Incorporation of labelled amino acids by chloroplast ribosomes

286

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 8313

I N C O R P O R A T I O N OF L A B E L L E D AMINO ACIDS BY C H L O R O P L A S T RIBOSOMES* ALVA A. A P P AND ANDRI~ T. JAGENDO1RF

McCollum Pratt Institute, The Johns Hopkins University, Baltimore, Md. (U.S.A.) (Received April i8th, 1963)

SUMMARY

I. Isolated spinach (Spinacea oleracea) chloroplasts are capable of incorporating ~*C-labeled amino acids. While no cofactor requirement was demonstrable, incorporation was enhanced by light, inhibited by chlorampimnicol, and unaffected by RNAase (EC 2.7.7.16 ). 2. A deoxycholate-insoluble fraction can also incorporate amino acids, but again no cofactor requirement was found. 3. Ribosomes are extracted from the chloroplast fraction by deoxycholate, and incorporation by these particles is enhanced by ATP, GTP, and a pH-5 fractio~, and inhibited by RNAase and chloramphenicol.

INTRODUCTION

As much as 5o % of the proteins of green leaves may be associated with chloroplasts in vivol, 2, and it seems likely a portion of this protein may be synthesized within the chloroplasts. This suggestion is reinforced by evidence for cytoplasmic inheritance of at least some of the chloroplast genetic determinants in both Euglena 3 and higher plants 4, and by recent experiments showing a relationship between RNA synthesis and chloroplast developmentS, e. Using intact leaves, HEBER7 showed a portion of photosynthetically fixed 1aco2 can be found in proteins of chloroplasts subsequently isolated. Tobacco chloroplasts have been shown to incorporate 14C-labelled leucine in vitro s, and an attempt has been made to demonstrate 14C02 incorporation into protein by spinach chloroplasts in vitro 7. In addition, there have been three reports of chloroplast-associated ribosomes 9-n, as well as a claim that ribosomes isolated from chloroplasts can actively incorporate ~aC-labelled amino acids 1~. In work reported here, we have extracted ribosomes from spinach chloroplasts, and examined their cofactor requirements for incorporation of labelled amino acids. Certain characteristics of amino acid incorporation by whole chloroplasts are also described. An abstract describing a portion of this work has appeared 13. Abbreviation: DOC, sodium deoxycholate. * Contribution n u m b e r 400 from the McCollum-Pratt Institute.

Biochim. Biophys. Acta, 76 (1963) 286-292

CHLOROPLAST RIBOSOME AMINO ACID INCORPORATION

287

MATERIALS AND METHODS

Chloroplasts were prepared by grinding spinach leaves in a Waring blender at 60 % line voltage in cold 0.02 M K H , P 0 4 (pH 6.7), 0.02 M MgC1, and 0.5 M sucrose. The slurry was filtered through cheese cloth, centrifuged briefly to remove cell debris, then centrifuged 7 min at 2000 ×g at 3 °. The chloroplast pellet was resuspended in 0.5 M sucrose, and recentrifuged at 2000 ×g for 7 min. It was then resuspended in 0.5 M sucrose, o.oi M MgCI~ and 0.02 M KH2PO 4 (pH 6.8), and termed "intact" chloroplasts. For extraction of ribosomes from chloroplasts, the 0.5 M sucrose pellet was resuspended in 0.02 M Tris (pH 7.6), 0.2 M sucrose, 6.5 mM MgCI,, 0.5 M KC1, and extracted with 0.5 % DOC. The preparation was gently homogenized with a glass homogenizer, allowed to sit for 20 rain, then centrifuged 15 min at 20 ooo × g at 3 °. The 20 ooo ×g supernatant was recentrifuged two times, then spun 4 h in a "30" Rotor at 30 ooo rev./min in a Model-L Spineo ultracentrifuge. The pellet was resuspended in 0.2 M sucrose, 6.5 mM MgC12, 0.02 M Tris (pH 7.6), gently homogenized, recentrifuged at 20 ooo x g for IO rain, then dialyzed against the sucrose-Mg2+-Tris buffer and assayed. A DOC-insoluble chloroplast fraction was prepared by extracting isolated intact chloroplasts with 0.5 % DOC in o.02 M K H , P 0 4 (pH 7.5). The insoluble fraction was centrifuged down at 20 o o o x g for 7 rain, washed three times in 0.02 M KH2PO 4 (pH 7-5), then dialyzed overnight against 0.02 M phosphate buffer. The fraction was then centrifuged and resuspended in o.oi M MgC12 and 0.02 M KH2PO , (pI-I 6.8). 14C incorporation by isolated ribosomes was determined by taking o.o5-ml aliquots of the reaction mix, plating on filter paper disks, and washing as described by MANS AND N'OVELL114. Zero time and background counts were substracted from total counts. Liver pH- 5 fraction was prepared according to HOAGLAND, KELLER AND ZAMECNIK15. A similar procedure was used to prepare a pH-5 fraction from intact spinach chloroplasts. RNA was estimated spectrophotometrically after extraction of interfering substances TM. Protein content was obtained by the LOWRY method iv. 14C incorporation b y intact or DOC-extracted chloroplasts was determined by killing the reaction with 5 % trichloroaeetic acid, washing the precipitate according to STEPHENSON, THIMANN AND ZAMECNIK 8, plating the protein on Whatman No. 542 filter paper, and counting the samples on a Nuclear-Chicago Model 186. All counts were corrected to infinite thickness. Spinach (Spinacea oleracea) was obtained from the local supermarket. Chloramphenicol was a gift from Parke Davis and Co. 14C-labelled algal protein hydrolysate was obtained from ~¢ew England Nuclear Corporation, Boston, Mass. Sodium salts of ATP, GTP, and PEP, and the RNAase (EC 2.7.7.16) and pyruvate kinase (EC 2.7.1.4o ) were from Sigma Chemical Co., St. Louis, Mo. RESULTS

Incorporation by intact chloroplasts and by a DOC-insoluble /raction The incorporation of labelled amino acids into intact spinach chloroplasts under certain conditions is stimulated by light (Fig. i). This confirms the original report of STEPHENSON, THIMANN AND ZAMECNIK8 w h o u s e d i n t a c t

tobacco

chloroplasts.

Biochim. Biophys. Acta, 76 (1963) 286-292

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5000 -

4000-

~o_3000-

o 20004

i 1000-

0,~ o

~

~o ~ eb Time (min)

~6o

Fig. I. I n c o r p o r a t i o n of 14C-labelled a m i n o acids b y i n t a c t c h l o r o p l a s t s in light (©) a n d d a r k ( • ) . R e a c t i o n m i x t u r e included: o.98 m g c h l o r o p h y l l / m l , o.o2 M MgC1v o.o2 IV[ K H 2 P O 4 (pH 6.8), o. 5 IV[ sucrose, 400 ooo c o u n t s / m i n 14C-labelled algal h y d r o l y s a t e . T h e t o t a l v o l u m e w a s 2.0 ml. T e m p e r a t u r e 23 °. T h e r e a c t i o n w a s p e r f o r m e d in i 2 - m l g r a d u a t e d t u b e s u n d e r air.

However, no stimulation could be found under the same conditions, by adding I mM ATP, or I mM ATP plus an ATP-generating system. This was true even after breaking chloroplasts open by hypotonic shock. In a further effort to demonstrate cofactor requirements chloroplasts were extracted with 0.5 % DOC. While over 80 % of the RNA was removed from the chloroplasts by this treatment, the residual insoluble fraction could still actively incorporate amino acids. Table I shows that none of the usual cofactors implicated in microsomal ribonucleoprotein particle protein formation system were effective in enhancing incorporation by this fraction. TABLE I COFACTOR REQUIREMENT FOR 14C INCORPORATION BY CHLOROPLAST DOC-INSOLUBLE FRACTION R e a c t i o n m i x t u r e i n c l u d e d 225 ooo c o u n t s / r a i n 14C-labelled algal h y d r o l y s a t e , 24 # g of e a c h of following L-amino acids, glutanaic acid, lysine, valine, alanine, arginine, leucine, p h e n y l a l a n i n e , a s p a r t i c acid, isoleucine, proline, t h r e o n i n e , tyrosine, serine, glycine, histidine, m e t h i o n i n e , cysteine, a s p a r a g i n e , g l u t a m i n e , t r y p t o p h a n , o.oi M MgClz, o.o2 M t i H 2 P O 4 (pH 6.8), io m g chlorop l a s t p r o t e i n , a n d w h e r e i n d i c a t e d i mlVi A T P , G T P , a n d 0. 5 m g liver p H - 5 fraction. T e m p e r a t u r e 20 ° . A dditions

None None

o 60

ATP

ATP+liver ATP+liver

Reaction (rain)time

p H - 5 fraction pH- 5 fraction+GTP

Counts/mg protein

24, 16o, 16o, 13o, 114,

25 167 162 15o 131

Biochirn. Bi ophy s . Acta, 76 (1963) 286-292

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CHLOROPLAST RIBOSOME AMINO ACID INCORPORATION

Although RNAase did not inhibit incorporation of amino acids by either whole or broken chloroplasts or the l)OC-insoluble fraction, chloramphenicol turned out to be a potent inhibitor (Table II). T A B L E II INHIBITION OF INTACT CHLOROPLAST INCORPORATION R e a c t i o n m i x t u r e i n c l u d e s 0.49 m g c h l o r o p h y l l / m l , 0. 5 M sucrose, o.oi IV[ MgClz, o.02 M K H 2 P O 4 (pH 6.8), 5o0 ooo c o u n t s 14C-labelled algal p r o t e i n h y d r o l y s a t e . T o t a l v o l u m e 2.0 ml. R e a c t i o n was p e r f o r m e d in I 2 - m l g r a d u a t e d t u b e s a t 20 ° u n d e r air in light. A dditions

Reaction (rain)time

Countslmg protein

o 90

12o, 132 7500, 6700, 6800 630, 690 7200, 61oo

None None I mM chloramphenicol 0.33 m g l~NAase

TABLE III COFACTOR REQUIREMENT FOR 14C INCORPORATION B Y CHLOROPLAST RIBOSOMES C o m p l e t e r e a c t i o n m i x t u r e i n c l u d e s I m M AT]?, i m M G T P , 200/zg p y r u v a t e kinase, 1.5 m g P E P , 600/zg liver p H - 5 p r o t e i n , 530 ooo c o u n t s / m i n 14C-labelled algal p r o t e i n h y d r o l y s a t e , 6 m M MgCI v 0.02 IV[ Tris (pH 7.6), a n d o. 7 m g r i b o s o m e p r o t e i n in a t o t a l v o l u m e of I.O ml. T e m p e r a t u r e 2o ~. Counts

Complete Minus pH- 5 fraction Minus ATP Minus GTP Minus ATP, phosphoenolpyruvate, pyruvate kinase, p H - 5 fraction Complete plus chloramphenicol Complete plus RNAase

per tube

520 80 28o 36o 2o0 14o ioo

Co[actor requirements/or amino acid incorporation by chloroplast ribosomes Table III shows enhancement of 14Cincorporation with added cofactors by ribosomes extracted with DOC from isolated spinach chloroplasts. The generally known cofactors of protein system seem to be involved, and this incorporation is sensitive to both chloramphenicol and RNAase. A value lower than the unsupplemented system for samples lacking only pH-5 enzyme was not repeatable. The amount of incorporation by supplemented ribosomes is small. Fig. 2 indicates a pH-5 fraction from spinach chloroplasts may substitute for liver pH- 5 fraction. The increase in chloroplast ribosome incorporation due to chloroplast pH-5 fraction, and inhibition of incorporation by chloramphenicol indicates ribosomal contamination in the liver pH- 5 fractions is not involved.

Ultracentri[ugal examination o[ extracted chloroplast ribosomes As shown in Fig. 3, the DOC-extracted ribosomal preparations contain at least 4 components, with sedimentation coefficients s::2,w corresponding to 18, 50, 71 and Bioc hi m . Bi ophy s . Acta, 76 (1963) 286-292

290

A.A.

APP, A. T. JAC-ENDORF

320 •

~_ 240

•

• 0 ~ 0

e 5

10

15

20

25

Time [min) Fig. 2. E n h a n c e m e n t of r i b o s o m a l i n c o r p o r a t i o n b y liver or c h l o r o p l a s t p H - 5 fractions. R e a c t i o n m i x t u r e included: 2. 5 m g ribosomal protein, I mSI A T P , I m M M G T P , 53o ooo c o u n t s / r a i n 14C-labelled algal p r o t e i n h y d r o l y s a t e , o.2 M sucrose, o.o2 M Tris (pH 7-5), a n d w h e r e i n d i c a t e d 6oo/~g liver p H - 5 ( A ) or 3 o o i i g chloroplasts p H - 5 ( © ) p r o t e i n in o.8 ml. T e m p e r a t u r e 2o °. O , control.

Fig. 3. U l t r a c e n t r i f u g e p a t t e r n of chloroplast ribosomal p r e p a r a t i o n . P h o t o g r a p h t a k e n 12 m i n a f t e r r e a c h i n g 42 ooo r e v . / m i n in Model-E Spinco u l t r a c e n t r i f u g e . B a r angle 6o °. 2. 4 m g r i b o s o m a l p r o t e i n p e r ml.

8o S (uncorrected to infinite dilution). The I8-S peak could conceivably be "Fraction I" protein, or a ribosomal subunit 1. Calculation of RNA and protein content by ultraviolet absorption at 26o m~ and 28o m/~is indicates the preparation contains approx. 4o-44 % RNA and 53-6o protein. Biochim. Biophys. Acla, 76 (i963) 286-292

CHLOROPLAST RIBOSOME AMINO ACID INCORPORATION

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DISCUSSION

Indirect evidence strongly suggests that protein synthesis takes place in chloroplasts b y means of a system similar to that of the microsomal ribonucleoprotein particle. Amino acids 19 and ATP 2° are known photosynthetic products. Activating enzymes seem to be associated with the chloroplasts ~1 as well as the cytoplasm ~2. RNA, and interestingly enough DNA, seem to be intimately associated with repeatedly recentrifuged chloroplasts '6. Ribosomes can be detached from the chloroplast fxaction b y breaking intact chloroplasts in water 9. Finally, we have found that DOC does not remove all RNA from the chloroplast fraction, and thus indicating that at least a portion of the RNA is very tightly bound. Amino acid incorporation by whole or broken spinach chloroplasts, or b y the DOC-insoluble fraction derived from them, is seen here to be insensitive to the addition of ATP, GTP, pH-5 fraction, or RNAase. These facts tend to cast doubt on the identification of the observed incorporation as protein synthesis. However, chloramphenicol does inhibit the activity (Table II), and chloramphenicol is known to be an inhibitor of protein s3mthesis in other systems 23. Thus we are tempted to conclude, at least tentatively, that protein is being synthesized in the intact chloroplasts, but that the requisite cofactors are bound in such a way that neither osmotic shock nor 0.5 % DOC extraction can remove them. Furthermore the functional nucleic acid for protein synthesis m a y be protected from RNAase attack by the chloroplast membranes in the same way as found for mitochondria 24. Previous work s has ruled out the possibility that amino acid incorporation by chloroplasts is really due to contaminating bacteria. Contamination of the chloroplasts with free or occluded ribosomes is highly unlikely, on several grounds. In the first place, we consistently find that from 75-8 5 o/ /o of the RNA is extractable with DOC. This consistency is not likely to be due to contamination. Secondly, if there were considerable ribosomal contamination one would expect RNAase to reduce incorporation substantially; and this does not occur. Furthermore if the amino acid corporation were due to contaminating microsomes one would expect to see stimulation due to the addition of ATP. To date, this has not been possible. Ribosomes extracted from the chloroplasts also incorporate amino acids, and as far as the evidence goes appear to have a mechanism similar to that found for the microsomal ribonucleoprotein particles. ATP, GTP and the pH-5 fraction all stimulate the incorporation, and RNAase addition is now inhibitory. This latter observation suggests that the nucleic acid of this fraction was protected from RNAase attack while present in the whole or broken chloroplasts. Unfortunately the significance of protein synthesis by the isolated ribosomes for reactions of the whole chloroplasts is obscured by the low incorporation rates found with the ribosome preparation. The low rates are probably due to degradation of the ribosomes during the isolation procedure. Thus most of the protein in the ribosome preparation is seen to have a sedimentation constant of 18 S (Fig. 3); while it is known from recent work that only intact and aggregated ribosomes, with sedimentation coefficients of greater than 70 S are actually active in protein synthesis 25. Whole leaves, and isolated chloroplasts, are known to contain proteins with a sedimentation coefficient of 18 S (ref. I). These, however, are extracted easily in aqueous buffers, and either have very little or no nucleic acid. The RNA content of over 40 ~o found in our ribosome preparation therefore indicates that the major I8-S Biochim. Biophys. Acta, 76 (1963) 286-292

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component observed in the ultracentrifuge consists of ribosomal subunits, rather than "Fraction I". Modification of the preparation procedures will be required to obtain a higher proportion of intact ribosomes, and more substantial rates of protein synthesis. None of our evidence demonstrates that the extracted ribosomes are responsible for amino acid incorporation byintact chloroplasts. This question is further complicated by the remaining significant activity in the DOC-insoluble fraction. Further work will be needed to clarify the mechanism for amino acid incorporation by whole or broken chloroplasts. ACKNOWLEDGEMENTS The authors would like to thank Mrs. A. HILKEN for technical assistance, and Miss T. CARR for assistance with the ultracentrifuge. This work was supported in part by Grant No. Gio87I from the National Science Foundation. REFERENCES 1 2 3 4 5 * 7 s 9 10 11 12 13 1, 15 16 17 18 19 20 21 22 23 24 2s

S. W'ILDMAN AND A. T. JAGENDORF, Ann. J~ev. Plant Physiol., 3 (1952) 131. C. R. STOCKING, Plant Physiol., 34 (1959) 56. ]t. LYMAN, H. EPSTEIN AND J. A. SCHIFF, Biochim. Biophys. Acta, 5 ° (1961) 3Ol. }Z. M. SMILLI}~, Can. J. Botany, 41 (1963) 123. A. O. POGO, G. BRA~VERMAN AND •. CHARGAFF, Biochem., I (1962) 128. W. R. EVANS AND 1~. ~¥i. SMILLIE, Proc. Ann. Meeting Am. So& Plant Physiol., (1962) x x x v i i i . U. HKBER, Nature, 195 (1962) 91. M. L. STEPHENSON, K. V. THIMANN AND P. C. ZAMECNIK, Arch. Biochem. Biophys., 65 (1956) 194. j . \¥. LYTTLETON, Exptl. Cell Res., 26 (1962) 312. E. MIKULSKA, M. S. ODINTSOVA AND N. •. SISSAKIAN, Naturwissenscha]ten, 49 (1962) 23. G. BRA'~,~ERMAN, Biochim. Biophys. Acta, 61 (1962) 313 . N. M. SISSAKIAN, I. ][. FILIPPOVlCH AND E. ~]'. SVETAITO, Dokl. Akad. Nauk S S S R , 147 (1962) 488. A. APP AND A. T. JAGENDORF, Federation Pro&, 22 (1963) 302. ]Z. J. MANS AND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 3 (196o) 54 o. M. B. HOAGLAND, E. B. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 218 (1956) 345. y . CHIBA AND K. SUGAHARA, Arch. Biochem. Biophys., 71 (1957) 367. O. H. LOWRY, lXT. J. ]~.OSEBROUGH, A. L. FARR AND l~. J. RANDALL, J . Biol. Chem., 193 ( I 9 5 I ) 265 . O. WARBURG AND \ ¥ . CHRISTIAN, Biochem. Z., 31o ( I 9 4 I ) 384 . I). C. SMITH, J. A. BASSHAM AND M. KIRK, Biochim. Biophys. Acta, 48 (1961) 299. D. I. ARNON, F. ~c~. WHATLEY AND M. B. A.LLEN, J. A~I~. Chem. Sot., 76 (1954) 6324. j . l~ovE AND I. D. ~C~AACKE,Arch. Biochem. Biophys., 85 (1959) 521. A. MARCUS, J. Biol. Chem., 234 (1959) 1238. }~. RENDI AND S. OCHOA, J . Biol. Chem., 237 (1962) 3711. D. B. ROODYN, P. J. REIS AND T. S. WORK, in tZ. J. C. HARRIS, Protein Biosynthesis, A c a d e m i c Press, N e w Y o r k , 1961, p. 37. j . R. \YARNER, P. M. KNOPF AND A. RICH, Pro& Natl. Acad. Sci. U.S., 49 (1963) 122.

Biochim. Biophys. Acta, 76 (1963) 286-292