the L protein band repre- sents a protein or proteins associated with the presence of the photosynthetic lamellae. In order to test this conclusion further, greening experiments were performed and the membrane proteins from Fraction 5 of the gradient were analyzed by electrophoresis. It was found that the electrophoretic pattern of the proteins from the dark-incubated cells gradually changed during the greening and became similar to those of the green cells (Fig. 4). A more accurate picture of the changes can be obtained from densitom- eter tracings (Fig. 5). The staining intensity by Amido black of the proteins from light-exposed cells (optical density per mg of protein per gel) was higher than that of the proteins from the dark-grown cells by a factor of about two. No explanation could be found for this phenomenon. Since no exact correlation can be made between the intensity of a peak and its absolute content in protein (17), no attempt to compare specific activity (counts per min per optical density of the Amido black-stained protein) was made. The absorption spectrum of both types of gels or of different protein bands from the same gel was similar. Therefore, the optical density tracings for all gels were carried out at the same wave length (600 mp). The peaks present on the densitometer tracings were numbered sequentially according to their migration distance from the origin of the gel and will be further referred to by their numbers (Fig. 5A). Examination of

FIG. 4. Changes in the electrophoretic pattern of membrane proteins during the greening process. Dark-grown cells were exposed to the light or dark for different periods of time. The cells were homogenized, fractionated on the urografin gradient, and the membrane proteins (200 pg) of Fraction 5 were analyzed by electrophoresis as described under “Materials and Methods”; d and I indicate dark- and light-incubated cells, respectively. The numbers indicate the time of incubation in hours. Numbers near arrows indicate the peak numbers as defined in Fig. 7. 0 denotes origin. The chlorophyll levels (micrograms per 10’ cells) for the different samples were as follows: do and de, 0.8; 12, 2.0; la, 4.5; 14, 7.0; 15, 11.0; 16, 12.7.

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Issue of September 10, 1970 G. Eytan and I. Ohad 4301

1

d0 0

-LIGHT-l HOUR

10 20 3.0 40 5.0

2.0

lo-

o- 1.0 2.0 3.0 40 5.0

DISTANCE FROM ORIGIN (cm)

FIG. 5. Changes in densitometer tracings of the electrophoretic patterns of membrane proteins as a function of greening in ab- sence or presence of inhibitors of protein synthesis. Dark-grown cells were incubated in the dark, light, or light with addition of 100 pg per ml of chloramphenicol or 0.2 pg per ml of cycloheximide. Samples were taken at different times as mentioned in the figures, and membrane proteins of Fraction 5 of gradients were analyzed by electrophoresis. For details see “Materials and Methods.” The chlorophyll levels (micrograms per 10’ cells) for the different samples were as follows: dark 7 hours, 0.8; light 1 hour, 1.2; light 3 hours, 4.4; light 5 hours, 10.2; light 7 hours, 14.7; chloramphenicol

(CAP) 3 hours, 2.6; cycloheximide (CHI) 3 hours, 1.6. Notic the similarity between the pattern of samples from cells incubated for 7 hours in the dark, 1 hour in the light (A), or 3 hours in the light with cycloheximide (C). In all three, one can easily dis- tinguish Peaks 11 and 12, characteristic of cells with a low chloro- phyll content. Also the relative heights of Peaks 7, 11, 18, and 16 are similar. The pattern is different in samples from cells incubated for 3 hours or more in the light (B) or in the light with addition of chloramphenicol (C). In all these samples the L protein peak is clearly evident.

FIG. 6. Parallel increase in the relative amount of the L protein and chlorophyll synthesis during greening. Dark-grown cells were incubated in fresh growth medium (in this experiment also containing 7 PCi per pmole of 3H-acetate) either in the dark or light. Cells were fractionated on gradients as described and the membrane proteins from Fraction 5 processed for electrophoresis. Densitometer tracings were obtained as described under “Ma- terials and Methods.” 0, optical density ratio of Peaks L to 7 of samples incubated in the light; l , optical density ratio of Peaks L to 7 of samples incubated in the dark. (Since the L pro- tein peak was absent here, the optical density ratio was calcu- lated for Peak 12 which occupies about the same region.) A, chlorophyll content of light-incubated samples; A, chlorophyll content of samples incubated in the dark. Peak numbers are as in Fig. 5A.

I I 1 I 1 1

o I 2 3 4 5 6

T I M E (hours)

the densitometer tracings showed that Peak 7 did not change in system of the chloroplast, cells were continuously labeled with intensity relative to the other peaks, and that it could be used as 3H-acetate during a greening experiment, and the radioactivity of an internal reference for the increase in intensity of the peak of L the L peak and Peak 7 was measured in cells incubated either in protein during the greening process. Using this technique, it the light or in the dark. The results of this experiment showed was found that the L protein increases parallel to the increase in that the incorporation of Sacetate into proteins of Peak 7 is chlorophyll during the greening, whereas no change was ob- similar in dark- and light-incubated samples. The radioactivity served in this peak relative to Peak 7 in the dark-incubated cells of Peaks 11, 12, and 13, which occupy the region of the L peak (Fig. 6). In order to further assess the possibility that the L in samples from dark-incubated cells, was much lower than that protein represents a class of proteins associated with the lamellar of the L peak in samples of light-incubated cells. The radio-

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4302 Biogenesis of Chloroplast Membranes. VI Vol. 245, No. 17

0 1 2 3 4 5 6

TIME (hours)

FIG. 7. Incorporation of 3H-acetate in membrane proteins during the greening process. Same experiment as in Fig. 6. The gels were cut in 0.75-mm slices, and radioactivity in each slice was determined as described under “Materials and Methods.” Loca- tion of the peaks was done prior to the slicing of the gels by densi- tometer tracing. 0, L protein of light-incubated cells; l , L protein of cells incubated in the dark; 0, protein in Peak 7 of light-incubated cells; n , protein in Peak 7 of cells incubated in the dark; A, chlorophyll of light-incubated cells; A, chlorophyll of cells incubated in the dark. The incorporation of acetate into the L protein peak is the highest in the light-incubated cells and parallels the synthesis of chlorophyll.

0 i I 02 04 06 08 10

ol! i. 80 120 160 200

CYCLOHEXIMIDE CMJCENTRATION (),g/ml) CHLORAMPHENICOL CONCENTRATION(JI~/~O

FIG. 8. Inhibition of chlorophyll synthesis and incorporation of aH-acetate into protein during greening as a function of the inhibitor concentration. Dark-grown cells were incubated in fresh growth medium in the presence of different inhibitor con- centrations. Chlorophyll was determined for each system at l- hour time intervals and the inhibition (%) calculated for the rate of synthesis in the linear phase of greening. Inhibition of pro- tein synthesis was measured by the determination of incorpora- tion of 3H-acetate in total protein during a pulse of 15 min given after 3f hours exposure of the cells to the light in the presence of the inhibitor (A). Chloramphenicol was given to the cells after a preliminary incubation in the light for 3 hours (B). Similar results were obtained when the inhibitor was given at the begin- ning of the greening. Cycloheximide causes complete inhibition at concentrations above 0.5 pg per ml (A) whereas the maximal inhibition by chloramphenicol does not exceed 50y0 (B). 0, chlorophyll; l , protein.

600 -

20 40 60 SLICE No.

0 2345 79 L 14 15 16 17 18 19

@

600

CAP ‘. :: ,.., :.CH,

20 40 60 SLICE No. 3

E,- ,3 0 2345 73 L ‘4 15 1617 18 !9

FIG. 9. Pulse labeling of the membrane proteins during green- ing in the presence of cycloheximide (CHZ) and chloramphenicol (CAP). Dark-grown cells were incubated either in the dark, light, or light with addition of protein synthesis inhibitors. Pulse was given with 3H-acetate (20 pCi per rmole, 5 X lo+ M) for 20 min, 3 hours after the beginning of the incubation. The chloro- phyll content (micrograms per lo7 cells) in the different samples was similar to that of the corresponding samples in Fig. 5. Cells were fractionated on urografin gradients, and membrane pro- teins from Fraction 5 were analyzed by electrophoresis as de- scribed under “Materials and Methods.” Chloramphenicol concentration was 100 fig per ml and cycloheximide, 0.2 pg per ml. A, cells labeled in the dark or light; B, cells labeled after incuba- tion in the light in presence of inhibitors. Notice the relatively even radioactivity in all protein peaks of samples from dark- incubated cells as compared with the high incorporation in Peaks 9, 10, and especially L protein in samples from light-incubated cells (A). Also the labeling of Peak 10 is higher in samples from cells incubated with cycloheximide as compared with those from cells incubated with chloramphenicol (B).

activity of this peak followed closely the kinetics of the chloro-

phyll synthesis (Fig. 7). InJluence of Inhibitors of Protein Synthesis on Labeling of L

Peak during Greening-It was previously shown that protein synthesis inhibitors such as chloramphenicol or cycloheximide inhibit the greening process in Chlamydomcmas cells (7, 18). In

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Issue of September 10, 1970 G. Eytan and I. Ohad 4303

TABLE II Inhibition of appearance of proton pump activity by chloramphenicol during greening process and recovery ajter removal of inhibitor

I)ark-grown cells were incubated in fresh growth medium in the light in the absence or presence of chloramphenicol (100 pg per ml). After 4 hours the cells were washed by centrifugation, and incubation continued for an additional 4 hours in the light, dark, or light with addition of chloramphenicol (166 pg per ml). The proton pump activity was measured in a system containing 50 and 100 pg per ml of chlorophyll, 0.1 M KCl, and 50 mM pyocyanine at an initial pH of 6.4 at 25”. Specific activity is defined as micromole of H+ per mg of chlorophyll per min. For details see “Materials and Methods.”

Before transfer I

After transfer

Incubation Specific activity Incubation

Light. .. .............. .. ....... Light + chloramphenicol. ......... Light + chloramphenicol. ...... Light + chloramphenirol. ........ .. Light + chloramphenicol. .. .......

1.53 0.20 0.20 0.20 0.20

Light. Light + chloramphenicol.. Light without chloramphenicol. Dark..... Light + cycloheximide.

Specific activity

-

2.06 0.23 0.86 1.03 1.40

2 47 60 88

a The specific activit,y of the control system (1.53) at the time of transfer (4 horirs of incrtbation) was considered as the lOOy, vslrie for the calculation of percentage of recovery.

order to test further the correlation between the appearance of the L peak, its labeling, the formation of the photosynthetic lamellae, and their photosynthet,ic activity, greening was per- formed in the presence of chloramphenicol and cycloheximide. The cells were continuously labeled or pulse labeled with 3H- acetate, and the distribution of the radioactivity in the gels was compared to the densitometer tracings of Amido black-stained gels. In parallel experiments, the photosynthetic proton pump activity was measured. Significant results can be obtained for such experiments if sufficient protein would be synthesized during the greening in the presence of each specific inhibitor to allow recognition and location of each protein peak in the gel after staining with Amido black. Such a situation would be achieved by partial inhibition. In order to standardize all the experi- ments, we have chosen to select proper conditions for about 50% inhibition for each inhibitor. The degree of inhibition as a function of cycloheximide or chloramphenicol concentration was measured and the results indicated that inhibition of greening is incomplete when the latter agent is used. A maximal inhibition (--6Oyc) could be obtained at a concentration of 200 pg per ml. However, cycloheximide inhibited the greening completely at concentrations of about 1 pg per ml (Fig. 8). To obtain a 50% inhibition of the greening, as measured by synthesis of chloro- phyll or incorporation of 3H-acetate into proteins, chloram- phenicol was used at 100 pg per ml and cycloheximide at 0.2 c(g per ml in all further experiments. The electrophoretic pattern of proteins, from Fraction 5 of the gradient, was compared for samples of cells incubated in the light, dark, or light with addition of inhibitors. It was found that the L protein was present in all the samples from light-incubated cells. However, in samples from cells incubated in the presence of chloramphenicol the intensity of the L band is somewhat higher than in the control cells incubated in the light, whereas in the presence of cyclo- heximide its intensity is lower and parallels the extent of chloro- phyll inhibition (Fig. 5C). Since the inhibition of the chlorophyll

synthesis and incorporation of 3H-acetate into protein became evident only after about 3 hours of illumination, the differences found in the densitometer tracings or distribution of radioactivity in the gels of samples from continuously labeled cells are expected to be small during the early phase of greening. In order to increase the difference between controls and inhibited systems,

the cells were pulse labeled for 20 min at different times during

the greening, and the distribution of the radioactivity in the gels was analyzed. The results of such an experiment, in which the pulse was given 3 hours after onset of illumination, is shown in Fig. 9. Radioactivity seems to be evenly distributed in the different peaks of proteins from dark-incubated cells (Fig. 9A). In contradistinction, radioactivity was unevenly distributed in the different protein bands in all samples incubated in the light. A broad region of high incorporation was found from Peaks 7 to 13, the highest radioactivity peak corresponding to the L protein band in samples incubated for 3 hours in the light (Fig. 9A). B somewhat similar situation was obtained when greening was inhibited by chloramphenicol or cycloheximide. In the chlor- amphenicol-treated cells the highest incorporation was found in a peak corresponding to Peak 12 and a shoulder overlapping the region of L protein. In cycloheximide-inhibited cells, incorpora- tion in these peaks was much lower while a sharp high radio- activity peak was found on the left edge of the L protein band corresponding to protein Peaks 10 and 11 (Fig. 9B). The differ- ence in the incorporation pattern between light- and dark- incubated samples is evident although the pulse was given in the early phase of the greening process during the transition from the lag to the linear phase of chlorophyll synthesis.

Labeling of Membrane Proteins during Recovery from Inhibition of Greening Process by Chloramphenicol-It is known that chloro- plast lamellae synthesized in the presence of cycloheximide are photosynthetically active whereas inhibition by chloramphenicol causes formation of inactive lamellae (7)?

As a measure of photosynthesis we have tested in the present work the photosynthetic proton pump activity which indicates the intactness of the electron transfer system as well as that of the energy coupling. This activity is very low in cells greening in the presence of chloramphenicol. When the latter agent is removed from the incubation system and cells are further incu- bated in fresh growth medium, the activity is recovered. This recovery is enhanced by addition of cycloheximide to the system incubated in the light which only partially prevents further chlorophyll synthesis (see also Reference 7). However, the synthesis of additional chlorophyll is not required since photo- synthetic activity can be recovered by further incubation of t’he cells in the dark after removal of the chloramphenicol. Results of such an experiment are shown in Table II.

2 Similar results were obt.ained by Weinhaus (19).

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4304 Biogenesis of Chloroplast Membranes. VI Vol. 245, No. 17

AP+CHI- 0.5 HOURS

CAPXHI-1.5 HOURS

20 40 60 80 SLICE No.

0 23 I, 56 789 1 !A I5 16 17 18 '9

FIG. 10. Pulse labeling of membrane proteins with 3H-acetate in cells incubated first in the presence of chloramphenicol (CAP), then transferred to cycloheximide (CHZ). Dark-grown cells were incubated in the light in fresh growth medium containing 100 pg per ml of chloramphenicol for 5 hours. The cells were washed by centrifugation and then transferred to fresh medium containing 0.2 pg per ml of cycloheximide and further incubated in the light. Samples of cells were pulse labeled for 20 min with ZH-acetate (20 pCi per rmole, 5 X lo-’ M) before transfer to cyclo- heximide and 3 hour and 13 hours after transfer. Cell fractiona- tion, electrophoresis of membrane proteins of Fraction 5 of gra- dient, densitometry, and analysis of radioactivity distribution in the gels were as described under “Materials and Methods.” A, B and C, labeling before the transfer and after ) and 13 hours after transfer of cells from chloramphenicol to cycloheximide, respectively. The chlorophyll content at the time of transfer from chloramphenicol to cycloheximide was 5.3 pg/107 cells and represented a 50% inhibition as compared to a control system incubated in the absence of chloramphenicol. Notice the pro- gressive reduction of incorporation in the L peak (B and C) after transfer to cycloheximide as compared with the high incorporation in this peak in the presence of chloramphenicol (A). Immediately after transfer to cycloheximide a high incorporation is found in the region containing Peaks 3, 4, 10, and 11 (B) and later in the region of Peaks 8, 9, 10, and especially 15 to 15 (C).

The pattern of radioactivity distribution in proteins labeled by a pulse of 3H-acetate prior to and after transfer from a medium containing chloramphenicol to one containing cycloheximide is shown in Fig. 10. It is evident that after 5 hours of incubation

” : 8.0- t- 0 c / .i f 60- 0

A. .A

/ 20(

I I / I I I 0 1 2 3 4 5 6

TIME (hrs)

FIG. 11. Effect of different concentrations of cycloheximide on chlorophyll synthesis in cells greening in the absence or presence of chloramphenicol. Dark-grown cells were incubated in fresh growth medium in the light in absence or presence of 100 pg per ml of chloramphenicol. After 4 hours the cells were washed by centrifugation, transferred to fresh medium, and further incu- bated in the absence or presence of different concentrations of cycloheximide. After an additional incubation for 1) hours the cells were pulse labeled with 3H-acetat.e (10 #Zi per pmole, 5 X lo+ M) for 20 min, and the incorporation of 3H-acetate into total protein was measured (see Table III). Control cells greening in the light and transferred at the time marked by the arrow to different cycloheximide concentrations: 0, no cycloheximide; 0, cycloheximide, 0.2 rg per ml; A, cycloheximide, 2.0 pg per ml. Cells greening in the light with addition of chloramphenicol and transferred at the time indicated by the arrow to different con- centrations of cycloheximide: l , chloramphenicol, 100 pg per ml; n , cycloheximide, 0.2 pg per ml; A, cycloheximide, 2.0 rg per ml. Notice the complete inhibition of chlorophyll synthesis by cyclo- heximide in the control cells as compared with the reduced effect at both concentrations used on the cells previously incubated with chloramphenicol.

in the presence of the former substance the highest incorporation is found in the L protein peak. After transfer to cycloheximide, a reduction of incorporation in this peak is observed already after ) hour (Fig. lOB), and very little radioactivity is incorporated in this peak when the pulse is given l+ hours after transfer to cycloheximide. However, other regions of the gels are highly labeled under these conditions, especially those corresponding to Peaks 8, 9, 14, 15, and 16 (Fig. 1OC).

The results of the above experiments indicated that synthesis of the L protein does not require concomitant synthesis of pro- teins sensitive to chloramphenicol and related to recovery of photosynthetic activity. However, it seemed that these pro- teins are required for the synthesis of chlorophyll since in chlor- amphenicol-inhibited systems the level of L protein is unaffected or even slightly increased, whereas the chlorophyll level is reduced by 40 to 50%. It was attractive to think that the synthesis of chloramphenicol-sensitive proteins requires the presence of free L proteins in the greening cells. If this were the case, it would be expected that cycloheximide would completely inhibit protein and chlorophyll synthesis in a normal greening system where no reserve of L protein is present but would have only a partial effect on a greening system previously inhibited by chloramphenicol, in which presumably free L protein has ac- cumulated. The results of an experiment devised to test this

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Issue of September 10, 1970 G. Eytan and I. Ohad 4305

TABLE III

Inhibition of pulse labeling of total proteins by different concentrations of cycloheximide in greening cells previously

incubated in absence or presence of chloramphenicol

The same experimental conditions are present as in Fig. 11. For details see “Materials and Methods.”

Preincubation

Light Light + 100 pg per ml of chloramphenicol

cg cyclo~midef cpm/lo’ cells 70 inhibition cpmflo~ cells 70

inhibition

hyl)othesis are shown in Fig. 11 and Table III. They indicate that indeed prior incubation of greening cells for 4 hours with chloramphenicol reduces the degree of inhibition of chlorophyll and protein synthesis by subsequent addition of cycloheximide.

Electrophoresis of proteins on acrylamide gels at acid pH has been used in the past for the preparation and identification of membrane proteins (10, 20). The use of this method was especially suitable in this work since no other criteria such as enzymatic activity could be used for the identification and quantitative determination of membrane proteins, enzymatic activity being lost during the process of solubilization. However, treatment of the membrane proteins with a high concentration of acetic acid and phenol, in the presence of a detergent and fl- mercaptoethanol, is so far the only method of rendering the chloroplast membrane soluble and permitting a relatively good resolution by electrophoresis. The slight modification of the method described by Takayama et al. (10) used in this work en- abled us to obtain reproducible electrophoretic patterns.

A major problem frequently encountered with this method is that some of the proteins do not penetrate the gel (20). Rou- tinely about 70% of the protein radioactivity layered on the gel did migrate. It is very probable that the rest of the proteins which precipitated at the start of the gel were a mixture in which all t,he proteins were represented in proportion to their concentration in the original solution. That this might be the case is indicated by preliminary experiments of fractionation of those proteins on Bio-Gel columns equilibrated with the same solvent as the one used for the solubilization of the membranes. When fractions obtained from such columns were analyzed on acrylamide gels it was found that it was possible to separate the membrane proteins by column chromatography in a pattern similar to that obtained by the gels. Yet, a small amount of precipitated protein was found at the start of the gels from each fraction obtained from the column. Thus, it might be possible that the protein plug at the start of the gels is a mixture of all the proteins. It should be mentioned that results obtained by Green et al. (20) and Raein clearly indicated that the protein plug is a mixture of all the migrating proteins.

Quantitation of protein content of the different peaks by densitometry and its difficulties have been considered by several authors (cf. Pert and Pinteric (17)). In this work we did not

3 S. Razin, personal communication.

attempt to measure the absolute amounts of the protein in the different bands on the gel since the extinction coefficient of the Amido black-protein complex for the different proteins is variable and not known. Thus we have only attempted to see whether there is a relative increase of some of the density of the bands during the greening as compared to an internal standard. In order to minimize the errors we have worked at conditions of proportionality between the optical density and the amount of protein layered on the gel. The arbitrary choice of Peak 7 as the internal standard was justified by its Constance in the gels of the proteins obtained from greening cells. It is possible that this peak represents proteins of mitochondrial origin. This would be in agreement with the fact that the mitochondrial content does not change during the greening (3). It would also imply that the mitochondrial contamination in the gradient Fraction 5 is rather constant. It is not yet possible to dis- tinguish among the peaks present in the gels from samples ob- tained from dark-grown cells which proteins belong to the chloro- plast and which to the mitochondria or membranes of other cellular origin. At least some of the peaks must belong to the chloroplast since the membrane-bound photosynthetic cyto- chromef is present in the cells (3,4,9). ,4n acidic electrophoretic pattern similar to that obtained in our work Ivas reported by Schmitz, Bauer-Staeb, and Braunitzer (21) for chloroplasts isolated from a variety of sources. Use of mutants lacking protein components of the photosynthetic electron chain such as those described by Levine (1) might help in the ident’ification of the chloroplast membrane proteins. The L protein peak can be clearly identified as a chloroplast lamellar protein. It is certain that this peak represents a protein or proteins formed de novo during the greening. The possibility that it represents a drastic increase in Peaks 11 and 12 causing their fusion and overlapping is very improbable since in this case one would expect these to be resolved again as two separate peaks when the amount of protein layered on the gel is gradually reduced. However, Peaks 11 and 12 in samples from dark-grown cells or the L protein peak in samples from light-incubated cells retain their identity at all protein concentrations between 50 pg and 200 pg per gel. The possibility should be considered that the L protein is present in the dark-grown cells but not associated with the membranes in Fraction 5 of the gradient and thus lost during the fractionation. The L protein could not be detected by gel electrophoresis in the total protein fraction sedimented at 100,000 X g per hour ob- tained from dark-grown cells. However, it was easily detected in a similar protein fraction obtained from greening cells in spite of the abundance of protein bands in the gels. Thus it seems unlikely that the L protein is present in the dark-grown cells and becomes associated with the photosynthetic lamellae during the greening process. The results of experiments of continuous labeling and pulse labeling are in agreement with the assumption that the L protein is formed de nova during the greening. Whether the L protein represents a single protein or a group of similar proteins cannot be ascertained at the present time. The broad shape of the L protein peak might indicate that it indeed represents a group of closely migrating proteins. One should also mention that occasionally the correlation between the radioactivity and optical density peaks is not exact as shown in Fig. 9B. This might be due partially to difficulties in the exact alignment of the gels during slicing for radioactivity analysis and to the lack of resolution of this technique since certain protein bands are quite narrow relative to the width of the slices (0.7

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4306 Biogenesis of Chloroplast Membranes. VI Vol. 245, No. 17

LIGHT

”

PHOTOSVNTKTIC

FIG. 12. Schematic representation of the proposed control pat- tern involved in the formation of chloroplast lamellae in C. reinhardi y-l mutant cells. Heavy arrows indicate synthesis of building blocks (enclosed in circles); solid or dotted arrows desig- nate stringent or partial control by the presence of one component on the synthesis of other components; crossed arrows indicate site of inhibitors action. For explanation see text. CHI, cyclo- heximide; CAP, chloramphenicol.

mm). It is also possible that when a protein band consists of a group of closely migrating proteins one should not expect an

exact matching between the optical density and the radioactivity peak since we cannot infer an exact correlation between the staining intensity and actual amount of protein (i.e. radioac- tivity).

The results of the experiments in which greening and con- tinuous or pulse labeling were done in the presence of inhibitors of protein synthesis indicate that the synthesis of L protein is sensitive to cycloheximide but not to chloramphenicol, and thus one can correlate t.he synthesis of this protein with the activity of the cytoplasmic ribosomes (22). The distribution pattern of radioactivity in the different protein peaks during pulse labeling after transfer of the cells from chloramphenicol to cycloheximide indicates again that the L protein synthesis diminishes in the presence of the latter agent. The results of this experiment also clearly show that other proteins such as Peaks 8, 14, 15, and I6 are not labeled in t,he llresence of chlor- amphenicol. However, radioactivity is incorporated in these proteins when chloramphenicol is removed even in the presence of cycloheximide thus indicating that the synthesis of these proteins is associated with chloroplast ribosomes4

4 The peaks labeled during the transfer of greening cells from chloramphenicol to cycloheximide might not be associated with the chloroplast membranes only since it is reasonable to assume that mitochondrial ribosomes can contribute in a similar way to the labeling of mitochondrial proteins present as a contamination

From these considerations it can be concluded that two classes of proteins are involved in the formation of the chloroplast lamellae, those which are synthesized by the cytoplasmic ribo- somes as well as those synthesized by the chloroplast ribosomes (cf. Hoober, Siekevitz, and Palade (7)). In addition the process requires transport of cytoplasmic “insoluble” proteins such as the L protein6 across the chloroplast envelope and their specific association with the growing photosynthetic lamellae. The mechanism through which this transport and “target recognition” is achieved remains so far completely unknown.

Concurrence of proteins from cytoplasmic and mitochondrinl origin in the formation of the mitochondrial membranes has also been reported (23,24) and the results of our work stress again the similarity between these two types of organelles.

The photosynthetic specific activity of membranes synthesized in normal greening systems is equal to that of membranes formed in the presence of cycloheximide at all levels of inhibition up to 100%. However, the specific activity of membranes, synthesized in the presence of chloramphenicol is exceedingly lower even at low degrees of inhibition as shown in this work for the proton pump activity (see also Hoober et al. (7), Wallach,’ and Weinhau9). The L protein is present in normal amounts in chloramphenicol-inhibited greening cells which do contain rather large amounts of photosynthetically inactive lamellae in the chloroplasts. Removal of chloramphenicol inhibition induces repair of the photosynthetic electron transfer activity and energy coupling as demonstrated by the reactivation of the proton pump simultaneously with synthesis of certain proteins (Peaks 8, 14, 15, and 16) related to activity of chloroplast ribosomes. Thus a basic conclusion we can draw from this study is that the chloro- plast lamellar proteins synthesized by the cytoplasmic ribosomes (L protein) can be related with the appearance of the membrane structures while the proteins of chloroplast origin seem to be associated with the photosynthetic activity (cf. Smillie et al. (25) and Margulies (26)).

The question arises as to what degree the synthesis and as- sembly of these proteins are correlated and to what extent a mutual control exists between these two types of proteins.

Since the L protein and lamellar structures are not formed in absence of chlorophyll synthesis (i.e. in the dark) we might assume that chlorophyll formation is required for the synthesis of L protein. However, there is no stringent cont,rol between the level of chlorophyll and that of the L protein since in chlor- amphenicol-inhibited greening cells the level of L protein is similar or even higher than that of the control cells whereas t,he chlorophyll synthesis is only half of the normal level. The synthesis of the L protein by the cytoplasmic ribosomes SCCI~S to require neither presence nor concomitant synthesis of nct.iva- tion proteins by the chloroplast ribosomes. On the other hand, the synthesis of protein by the chloroplast ribosomes requires either concomitant synthesis of the L protein or its presence in excess amounts over the normal counterpart of activation pro- proteins. This is indicated by the fact that cyclohesimide which inhibits the synthesis of lrrotein by the cytoplasmic ribosomes (L protein) (22) can block completely synthesis of

in the gradient Fraction 5. However, this contribution is prob- ably very small since most of the biosynthetic activity in the greening cells is associated with the formation of chloroplnst material (6).

5 It is possible that these proteins are initially soluble and are subsequently modified and rendered insoluble while becoming associated with the photosynthetic lamellae.

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Issue of September 10, 1970 G. Eytan and I. Ohad 4307

protein by both cytoplasmic and chloroplast ribosomes when added at appropriate concentration at all times during the greening process. This would result also in the complete block of chlorophyll synthesis (see Fig. 12). However, incubation of greening cells with chloramphenicol which allows accumulation of excess L protein, protects the cells against the inhibitory effect of cycloheximide on the synthesis of the active proteins by the cytoplasmic ribosomes.

In addition, the synthesis of the activation proteins can occur in the dark and therefore it does not require concomit,ant synthe- sis of chlorophyll. However, it seems that the synthesis of chlorophyll which is light dependent in the y-l mutant cells (2, 3) is controlled to a certain degree by the level of the activation proteins. This is indicated by the fact that chloramphenicol which allows synthesis of normal levels of the L protein partially inhibits the synthesis of rhlorophyll. Thus it is reasonable to assume that in the greening Chlamydomonas y-l cells a kind of feedback control exists between chlorophyll, cytoplasm, and chloroplast-synthesized lamellar proteins, all of which is tenta- tively represented in the scheme shown in Fig. 12.

On the basis of the available data it is premature to ascribe exact roles to each of the components of these schemes or to define the exact extent of control exerted by each component on the rest of the system. The actual control pattern might indeed be much more complex. The scheme is intended only to summarize the data so far available and as a guideline for future work.

Two possible types of assembly mechanisms have been con- sidered as operative during formation of biological membranes. (a) A simultaneous or single step mechanism involving mutual comrol on the synthesis and assembly of the different com- ponents forming a functional membrane (3, 4) and (5) a multi- step mechanism by which different components might enter the membranes at different times independently (7, 27-29). The results of this work as well as data previously published on the biogenesis of the chloroplast membranes in Chlamydomonas (3, 6, 7) suggest that in this organism the different building parts of the photosynthetic lamellae might be added sequentially, but a certain degree of control is exerted by the different components resulting in a feedback control mechanism, the extent and accuracy of which remains to be studied.

;lcknowledgnlents--We would like to thank Mr. I>. Wallach, who did the measurements of the proton pump activity, Mr. I. Goldberg, who permitted us to use some of his data cited in Fig. 8, and Dr. M. Schramm for his valuable suggestions and for reading the manuscript.

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Gera Eytan and Itzhak OhadREINHARDI y-1

GREENING PROCESS IN A MUTANT OF CHLAMYDOMONAS OF PHOTOSYNTHETIC LAMELLAR PROTEINS DURING THE

CYTOPLASMIC AND CHLOROPLAST RIBOSOMES IN THE SYNTHESIS Biogenesis of Chloroplast Membranes: VI. COOPERATION BETWEEN

1970, 245:4297-4307.J. Biol. Chem. 

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