Proteins of cytoplasmic, chloroplast, and mitochondrial ribosomes of some plants

492

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96681

P R O T E I N S OF CYTOPLASMIC, CHLOROPLAST, AND MITOCHONDRIAL RIBOSOMES OF SOME PLANTS A. C. L. V A S C O N C E L O S * AND L A W R E N C E B O G O R A D

The Biological Laboratories, Harvard University, Cambridge, Mass. and The Department o/ Botany, University of Chicago Chicago, Ill. (U.S.A.) (Received J u l y i o t h , 197 o)

SUMMARY

The principal objective of this work was to compare the electrophoretic behavior of basic proteins of ribosomes from parts of the same cell. Ribosomes were isolated from maize chloroplasts and cytoplasm as well as from chloroplasts, mitochondria and cytoplasm of mung beans. Using acrylamide gel electrophoresis, basic proteins of these types of ribosomes were compared with those of ribosomes from Escherichia coli and from the blue-green alga Phormidium luridum. In part in agreement with much other work, ribosomes from the cytoplasm of the eukaryotic plants studied were found to be about 80 S and to contain RNA's with molecular weights of about 1.2-lO 6 and 0.7" lO 6• The ribosomes of the various chloroplasts and mitochondria, as well as of P. luridum, sediment as approx. 7o-S particles and contain RNA's with molecular weights of about I.I. lO 8 and o.58"Io ~. However, each type of ribosome studied here could be clearly distinguished from any other, including those from E. coli, because of easily observed differences in the electrophoretic behavior of its proteins.

INTRODUCTION

The earliest studies of leaf ribosomes l, ~ revealed that two types were present with regard to sedimentation characteristics and size. Chloroplast ribosomes were shown to be smaller and to sediment less rapidly (about 70 S) than cytoplasmic ribosomes (about 80 S) in spinach and maize. In addition, mitochondria of leaf cells of swiss chard have been shown to contain ribonuclease digestible particles which appear to be ribosomes 3. Mitochondria of yeast, rats and some other eukaryotes have been shown to contain ribosomes of about 7 o S (ref. 4) but initochondrial ribosomes from green eukaryotic plants had not been isolated prior to the present work. The antibiotic chloramphenicol inhibits protein synthesis by ribosomes in prokaryotes as well as in chloroplasts and mitochondria but appears to not affect synthesis by cytoplasmic ribosomes. Conversely, cyclohexamide seems to inhibit protein synthesis by cytoplasmic but not by chloroplast, mitochondrial or prokaryote * P r e s e n t address: R u t g e r s , T h e S t a t e U n i v e r s i t y , D e p a r t m e n t of B i o c h e m i s t r y a n d Microbiology CAES, N e w B r u n s w i c k , N . J . o89o3, U.S.A.

Biochim. Biophys. Acta, 228 (1971) 492-502

RIBOSOMAL PROTEINS OF SOME PLANTS

493

ribosomes. The locations of the genomes for chloroplast and mitochondrial ribosomes have not been determined and there has been much speculation about the phylogenetic and ontogenetic origins of chloroplasts and mitochondria. In view of all these problems it appeared profitable to compare sedimentation properties and the nature of the proteins in ribosomes from different species (e.g. maize and mung bean cytoplasmic ribosomes; 7o-S ribosomes from these plants as well as from Escherichia coli and from the blue-green alga Phormidium luridum) and in ribosomes from different parts of the cell of a single species (e.g. mung bean cytoplasmic, mitochondrial and chloroplast ribosomes). We were particularly interested in whether mung bean mitochondrial and chloroplast ribosomes might differ despite the similarities in sedimentation properties which were expected. After it was established that they were both about 70 S, the acrylamide gel electrophoresis of their proteins estabhshed their individuality although there were some bands which migrated similary.

EXPERIMENTATION AND RESULTS

Isolation o[ ribosomes As noted above, the sedimentation coefficients of both chloroplast and cytoplasmic ribosomes of leaves of some species have been determined. These characteristics were confirmed here incidental to the development of procedures for the purification of ribosomes from these sources for examination of ribosomal proteins. Detailed procedures for the purification of ribosomes are given in order to establish the qualify of the preparations used for analysis of ribosomal proteins. Ribosomes from isolated chloroplasts or mitochondria or from supernatant solutions from which the organdies were prepared were purified by sedimentation through sucrose followed by one or more cycles of sucrose density gradient centrifugation. Analysis of rRNA, generally by gel electrophoresis, was used to check cross-contamination of ribosomal types and to insure further against errors which might have arisen in the selection of bands from preparative sucrose gradients of ribosomes.

Maize plastid ribosomes Plastids were prepared from maize plants (Zea mays), WF9Tms × B37 (Illinois Foundation Seeds, Inc. Champaign, Ill.) grown in darkness at 28 ° for 7-9 days and exposed to light for 3 h before harvest. Cleaner plastid ribosomes could be obtained more easily from this material than from light-grown fully green plants. Chilled leaves were ground with I ml of 0. 5 M sucrose-Tris-25 mM MgCl~-spermidine (o.I M Tris-HC1-25 mM MgC12-I mM spermidine; pH 8.0) medium per g of tissue. The homogenate was filtered through muslin and centrifuged for 5 rain at 121 ×g at 2 ° to remove large particles. The supernatant fluid was centrifuged at IOOOx g for IO rain. The pellet, containing mainly chloroplasts, was resuspended in o.5 M sucrose-Tris-25 mM MgCl~-spermidine buffer and centrifuged through a o-65 % sucrose (in Tris-25 mM MgCl~-spermidine buffer) gradient in an International B-6o centrifuge using the B XV zonal rotor. Separation was achieved by centrifugation for 45 rain at 15 ooo rev.]min. The chloroplast fractions were collected from the zonal centrifuge and diluted with Tris-25 mM MgC12-spermidine buffer to approx. 0.5 M sucrose and then concentrated by centrifugation. Bioch~m. Biophys. Acta, 228 (1971) 492-5o2

494

A . C . L . VASCONCELOS, L. BOGORAD

Ribosomes were extracted from the chloroplasts b y suspending the pellet in Tris-25 mM MgC12-spermidine buffer containing 5 ~o Triton X-Ioo. After IO rain at o ° the broken plastid suspension was centrifuged at 12 o o o × g for 15 rain. The supernatant fluids from these centrifugations were pooled and approx. 8.5 ml of extract was layered on to 5 ml of I M sucrose-Tris-5 mM MgC12-spermidine buffer contained in a cellulose nitrate centrifuge tube (Tris-5 mM MgC12-spermidine buffer is 5 mM rather than 25 mM with regard to MgC12 but is otherwise the same as Tris25 mM MgC12-spermidine buffer). The ribosomes which were sedimented through the I M sucrose by centrifugation at 164 905 × g for 5 h (in a Ti-5o rotor of a Spinco model L2-B65 centrifuge) were resuspended in 0. 5 ml of Tris-5 mM MgCl2-spermidine buffer. The suspension was clarified by centrifugation at 17 300 × g for IO rain and the concentration of RNA in the solution was estimated speetrophotometrically. An absorbance of 20 at 260 nm was taken to be that of a solution containing z m g of RNA per ml. Ribosomes were then further purified by centrifugation in sucrose density gradients. Ribosome suspensions containing about 1.2 mg of RNA were layered on lO-34 ~o linear gradients of sucrose in Tris-5 mM MgC12-spermidine buffer formed in Spinco SW 27 type cellulose nitrate centrifuge tubes. Centrifugation was for 8-1o h at 4 ° and at 26 ooo rev./min (IOO ooo ×g). Gradients were analyzed using an ISCO density gradient fractionating apparatus and 254 nm ultraviolet flow analyzer. Fig. I a shows that the preparation contained essentially one size of ribosome. Maize plastid ribosomes for analyses of proteins were obtained by fractionating these gradients. Ilb

]a

0.6

0.6

~

04 0.3

O0.4 0.2 0.2 O.I J

I

(Bo,o5~ 15

,

25

I

,

35 45

FraCtion NO,

2 {Top)

4

6

8

10

Cm

Fig. I. Maize chloroplast ribosomes: (a) The distribution of 254-nm absorbing material after centrifugation of the chloroplast ribosome preparation in a lO-34 ~o sucrose density gradient. (b) Profile of 26o-nm absorption after polyacrylamide gel electrophoresis of RNA prepared from band "'x" of (a).

Maize plastid ribosomes moved in a sucrose gradient together with ribosomes (7° S) of tritiated E. coli. In addition, they were found to be about 7 ° S in studies with the Spinco Model E centrifuge. The RNA's of maize plastid ribosomes recovered from sucrose gradients were analyzed by gel electrophoresis as a further check on the freedom of preparations from ribosomal cross contamination. Biochim. Biophys. Acta, 228 (I971) 492-502

495

RIBOSOMAL PROTEINS OF SOME PLANTS

rRNA was prepared b y treatment of ribosomes with 2 % sodium dodecylsulfate at room temperature for 20 min. Then the samples were clarified b y centrifugation for 15 min at 12 o o o x g . 2 vol. of chilled (--20 °) 95 % ethanol were added to the supernatant fluid; after 20 min at --20 ° the precipitate was collected by centrifugation at 27 o o o × g at --20 ° and dried under vacuum. The RNA was dissolved to a concentration of I mg/ml in the electrophoresis buffer to which IO % sucrose had been added. The gels were prepared, run and analyzed essentially following the method of LOENING6. E. coli rRNA was used as a standard to estimate sizes of other rRNA's analyzed b y acrylamide gel electrophoresis. The data in Fig. I b show that the maize chloroplast ribosome preparation shown in Fig. I a contains two major electrophoretically separable rRNA's; these have molecular weights of 1.o 9. IOe+O.O2.IO e and o.58.IO e ±0.02. lO e. (LOENING AND INGLEv observed that when RNA is extracted from either whole leaves or from isolated plastids of a number of species, acrylamide gel electrophoresis shows the ratio of the approx, o.6.1o 6 to the approx. I . i . I O e molecular weight chloroplast rRNA to be one or greater. In all cases examined here, analysis of rRNA's prepared from isolated "whole" ribosomes shows the two types to be present in about the expected proportions. This suggests that the relationships observed by LOENING AND INGLEv m a y not result simply from breakdown during isolation of RNA.)

Maize cytoplasmic ribosomes Maize cytoplasmic ribosomes were prepared from the supernatant fluid of the iooo × g pellet from which maize chloroplasts had been removed. After centrifugation at 34 ooo × g for 20 min to remove broken plastid material from the solution, aliquots were layered over I M sucrose in Tris-5 mM MgC12-spermidine buffer and sedimented as described for chloroplast ribosomes. Further purification was achieved b y centrifugation twice in lO-34 °/o sucrose density gradients of the kind described above. Ribosomes in the most prominent band of the second gradient (Fig. 2a) were found to be approx. 80 S from experiments with the Spinco Model E centrifuge and b y co-centrifugation in sucrose density gradients with 32p-labeled ribosomes from , 2o

0,8

0.6

0.4

0.2

~ ~b 2 0

06

2b

0.5 Ec 0.4

$ , ~ 0.21 0.2

j

~o' ~o'

Fraction No.

1.0

2

4

fi

B

10

Cm

Fig. 2. Maize c y t o p l a s m i c ribosomes: (a) T h e d i s t r i b u t i o n of 2 5 4 - n m a b s o r b i n g m a t e r i a l after sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of a c y t o p l a s m i c r i b o s o m e p r e p a r a t i o n for t h e second time. (b) P o l y a c r y l a m i d e gel electrophoresis of t h e R N A e x t r a c t e d f r o m t h e r i b o s o m e s c o n s t i t u t i n g t h e m o s t p r o m i n e n t b a n d in (a).

BiocMm. Biophys. Acta, 228 (1971) 492-502

496

A.C.L.

VASCONCELOS, L. BOGORAD

anaerobically grown yeast cells. Ribosomes in this band were collected for analysis of ribosomal proteins; the preparations were found to contain two RNA's by gel electrophoresis (Fig. 2b) of molecular weight 1.26.IO8~O.O2-io 6 and o.68.1o6± o.o2.1o ~. (The molecular weight values corresponding to the "25-S" and "I8-S" cytoplasmic rRNA's in Pisum, Phaseolus, Zea mays, and Raphanus were judged to be approx. 1.27. IoS-I.3 I. lO 6 and 0. 7. lO6, respectively by LOENINGS.)

Mung bean chloroplast ribosomes Mung beans (Phaseolus aureus) were grown in continuous light at 25 ° in the greenhouse for 6- 7 days. The leaves were harvested, chilled and ground in a Waring Blendor for 30 sec using 3 ml of a grinding medium (o.5 M Tris-HC1-5o mM MgC]225 mM KC1-4 mM mercaptoethanol-o.5 M sucrose; pH 8.0) per g of tissue. The homogenate was filtered through muslin. The chloroplasts which sedimented at IOOOXg were washed 3 times by resuspending them, each time in 30 ml of grinding medium per tube; each tube contained chloroplasts from about 5 ° g of leaves. The chloroplasts were treated with Triton X-ioo and ribosomes were extracted from the plastids and purified as described in the maize chloroplast experiments, except that Tris-25 mM MgCl2-spermidine buffer was used instead of Tris-5 mM MgCl~-spermidine buffer throughout and purification was through a series of two sucrose density gradient centrifugations. Fig. 3a shows the 254-nm absorption profile in a sucrose density gradient after the first centrifugation. The fraction under the peak indicated by "x" was collected, the ribosomes were concentrated by centrifugation and were then run in a second linear lO-34 ~o gradient of sucrose in Tris-25 mM MgCl2-spermidine buffer (Fig. 3b). 1.8 i 3c ]

3a

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35

45

c 1,0.

.-q

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I

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0.2

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~ 08

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0.4

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02

"~ 30 .

.

.

.

2

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10

Fig. 3. M u n g b e a n chloroplast ribosomes: (a) T h e 2 5 4 - n m a b s o r p t i o n profile after t h e first d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of t h e r i b o s o m e p r e p a r a t i o n . T h e m a t e r i a l u n d e r t h e b a n d m a r k e d "'x" was collected, c o n c e n t r a t e d b y centrifugation, r e s u s p e n d e d , a n d r u n in a second sucrose d e n s i t y gradient. (b) T h e 2 5 4 - n m a b s o r p t i o n profile after t h e second sucrose d e n s i t y g r a d i e n t centrifugation. T r i t i a t e d E. coli r i b o s o m e s were included. T h e r a d i o a c t i v i t y profile of t h e E. coli r i b o s o m e s (- - -) is seen to coincide w i t h t h e a b s o r b a n c e profile of t h e m u n g b e a n chloroplast ribosomes. (c) T h e b e h a v i o r on p o l y a c r y l a m i d e gel electrophoresis of R N A e x t r a c t e d from t h e m a j o r p e a k of sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n s of t h e kind s h o w n in (b).

The sedimentation properties of mung bean chloroplast ribosomes were studied in the Spinco Model E centrifuge as well as by centrifugation in sucrose gradients together with tritiated E. coli ribosomes as shown in Fig. 3b. They were Biochim. Biophys. Acea, 228 (1971) 492-5o2

497

RIBOSOMAL PROTEINS OF SOME PLANTS

found to be of the "7o-S '' type. (The slower moving minor band in Fig. 3b is probably composed of 5o-S and/or 6o-S ribosomal subunits from 7o-S or 8o-S ribosomes, respectively.) Ribosomes prepared and purified by centrifugation through two sucrose gradients were collected and used for gel electrophoretic analyses of ribosomal proteins. The two rRNA's in these preparations migrated like 23-S and I6-S species (Fig. 3c) judging from simultaneous electrophoresis with E. call rRNA's. M u n g bean mitochondrial ribosomes

Mitochondria were isolated from hypocotyls of etiolated 5-day-old P. aureus seedlings ("bean sprouts" bought from Hung Food Products, Brighton, Massachusetts) by a modification of a method used by PARSONS et al. ~ for isolating liver mitochondria. After removing the cotyledons and primary leaves, the hypocotyls were chilled and ground in an automatic mortar and pestle, using 2 ml of grinding medium (0.25 M Tris-HC1-25 mM MgC12-o.I mM EDTA-4 mM mercaptoethanol-o.28 M sucrose; pH 7.2) per g of tissue. In a typical preparation, the homogenate of about 75° g of hypocotyls was filtered through muslin and centrifuged at 650 xg for IO rain. The supernatant fluid was collected and spun at 14 600 ×g for 12 min. The supernatant fluid of the second centrifugation was removed by suction and care was taken to wash out the "fluffy" layer on top of the pellet. The pellets were resuspended ip 320 ml of grinding medium to which i mM spermidine had been added. The resulting suspension was centrifuged at 5o0 xg for Io rain. The supernatant fluid was removed with a hypodermic syringe and centrifuged first at I9ooxg for 8min and then continued at IO 8ooxg for 2 rain. The supernatant fluid was discarded and the pellet washed twice using 320 ml of solution each time. This procedure gave about 75 mg of mitochondria per kg of hypocotyls. Ribosomes were liberated from the mitochondria by suspension in Tris-25 mM MgC12-spermidine buffer (pH 7.8) containing 5 % Triton X-Ioo. The ribosomes were purified by centrifugation through I M sucrose and by density gradient centrifugation as described above. The absorbance profiles (254 nm) of two successive sucrose gradients are shown in Figs. 4a and 4b.

~2 4, ,

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No.

Fig. 4. M u n g b e a n m i t o c h o n d r i a l ribosomes: (a) T h e 2 5 4 - n m a b s o r p t i o n profile after sucrose d e n s i t y c e n t r i f u g a t i o n of t h e r i b o s o m e p r e p a r a t i o n . T h e m a t e r i a l in t h e b a n d m a r k e d " x " w a s collected, c o n c e n t r a t e d , a n d c e n t r i f u g e d in a second sucrose d e n s i t y gradient. (b) T h e 2 5 4 - n m a b s o r p t i o n profile of t h e m a t e r i a l f r o m b a n d " x " after a second sucrose d e n s i t y g r a d i e n t c e n t r i f u g a tion. (c) As in (b) b u t t r i t i a t e d E. call r i b o s o m e s were included (- - -).

BiocMm. Biophys. Acta, 228 (1971) 492-502

498

A.C.L.

V A S C O N C E L O S , L. B O G O R A D

The sedimentation properties of mung bean ribosomes recovered from gradients like the one shown in Fig. 4b were determined in the Model E Spinco centrifuge and also by centrifugation in sucrose density gradients together with tritiated E. coli ribosomes (Fig. 4c). The mung bean mitochondrial ribosomes were found to be approx. 7 ° S. The rRNA from mung bean mitochondria separated into ~wo classes on a 15-3o ~/o sucrose-sodium dodecylsulfate gradient. The samples were run for 5 h in the SW 39 rotor of the Spinco centrifuge at IOO ooo x g at 4 °. The gradients indicated that we were isolating the "7o-S '' ribosomes as a unit and not the 5o-S subunit alone or some possible aggregates of ribosomal subnnits. Ribosomes, collected in a band from gradients like the one shown in Fig. 4 b were used for analyses of proteins. Mung bean cytoplasmic ribosomes Cytoplasmic ribosomes of mung beans were prepared from the sup~rnatant fluid obtained in the mitochondrial isolation step which yielded the IO 800 x g pellet. These ribosomes were purified and concentrated by centrifugation through I M sucrose and further purified by a series of two sucrose density gradients (as described for maize cytoplasmic ribosomes) before use for analysis of ribosomal proteins. Fig. 5a is a profile of absorbance at 254 nm of a "second" gradient tube. The RNA of material from the major band of Fig. 5a was examined by gel electrophoresis (Fig. 5b) and found to consist of RNA's with molecular weights of 1.31" IO~4-o.o2 •IO~ and o.68. IO6~O.O2 •lO -6.

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No.35

45

2 Cm

Fig. 5. Mung bean cytoplasmic ribosomes: (a) The distribution of 254-nm absorbing material after a "second" density gradient centrifugation. (b) Polyacrylamide gel electrophoresis of RNA recovered from the major band shown in (a). Phormidium luridum ribosomes Ribosomes were prepared from Phordimium luridum by fiist washing cells in Tris-25 mM MgC12-spermidine buffer to which an extra I mmole of spermidine had been added per 1. Cells were collected by centrifugation at 30 000 × g for 15 min and were broken by 5 cycles of freezing in liquid nitrogen and thawing. To achieve breakage, I g of cells was suspended in I0 ml of the washing solution. After every second cycle the cells were centrifuged down, the supernatant fluid was collected, and 5 ml of additional medium was added to the pellet. Biochim. Biophys. Acta, 228 (~97~) 492-5°2

499

RIBOSOMAL PROTEINS OF SOME PLANTS

Ribosomes were purified as in the other cases, by centrifugation through I M sucrose-Tris-5 mM MgCl~-spermidine buffer and then through lO-34 % sucroseTris-5 mM MgCl~-spermidiue buffer gradients before being used for analysis of rRNA or protein. The profile of absorbance at 254 nm within a gradient after centrifugation is shown in Fig. 6a. Ribosomes from P. luridum sediment in sucrose gradients together with E. coli ribosomes which are 7 ° S (Fig. 6a). Acrylamide gel electrophoresis of the P. luridum rRNA's (Fig. 6b) showed them to be similar in size to those of E. coli rRNA's. 0.71

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35

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4

5

8

10

cm

Fig. 6. P. luridum ribosomes: (a) The distribution of 254-nm absorbing material from a ribosome preparation together with t h e radioactivity profile of a d d e d tritiated E. coli ribosomes (- - -). The m a j o r b a n d from gradients of this sort from which E. coli was o m i t t e d was collected and 1RNA was e x t r a c t e d for analysis of polyacrylamide gel electrophoresis. (b) The results of t h e polyacrylamide gel electrophoresis of t h e 1RNA p r e p a r e d as in (a).

Analyses o/ribosomal proteins Ribosomes purified by one or two cycles of sucrose density gradient centrifugation, as described in each case, were collected by centrifugation and the methods of GESTELAND AND STAEHELIN9 were used to obtain ribosomal proteins and to analyze the proteins by acrylamide gel electrophoresis at pH 4.5 in 8 M urea. The electrophoresis was carried out at 4 °. The gels were stained with i °/o Coomassie blue in 7.5 % acetic acid and IO % methyl alcohol, for 3 h, and then destained with 7.5 % acetic acid, IO °/o methyl alcohol, and a bed of AG 5oI-X8(D) 20-50 mesh (analytical grade mixed red resin from Calbiochem) was used to speed up the destaining. The destained gels were stored in the dark in 7.5 % acetic acid. The basic proteins derived from each type of ribosome separated to provide a recognizable pattern of bands in the gel (Figs. 7a, 7b). Data on the number of protein bands formed during electrophoresis of the ribosomal proteins from the various sources studied here are summarized in Table I. The table also indicates the number of similarly migrating bands present in more than one type of ribosome; this information only indicates the maximum possible number of similarly migrating bands. Since each band could include more than one protein and even identical (as opposed to apparently similar) migration does not prove identity of proteins, it did not seem profitable to pursue similarities at this time by using split gel techniques, etc. The data do show clearly that the pattern of basic proteins of each type of ribosome studied -- including mitochondrial and chloroplast ribosomes of mung beans -- is distinctively different. Biochim. Biophys. Acta, 228 (i97 x) 492-5o2

500

A . C . L . VASCONCELOS, L. BOGORAD

~Ta

=, =, ,_

~=_

=,

M u n g bean mrtochondr[a

Mung bean chloroplast

Maize chlor'oplast

E. col/

R lur/dum

Maize cytoplasm

M u n g bean cytoplasm

Fig. 7. (a) Polyacrylamide gel electrophoresis patterns of basic ribosomal proteins. (i) mung bean mitochondria; (2) mung bean chloroplast; (3} E. coli; (4} maize chloroplast; (5) mung bean cytoplasm; (6) maize cytoplasm; (7) P- luridum. Conditions used in electrophoresis, staining, etc. are described in the text. (b) Diagrams of ribosomal protein patterns based on the acrylamide gels shown in (a). Biochim. Biophys. ~4cta, 228 (i971) 492-5o2

RIBOSOMAL PROTEINS OF SOME PLANTS

501

TABLE I A SUMMARY OF THE RESULTS OF GEL ELECTROPHORESIS OF PROTEINS FROM VARIOUS TYPES OF RIBOSOMES T h e m a x i m u m n u m b e r of b a n d s w h i c h m a y b e c o m m o n t o v a r i o u s t y p e s of r i b o s o m e s . M a x i m u m n u m b e r of b a n d s p o s s i b l y c o m m o n t o all 7o-S r i b o s o m e s = 3. M a x i m u m n u m b e r of b a n d s p o s s i b l y c o m m o n t o all 7o-S a n d 8o-S r i b o s o m e s = 2.

Sources of ribosomes

Mung bean mitochondria

Mung bean mitochondria

Mung bean cytoplasm

5

Mung bean cytoplasm

Mung bean chloroplast

Maize cytoplasm

Maize chloroplast

P. luridum

E. coli

5

5

7

3

io

4

8

4

4

12

2

7

3

5

Mung bean chloroplast Maize c y t o p l a s m

9

Maize c h l o r o p l a s t

4

8

5

9

P. luridum

7

E. coli Total number of b a n d s

25

25

18

18

21

16

30

DISCUSSION

WALLER AND HARRIS1°, who studied E. coli ribosomal proteins by starch gel electrophoresis, first showed that many different proteins are associated in a single kind of ribosome. The findings have been confirmed by many others using different techniques. Furthermore, the complicated band patterns obtained have been shown to be due to the presence of numerous proteins rather than to reversible proteinprotein interaction, isomerization or protein-buffer interaction. In the present work, basic proteins from ribosomes with approximately the same sedimentation coefficient but from different organisms (e.g. mung bean and maize cytoplasm; mung bean plastids and mitochondria, maize plastids, E. coli, P. luridum) or from different organelles of a single organism (mung bean cytoplasm, mitoehondria, and chloroplasts; maize cytoplasm and chloroplasts) were frequently found to separate into different numbers of differently migrating bands on acrylamide gel electrophoresis. For example, 7o-S ribosomes of maize chloroplasts showed 21 bands, while the proteins of 7o-S ribosomes of mung bean chloroplasts separated into 18 bands. The same sorts of observations have been made in comparisons of chloroplast ribosomes of other plants n-13. Proteins which migrate separately in acrylamide gel electrophoresis are undoubtedly unlike and may differ in amino acid sequence, in conformation or in state of aggregation. However, two different proteins could be indistinguishable on the basis of their mobility in acrylamide gels. Thus, the number of bands into which the proteins of a particular ribosomal type separate may be smaller than the actual Biochim. Biophys. Acta, 228 (1971) 4 9 2 - 5 0 2

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A . C . L . VASCONCELOS, L. BOGORAD

number of different proteins in the ribosome. From these considerations it seems unprofitable to t r y to ascribe a n y particular significance to differences in the number of protein bands found when various types of ribosomes are analyzed; attempts to infer degrees of relatedness of ribosomes on the bases of relative numbers of bands in common among them also seems of questionable value. On the other hand, since variations in migration do undoubtedly reflect differences among proteins some conclusions can be drawn from differences observed here. The ribosomes of mung bean chloroplasts and mitochondria are indistinguishable on the basis of their sedimentation coefficients but most, if not all, of the proteins of ribosomes from these two organelles are distinctly different. The different proteins are coded for by different genes but we do not know whether these genes are within one or more genomes. However, these observed differences in proteins argue that, at least in P. aureus, mitochondria and chloroplasts do not originate anew each generation from nuclear material as has been suggested for some ferns 14. No evidence is presel~ted here that a n y two similarly migrating bands are identical, let alone that any two kinds of ribosomes contain a common protein. However, the occurrence of bands which seem to migrate about the same suggests the possibility that ribosome formation and/or function in one cell compartment might be controlled b y some information and protein synthesis in another part. If, for example, mung bean mitochondrial, chloroplast and cytoplasmic ribosomes in fact contain one identical protein and the gene for this particular protein occurs in only one of the three genomes (regardless of the site of the bulk of the genome for the proteins of any ribosomal type) the formation of ribosomes throughout the cell could be affected by the availability of the product of one gene in one organelle. Furthermore, along this same line, this protein might be produced on only one type of ribosome for incorporation into all ribosomes within the cell.

ACKNOWLEDGEMENT This work was supported in part b y a grant from the National Institute of General Medical Science of the U.S. Public Health Service.

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