Planta 9 by Springer-Verlag 1978
Planta 142, 153-160 (1978)
Effects of Lysine, Threonine and Methionine on Light-driven Protein Synthesis in Isolated Pea (Pisum sativum L.) Chloroplasts * W. Ronald Mills** and Kenneth G. Wilson Department of Botany, Miami University, Oxford, OH 45056, USA
Abstract. Light-driven incorporation of [14C]leucine (LEU) into protein in isolated pea chloroplasts was inhibited by 0.1 and 1 m M lysine (LYS), 0.1-10 m M threonine (THR) and 1 mM methionine (MET). Equimolar combinations of LYS plus T H R were inhibitory at both 0.1 and 0.5 mM. Incorporation of [I4C] aspartic acid (ASP) and [3H]tyrosine (TYR) was also reduced by 1 mM LYS or THR. In the cases tested, LYS and/or T H R inhibitions were partially or fully reversed by 0.1 m M MET. [35S]MET incorporation was unaffected or stimulated by LYS and THR. These data are consistent with the hypothesis that MET is biosynthesized in isolated chloroplasts and that its synthesis is regulated by LYS and/or THR. Of 16 other amino acids tested at 1 mM, isoleucine, phenylalanine, tryptophan, tyrosine and valine inhibited protein synthesis. Key words: Amino acids - Biosynthetic regulation - Chloroplasts (protein synthesis) - P i s u m Protein synthesis.
Introduction Increasing evidence indicates that chloroplasts are a major site of amino-acid biosynthesis as well as sulfur and nitrogen reduction (see Miflin and Lea, 1977). Recent studies have shown several enzymes involved in the biosynthesis of the nutritionally important aspartate-family amino acids to be associated with plas* A portion of this work was included in the Doctoral Dissertation of W.R.M. (Mills, 1977) ** Present address: Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ, U.K. Abbreviations." DCMU= 3-(3,4-dichlorophenyl)-1,1-dimethylurea; ASP=aspartic acid; HS=homoserine; ILE=isoleucine; LEU= leucine; LYS =lysine; MET=methionine; THR=threonine; TYR = tyrosine
tids. These include: aspartate kinase (Miflin et al., in press), homoserine dehydrogenase (Bryan et al., 1977; Burdge et al., unpublished), diaminopimelate decarboxylase (Mazelis et al., 1976) and acetolactate synthetase (Miflin, 1974). Furthermore, Shah and Cossins (1970) reported that enzyme extracts from pea chloroplasts could catalyze the synthesis of methionine from homocysteine. Isolated intact chloroplasts incubated in light also appear capable of incorporating radio-labeled CO2 or aspartate into some or all of the aspartate-family amino acids (Kirk and Leech, 1972; Mills, 1977). Biosynthetic regulation of the aspartate-derived amino acids in higher plants has generally been investigated in two ways. One approach has been to study the properties of enzymes involved in the biosynthesis of these amino acids in vitro. Often these enzymes have been found to be affected in an allosteric manner by certain pathway end-products (see Miflin et al., in press). A second method has been to observe the influence of aspartate-derived amino acids on the growth of plants or plant cultures (see Miflin et al., in press). In some cases the effects of aspartate-family amino acids on protein synthesis and metabolism of labeled precursors have also been examined. For example, LYS and T H R have been shown to inhibit growth and protein synthesis in M a r c h a n t i a gemmalings (Dunham and Bryan, 1969, 1972) and M i m u l u s seedlings (Henke etal., 1974; Henke and Wilson, 1974). These effects have often been interpreted in terms of feedback control of enzymes occurring early in the pathway for the biosynthesis of LYS, MET, T H R and ILE from ASP, such as aspartate kinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) (see Fig. 1). Henke and Wilson (1974) have suggested that MET biosynthesis in M i m u l u s seedlings treated with LYS and/or T H R is insufficient to support normal rates of protein synthesis and growth. Presumably these compounds inhibit en-
0032-0935/78/0153/$01.60
154
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts
ASPARTATE ~-ASPARTYLPHOSPHATE ~'ASPARTYL~iMIALDEHYDE
~)
~>1
HOMOSERINE
O-PHOSPHOHOMOSERI )'1 NE~ THREONINE 4, ,b + +
ISOLEUCINE
METHIONINE
LYS|NE
Fig. 1. s c h e m e for the biosynthesis of LYS, T H R , M E T and ILE from ASP. The following numbers indicate enzymes which have been shown to be feedback-controlled in plants: 1 aspartate kinase ; 2 dihydrodipicolinic acid synthetase; 3 homoserine dehydrogenase; 4 threonine synthetase; 5 acetolactate synthetase. (Taken from Bryan, 1976; Miflin and Lea, 1977)
with an ISCO Model SR Spectroradiometer, Lincoln, Neb. U S A ; ca. 10,000 lx) for 11 14 d at 21 + 3 ~ C. Plants were treated on alternate days with water or Hoagland's version 2 nutrient solution (Arditti and D u n n , 1969). Prior to harvest, seedlings were placed in darkness 16 24 h to reduce starch in the chloroplasts. This was followed by 30 min in light to reduce starch further and enhance subsequent photophosphorylation in vitro (Nobel, 1968; Chrang et al., 1975; Anderson and Avron, 1976; Wolosiuk and Buchanan, 1977). Generally, 5 15 g of young leaves and stems were harvested, and washed 3 times with ice-cold, sterile distilled water. Chloroplasts were isolated essentially by the method of Bottomley et al. (1974). The leaves and stems were placed in a 50-ml homogenization vessel with 45 ml of ice-cold grinding medium. They were homogenized for 4 s with a Sorvall Omni-mixer (DuPont Instruments, Newton, Conn., USA) set at 7.5. The brei was filtered through two layers of cheesecloth and two layers of Miracloth (Calbiochem, San Diego, Cal., USA) and centrifuged in a refrigerated centrifuge with a swing-out rotor at 2,000 x g for 50 s. The supernatant was decanted, and the pellet washed with 45 ml of the isolating medium and centrifuged as above. The grinding medium contained 3 3 0 m M sorbitol, 5 0 m M N-tris(hydroxyethyl)methylglycine(Tricine)-KOH (ph 8.4), 2 m M E D T A (ethylendiaminetetraacetic acid), t m M MgCI~ and 4 m M 2-mercaptoethanol. All glassware and utensils were oven-sterilized while solutions were filter-sterilized. The intactness of chloroplast preparations was judged with phase-contrast microscopy (Spencer and Unt, 1965) as well as with transmission electron microscopy. In the latter, chloroplasts with an electron-dense stroma and with visibly unbroken envelopes were considered to be intact. Chlorophyll was determined by the method of A r n o n (1949).
Incubation of Chloroplasts
zymes common to the biosynthesis of aspartate-derived amino acids, ultimately blocking the flow of carbon from ASP to MET. Isolated, intact pea chloroplasts have been shown to incorporate labeled amino acids into protein when light is the sole energy source (Blair and Ellis, 1973). Neither exogenous amino acids nor cofactors are required for high rates of synthesis. If amino acids are biosynthesized in isolated chloroplasts they should be available for incorporation into protein. As in whole plants, LYS and/or T H R could regulate MET production. Consequently, MET would be reduced below the level required for high rates of protein synthesis. In this paper we report findings from experiments in which the effects of exogenous amino acids on light-driven protein synthesis in isolated pea chloroplasts were examined.
Material and Methods
Isolated choroplasts were resuspended in the KC1 incubation medium of Ramirez et al. (1968). This medium contained 200 m M KC1, 66 m M Tricine-KOH (pH 8.3) and 6.6 m M MgCI2. The chloroplast suspensions were placed in 10 m m diameter x 75 m m test tubes and radiolabeled LEU, ASP, M E T or T Y R was added to make the final volume. The tubes were incubated in a water bath at 20 _+ 1~ C. The partially submerged tubes (1 cm from the surface at the level of the sample suspension) were positioned at a 30~ angle to the water surface and shaken reciprocally at 80 cycles/rain. White light from two 500-W photoflood lamps (Photoflood E B U No. 2; General Electric, Cleveland, O., USA) provided an energy fluence rate at the level of the sample of ca. l0 s ~tW cm 2. Tubes for dark treatments were wrapped in aluminium foil. Samples of resuspended chloroplasts were periodically checked for bacterial contamination by plating aliquots on nutrient agar and counting colonies after incubation for 36 h at 37 ~ C. The nutrient agar was prepared by adding Difco (Difco Laboratories, Detroit, Mich., USA) Bacto Nutrient Broth (8 gfl) to 1%' Difco Bacto-Agar. The contamination of the chloroplast preparation by other organelles was estimated by utilizing marker enzymes. The mitochondrial marker, cytochrome oxidase, was assayed according to Hackett (1964) as modified by Miflin (1974), while catalase, the microbody marker, was assayed by the method of Aebi (1974).
Growth of Seedlings and Isolation of Chloroplasts
Determination of Radioactivity Incorporated into Protein
Plants of Pisum sativum L., cv. Alaska (W. Atlee Burpee Co., Warminster, Pa. USA), were grown in vermiculite in a growth room under continuous illumination (ca. 5 • 104 p c m - 2 ; measured
Following incubation, the chloroplast suspensions were rapidly cooled on ice, then spotted and dried on W h a t m a n (Whatman, Clifton, N.J., USA) 3-mm filter papers. The discs or squares
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts (2.5 cm diameter or 2.5 cm square) were prepared for liquid scintillation counting by the procedure of Mans and Novelli (1961). The processed discs or squares were placed in scintillation vials containing 5 ml of BBOT-toluene (8 g of 2,5-Bis[5'-tert-butyl-2benzoxazolyl]thiophene/1 toluene). The discs were counted in a Packard (Packard Instrument Co., Downers Grove, Ill., USA) TriCarb 3310 Liquid Scintillation Spectrometer at ca. 70% counting efficiency.
Chemicals All amino acids, EDTA, Triton X-100, 2-mercaptoethanol, Tricine, ribonuclease A, chloramphenicol and cycloheximide were obtained from Sigma Chemical Co., St. Louis, Mo., USA; actinomycin D from Merck, Sharp & Dohme, Rahway, N.J., USA; DCMU from ICN Pharmaceuticals, Plainview, N.Y., USA; and BBOT from Eastman Kodak, Rochester, N.Y., USA. Other unlabeled chemicals were obtained from Matheson, Coleman & Bell, Norwood, O., USA. The following radioactively labeled chemicals were acquired from New England Nuclear Corp., Boston, Mass., USA: L-[U14Claspartic acid, 203 mCi/mmol; L-[U-l*C]leucine, 325mCi/ mmot; L-l~hS]methionine, 511.9 Ci/mmot; L-[3,5-3H]tyrosine, 60.3 Ci/mmol.
155
Table 1. Characteristics of light-driven protein synthesis in isolated pea chloroplasts. A final volume of 0.1 ml contained 20 gg chlorophyll and 0.5 laCi of [~C]LEU; incubation was for t5 rain. The mean light-control value was 7570 cpm Treatment
Incorporation (% of light control)
Zero time Lysed Dark ATP (2 mM) Dark • ATP (2 mM) NH4C1 (1 mM) DCMU (2 ~tg/ml) D-Threo-Chloramphenicol (100 gg/ml) Cycloheximide (100 Fg/ml) Actinomycin D (10 lag/ml) Ribonuclease A (15 gg/ml)
8 9 27 93 38 19 36 37 93 126 99
9 Dark 9 Light
7.5
Results 7
Characteristics of Light-driven Protein Synthesis in Isolated Chloroplasts L i g h t a n d i n t a c t c h l o r o p l a s t s a r e r e q u i r e d for h i g h r a t e s o f s y n t h e s i s ( T a b l e 1). M a x i m a l r a t e s n e a r 6.0, 1.0, 0.1 a n d 0 . 1 n m o l m g 1 chlorophyll h-1 were o b s e r v e d w i t h [ 1 4 C ] L E U , [14C]ASP, [ 3 H ] T Y R , a n d [ 3 S S ] M E T , in this o r d e r . I n c o r p o r a t i o n o f [ I ~ C ] L E U i n t o p r o t e i n is r e d u c e d b y t h e u n c o u p l e r a m m o n i u m c h l o r i d e , by t h e e l e c t r o n - f l o w i n h i b i t o r D C M U , a n d the 70S-ribosome inhibitor chloramphenicol. However, n e i t h e r t h e 8 0 S - r i b o s o m e i n h i b i t o r c y c l o h e x i m ide, n o r t h e R N A - s y n t h e s i s i n h i b i t o r a c t i n o m y c i n D , n o r r i b o n u c l e a s e A r e d u c e d s y n t h e s i s at t h e c o n c e n t r a t i o n s tested, A T P at 2 m M was o n l y slightly s t i m u l a t o r y in t h e d a r k ( T a b l e 1). As noted by Jagendorf and colleagues, early studies on protein synthesis in isolated chloroplasts were complicated by bacterial contamination (App and Jagendorf, 1964; Bamji and Jagendorf, 1966). Therefore, the preparations of isolated chloroplasts were examined in a variety of ways to determine if contaminating bacteria affected the observed protein synthesis. Figure 2 illustrates a time course for the incorporation of radiolabeled LEU into protein. Incorporation is markedly light-dependent and the rate approaches zero after ca. 15-20 min. Incorporation by bacteria should be linear over this time interval (Bamji and Jagendorf, 1966). Furthermore, neither light dependence nor the sensitivity to photophosphorylation inhibitors evident in Figure 2 should be exhibited by bacteria. Treatment of the plastid suspensions with 2% Triton X-100 detergent, which solubilizes chloroplasts but not whole cells, bacteria or nuclei (Parenti and Margulies, 1967), left less than 1% of the radioactivity in the 10,000 x g pellet. Addition-
to 5.c
2.5
TIME (MIN)
Fig. 2. Time course for light-driven incorporation of [14C]LEU into protein by isolated pea chloroplasts. The final volume was 0.3 ml. Each tube contained 27.1 gg of chlorophyll and lgCi of [14C]LEU. At specified times, 0.05-ml aliquots were removed from each tube and processed for liquid scintillation counting
ally, neither plating on nutrient agar nor examination by transmission electron microscopy indicated bacterial contamination. Cytochrome oxidase and catalase activities have been used as markers for mitochondria and microbodies, respectively (Miflin and Beevers, 1974). After the isolation of the chloroplasts, the initial brei and the chloroplast fraction were checked with these markers for the presence of contaminating mitochondria and microbodies (Table 2). Less than 0.1% of the total catalase and cytochrome-oxidase activity was found in the chloroplast fraction, while 3% of the total chlorophyll was in this fraction. Thus, it is unlikely that results on light-driven protein synthesis in isolated pea chloroplasts have been affected by bacterial, mitochondrial or microbody contamination. T h e c h l o r o p l a s t p r e p a r a t i o n s w e r e f o u n d to be 45 8 5 % i n t a c t as j u d g e d by p h a s e - c o n t r a s t m i c r o s c o p y ( S p e n c e r a n d U n t , 1965) a n d t r a n s m i s s i o n electron microscopy.
156
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts
TaMe 2. Marker enzyme and chlorophyll distribution in chloroplast preparation. Chloroplasts were isolated as described in Materials and Methods. Aliquots were taken from the filtered brei and the resuspended chloroplast pellet and analyzed for chlorophyll content or catalase and cytochrome oxidase activity
100
LYS
Amount or activity
Organelle marker
Chlorophyll (gg/fraction) Catalase (gmol fraction 1 min- 1) Cytochrome oxidase (nmol fraction- l min + l)
Chloroplast pellet
Crude homogenate (cpm)
cpm
% of crude homogenate
7600 7600
200 5
3.0 0.07
2400
2
0.08
i 100 .~
.+,sI
oi ..I
Table 3.
Effect of aspartate-family amino acids on light-driven protein synthesis in isolated pea chloroplasts. In experiments with [i4C]LEU and [I+C]ASP label, the final volumes were 0.1 ml containing 5-20 gg chlorophyll and 0.5 gCi. Samples were incubated for 30 rain. With [3H]TYR, the final volume was 0.4 ml containing 16 lxg chlorophyll and 1.0 gCi. Incubation was for 15 min. The mean light control values were ll,099cpm with [a4C]LEU, 1039 cpm with [14CLASP and 1813 cpm with [3H]TYR Amino acid (raM)
Incorporation (% of light control) [i4C]LEU
None, dark LYS 1.0 THR 1.0 MET 0.1 MET 1.0 LYS 1.0+MET 0.1 THR 1.0+MET 0.l Amino acid mixture ~
21 58 69 103 . 70 92 95 95
[I+C]ASP
[3H]TYR
45 48 62 95 82 -
30 73 89 86 92 -
@ 0.
MET
75
Fig. 3. Effect of varying concentrations of LYS, THR and MET on light-driven incorporation of 1+C into protein by isolated pea chloroplasts. Incubation time was 15 rain. A final volume of 0,4 ml contained 0.1 gCi of [i4C]LEU
The amino acid mixture was composed of the following L-isomers, at 10 p.M each: alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glutamic acid, histidine, isoleucine, phenylalanine, proline, serine, tryptophan, tyrosine and valine
Effects of Aspartate-family Amino Acids on Light-driven Protein Synthesis in Isolated Chloroplasts In preliminary experiments, LYS, T H R or MET at 1 mM inhibited [I+C]LEU incorporation into protein, while 0.1 m M MET had no effect (Table 3). Addition of 0.1 m M MET reversed the LYS and T H R inhibition, while a mixture of the protein amino acids, excluding LYS, T H R , MET and LEU, had no effect. Similar experiments were conducted using [I+C]ASP and [3H]TYR as labels (Table 3). LYS, T H R and MET at 1 m M inhibited It+ClASP incorporation in a manner similar to that observed with labeled LEU. With [3H]TYR, 1 mM LYS or T H R reduced synthesis; however, the inhibition was less than that
observed with the other labeled amino acids. Again 0.1 mM MET partially reversed the inhibition. When the time course of inhibition of [t4C]LEU incorporation into protein by 1 m M LYS and 1 m M T H R was examined, the inhibition was found to be similar at 2.5, 5 and 15 min (data not shown). Therefore, a 15-rain incubation period was used in subsequent experiments since this is approximately the time when incorporation becomes asymptotic. A separate group of studies was initiated to determine the concentration of LYS, T H R and MET having the greatest effect on the incorporation of [14C]LEU into protein (Figure 3). Protein synthesis was reduced about 20% by 0,1 m M and 30% by 1 mM LYS. Lower concentrations had no effect. One and 10 raM T H R inhibited synthesis, while lower
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts Table 4. MET reversal of L Y S + T H R inhibition of light-driven protein synthesis in isolated pea chloroplasts Amino acid (raM) [14C]LEU~ incorporation cpm/ lag chl
% of light co,trot
[3H]TYRb incorporation cpm/ lag chl
% of light centre!
151+ 1-
-
None
774_+ 59 -
None, dark
116_+
7 15
46_+11
30
LYSO1 + T H R 0.1
574_+103 74
103_+18
68
LYS0.1 + T H R 0.1 + M E T 0.1
757+_ 21 98
149+_22
99
LYS 05 + T H R 0.5
158_+ 62 20
LYS 0.5 + T H R 0.5 + M E T 0.1
667+ 56 86
A final volume of 0.2 ml contained 5 lag chlorophyll and 0.5 gCi of [14C]LEU. Incubation time was 15 min b A final volume of 0.4 ml contained 12 ~tg chlorophyll and 1.0 gCi [3H]TYR. Incubation time was 15 min
Table 5. Effect of various combinations of LYS, THR and MET on the incorporation of [35S]MET into protein in isolated pea chloroplasts. A final volume of 0. l ml contained 7.9 lag chlorophyll and 1/aCi MET; incubation time was 15 rain. The mean lightcontrol value was 18,905 cpm Amino acid
Incorporation (% of light control)
LYS 0.1 mM LYS 1.0 mM THR 0.l mM THR 1.0raM MET 0.1 mM MET 1.0raM LYS 10laM+THR 10gJM LYS 0.1 m M + THR 0.1 mM LYS 0.1 m M + T H R 0.1 mM + MET 0.1 laM LYS 0.1 mM + T H R 0.1 m M + MET 0.1 ~tm LYS 0.1 m M + T H R 0.1 m M + MET 10 ~tM
89 103 104 158 22 26 131 115 116 96 37
Table 6. Effect of various amino acids on light-driven protein synthesis in isolated pea chloroplasts. A final volume of 0.4 ml contained 22.1 lag chlorophyll and 0.1 laCi of [Ir or the final volume of 0.5 ml contained 13 1 gg chlorophyll and 1 laCi of [3H]TYR. Incubation was for 15 rain. The mean light-control values were 2423 cpm with [14C]LEU and 1266 cpm with [3H]TYR Amino acid (1 raM)
c o n c e n t r a t i o n s h a d little effect. S i m i l a r l y , p r o t e i n synthesis was i n h i b i t e d by 1 m M M E T a n d u n a f f e c t e d by l o w e r c o n c e n t r a t i o n s .
Effect of Equimolar L Y S + THR on Light-driven Protein Synthesis in Isolated Pea Chloroplasts C o m b i n a t i o n s o f L Y S + T H R at 0.1 a n d 0.5 m M e a c h r e d u c e d l i g h t - d r i v e n p r o t e i n s y n t h e s i s in i s o l a t e d p e a chloroplasts when [~4C]LEU or [3H]TYR were the l a b e l s ( T a b l e 4). T h e d e g r e e o f i n h i b i t i o n was as g r e a t or g r e a t e r t h a n w i t h L Y S o r T H R a l o n e at a n y concentration tested. Furthermore, addition of 0.1 m M M E T p a r t i a l l y o r fully r e v e r s e d t h e i n h i b i t i o n in all cases. L Y S + T H R at 1.0 m M e a c h or g r e a t e r p r o d u c e d v a r i a b l e results, b u t g e n e r a l l y w e r e less inhibitory (data not shown).
Effect of L YS, THR and M E T on Light-driven Incorporation of [35S]ME T into Protein T a b l e 5 s h o w s d a t a f r o m a series o f e x p e r i m e n t s in w h i c h t h e effects o f L Y S a n d / o r T H R o n [ 3 5 S ] M E T incorporation into isolated pea chloroplast protein was o b s e r v e d . A l l c o n c e n t r a t i o n s o f L Y S , T H R a n d L Y S + T H R t e s t e d r e s u l t e d in levels o f [ 3 5 S ] M E T incorporation approaching or exceeding the control.
157
Incorporation (% of light control) [14C]LEU
Alanine Arginine Asparagine Aspartic acid Cystine Glutamic acid Glutamine Glycine Histidine Isoleucine Phenylalanine Proline Serine Tryptophan Tyrosine Valine
98 93 103 99 96 95 94 95 96 56 69 100 96 43 61 88
[3H]TYR 80 -
Effect of Other Protein Amino Acids on Light-driven Protein Synthesis in Isolated Pea Chloroplasts T h e effect o f 16 p r o t e i n a m i n o acids, o t h e r t h a n L Y S , T H R , M E T a n d L E U , o n l i g h t - d r i v e n p r o t e i n synthesis in i s o l a t e d p e a c h l o r o p l a s t s a r e s h o w n in T a b l e 6. A l a n i n e , a r g i n i n e , a s p a r a g i n e , a s p a r t i c acid, cystine, g l u t a m i n e , g l u t a m i c acid, glycine, h i s t i d i n e , p r o line a n d serine h a d little o r n o effect. V a l i n e was slightly i n h i b i t o r y , w h i l e I L E was m o r e effective. I L E inhibited incorporation of both [~C]LEU and
158
W.R. Millsand K.G. Wilson: AminoAcidsand Protein Synthesisin Pea Chloroplasts
[3H]TYR; however, the effect was more pronounced with [14C]LEU as the label. The aromatic amino acids, phenylalanine, tyrosine and tryptophan, all markedly inhibited [a4C]LEU assimilation into protein.
Discussion
Although several enzymes of the biosynthetic pathway(s) of the aspartate-famity amino-acids have been found in plastids (Miflin, 1974; Mazelis et al., 1976; Bryan et al., 1977; Miflin et al., in press, the question of LYS, THR, MET and ILE biosynthesis and its regulation in isolated chloroplasts has received little attention. One way to investigate the localization of amino acid biosynthetic capacity in isolated organelles is to examine the effect of potential feedback inhibitors on protein synthesis i n these organelles. LYS and/or THR inhibited protein synthesis in this system and these effects were reversed by MET. This is the expected result if MET is biosynthesized in isolated pea chloroplasts and its synthesis regulated b y LYS and/or THR. Similar inhibition of growth (see Miflin et al., in press) and protein synthesis (Dunham and Bryan, 1971; Henke and Wilson, 1974) by LYS and/or THR and reversal by MET have been observed in several plants. It is unlikely that competition for uptake between LYS and/or THR and the labeled amino acid caused the inhibition. First, similar results were obtained when the structurally different amino acids, ASP, LEU and TYR, were used as labels, and they have been reported to be transported by different carriers in isolated chloroplasts (Heldt and Rapley, 1970; Nobel and Cheung, 1972; McLaren and Barber, 1977). Second, in other experiments we found little difference in levels of free [I~C]LEU taken up by isolated pea chloroplasts in the presence of LYS or THR, even though typical inhibition of protein synthesis was observed (Mills, 1977). Aarnes and Rognes (1974) isolated one aspartate kinase and two homoserine dehydrogenases from 3-dold etiolated pea seedlings. The activity of the aspartate kinase and one of the homoserine dehydrogenases was inhibited in vitro by THR. These enzymes were unaffected by LYS. Therefore, the inhibition of protein synthesis in isolated pea chloroplasts by LYS was undxpected (Table 3). However, the reversal of LYS inhibition by MET indicates that some enzyme(s) i n the)pathway from ASP to MET may be inhibited by L~S. Davies (1977) isolated two aspartate kinases fromcarrot cell SUSlSensioncultures. One enzyme was inhibited by LYS and the other by THR. The relative concentrations o f these enzymes varied with culture
age. Thus, one possibility is that two aspartate-kinase isozymes exist in pea chloroplasts. If the LYS-sensitive enzyme is bound to plastid membranes, it could have been removed during enzyme isolation. Alternatively, if LYS and THR sensitive forms vary with tissue age, as in cultured carrot cells, 3-d-old etiolated pea seedlings may only have a THR-sensitive form, while older, light-grown plants could have both. Obviously, LYS may exert its effect on other enzymes of MET biosynthesis (see e.g. Wong and Cossins, 1976). Inhibition of light-driven protein synthesis by equimolar combinations (0.1-0.5 mM) of L Y S + T H R were observed in several experiments (Table 4). The effects were reversed by MET. L Y S + T H R at these concentrations often reduced rates of protein synthesis to the level of the dark control. However, higher levels of L Y S + T H R (1.0raM) were often less inhibitory than lower concentrations, although this effect was variable. These results are difficult to understand and are being studied further. The unusual inhibition pattern may be related to the unique accumulation of HS during the first few weeks of pea seedling growth (Larson and Beevers, 1965). As a further test of the hypothesis that MET biosynthesis occurs in isolated pea chloroplasts and that LYS + T H R regulates the synthesis, incorporation of [3~S]MET into protein was examined (Table5). If isolated pea chloroplasts are incubated with labeled MET, the exogenous [3SS]MET and the endogenous MET should compete for incorporation into protein. Under inhibitory conditions, i.e. in the presence of LYS and/or THR, lowered MET biosynthesis should reduce isotope dilution. Hence, although overall protein synthesis should be reduced, a concomitant decrease in measurable [35S]MET incorporation should not be observed. Indeed, with all LYS and/or THR treatments, [35S]MET incorporation approaching or exceeding the control was observed. Miflin (1969) studied the effect of exogenous amino acids on the growth of barley seedlings. He noted that the most inhibitory ones, LYS, THR, MET, HS, LEU, TYR and valine, are all produced by branched pathways from common precursors. Similarly, with isolated pea chloroplasts, the amino. acids inhibiting protein synthesis the most are all biosynthesized in branched pathways. It is unclear if these effects result from reduced amino-acid biosynthesis because of feedback inhibition. However, it is interesting that similar groups of amino acids inhibit both light-driven protein synthesis in isolated pea chloroplasts and growth of barley seedlings. The use of isolated pea chloroplasts capable of high rates of protein synthesis with light as the only
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts energy source has several a d v a n t a g e s . First, the p r e p a r a t i o n s c o n t a i n high p e r c e n t a g e s o f intact plastids, therefore s t r o m a l enzymes are n o t lost. Second, inc o r p o r a t i o n o f a m i n o acids into p r o t e i n s h o u l d facilitate c a r b o n flux t h r o u g h a m i n o - a c i d b i o s y n t h e t i c p a t h w a y s , as well as reduce the c o n c e n t r a t i o n o f end o g e n o u s a m i n o acids which c o u l d p o t e n t i a l l y act as f e e d b a c k inhibitors. T h i r d , when light is used to drive the reactions, it n o t only reduces the l i k e l i h o o d o f s p u r i o u s results c a u s e d by b a c t e r i a or o t h e r cont a m i n a n t s , b u t also eliminates the need for e x o g e n o u s A T P or r e d u c e d p y r i d i n e nucleotide. The d a t a p r e s e n t e d here are c o n s i s t e n t with the h y p o t h e s i s t h a t M E T is b i o s y n t h e s i z e d in i s o l a t e d p e a c h l o r o p l a s t s a n d t h a t this synthesis is r e g u l a t e d b y LYS and/or THR. First, LYS and THR alone and in specific c o m b i n a t i o n s inhibit l i g h t - d r i v e n p r o t e i n synthesis in p e a c h l o r o p l a s t s a n d this effect is r e v e r s e d b y M E T . S i m i l a r effects have been o b s e r v e d in whole p l a n t s a n d have been a t t r i b u t e d to i n h i b i t i o n o f M E T biosynthesis b y L Y S a n d / o r T H R . Second, i n c o r p o r a tion o f laSS]MET into p r o t e i n by i s o l a t e d p e a c h l o r o plasts is u n a f f e c t e d or s t i m u l a t e d by L Y S a n d / o r T H R while these c o m p o u n d s r e d u c e i n c o r p o r a t i o n o f o t h e r l a b e l e d a m i n o acids. This is e x p e c t e d if M E T p r o d u c tion occurs in i s o l a t e d c h l o r o p l a s t s a n d these c o m p o u n d s i n h i b i t M E T b i o s y n t h e s i s a n d thus reduce i s o t o p e dilution. Third, in p r e l i m i n a r y e x p e r i m e n t s we f o u n d t h a t r a d i o l a b e l e d M E T is p r o d u c e d f r o m [ l a C ] A S P a n d [35S]SO42-b y i s o l a t e d p e a c h l o r o p l a s t s i n c u b a t e d u n d e r c o n d i t i o n s for l i g h t - d r i v e n p r o t e i n synthesis (Mills, 1977). F u r t h e r m o r e , M E T b i o s y n thesis f r o m b o t h p r e c u r s o r s was i n h i b i t e d by LYS+THR at 0 . 5 r a M each. These d a t a , c o u p l e d with recent evidence showing t h a t a s p a r t a t e kinase, homoserine dehydrogenase, diaminopimelate decarbo x y l a t e a n d a c e t o l a c t a t e s y n t h e t a s e are f o u n d in plastids, i n d i c a t e t h a t LYS, T H R , M E T a n d I L E are all b i o s y n t h e s i z e d in c h l o r o p l a s t s . We wish to thank S.W.J. Bright and P.J. Lea (Rothamsted Experimental Station, Harpenden, Herts, U.K.), M.J. Muhitch and E.L. Burdge, III (Miami University), for critically reading the manuscript, and M.J. Powell (Miami University) for assistance with the electron microscopy. Financial assistance was provided in part by the Miami University Faculty Research Committee.
References Aarnes, H., Rognes, S.E. : Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum. Phytochemistry 13, 27175724 (1974) Aebi, H. : Catalase. Methods Enzym. Anal. 2, 673-676 (1974) Anderson, L.E., Avron, M.: Light modulation of enzyme activity in chloroplasts generation of membrane-bound vicinal-dithiol
159
groups by photosynthetic electron transport. Plant Physiol. 57, 209-213 (1976) App, A. A., Jagendorf, A.J.: [l*C]amino acid incorporation by spinach chloroplast preparations. Plant Physiol. 39, 772 776 (1964) Arditti; J., Dunn, A.: Experimental plant physiology Experiments in cellular and plant physiology. New York: Holt, Rinehart, Winston 1969 Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenyloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949) Bamji, M.S., Jagendorf, A.J. : Amino acid incorporation by wheat chloroplasts. Plant Physiol. 41, 764-770 (1966) Blair, G.E., Ellis, R.J. : Protein synthesis in chloroplasts. I. Lightdriven synthesis of the large subunit of fraction I protein by isolated pea chloroplasts. Biochim. Biophys. Acta 319, 223 234 (1973) Bottomley, W., Spencer, D., Whitfield, P.R.: Protein synthesis in isolated spinach chloroplasts: Comparison of light-driven and ATP-driven synthesis. Arch. Biochem. Biophys: 164, 106-117 (1974) Bryan, J.K. : Amino acid biosynthesis and its regulation. In: Plant biochemistry, pp. 525 560, Bonner, J, Varner, J.E., eds. New York: Academic Press 1976 Bryan, J.K., Lissik, E.A., Matthews, B.F.: Changes in enzyme regulation during growth of maize. III. Intracellular localization of homoserine dehydrogenase in chloroplasts. Plant Physiol. 59, 673-679 (1977) Chrang, R.E., Budd, G.C., Ashbaugh, P.H., Nobel, R.D.: Initial light effects on chloroplast ultrastructure in soybean. Proe. Electron Mieroscop. Soc. Amer. 33, 566-567 (1975) Davies, H. M.: Regulation of amino acid biosynthesis in carrot tissue culture. Ph.D. thesis, Univ: of London, U.K. (1977) Dunham, V.L., Bryan, J.K.: Synergistic effects of metabolically related amino acids on the growth of a multicellular plant. Plant Physiol. 44, 1601 1608 (1969) Dunham, V.L., Bryan, J.K.: Synergistic effects of metabolically related amino acids on the growth of a multicellular plant. II. Studies of 14C amino acid incorporation. Plant Physiol. 47, 91-97 (1971) Hackett, D.P.: Enzymes of terminal respiration. In: Modern methods of plant analysis, vol. 7, pp. 647-699. Linskens, H.F., Sanwal, B.D., Tracey, M.V., eds. Berlin-G6ttingen-HeidelbergNew York: Springer 1964 Heldt, W.H., Rapley, L. : Specific transport of inorganic phosphate, 3-phosphoglycerate and dihydroxyacetonephosphate, and of dicarboxylates across the inner membrane of spinach chloroplasts. FEBS Lett. 10, 143 I48 (1970) Henke, R.R., Wilson, K.G. : In vivo evidence for metabolic control of amino acid and protein synthesis by exogenous lysine and threonine in Mimulus cardinal&. Planta 121, 155-166 (1974) Henke, R.R., Wilson, K.G., McClure, J.W., Treick, R.W. : Lysinemethionine-threonine interactions in the growth and development of Mimu&s cardinal& seedlings. Planta 116, 333-345 (1974) Kirk, P.R., Leech, R.M.: Amino acid biosynthesis by isolated chloroplasts during photosynthesis. Plant Physiol. 50, 228-234 (1972) Larson, L.A., Beevers, H. : Amino acid metabolism in young pea seedlings. Plant Physiol. 40, 424436 (1965) Mans, R.J., Novelli, G.K. : Measurement of the incorporation of radioactivity amino acids into protein by filter-paper disk method. Arch. Biochem. Biophys. 94, 48-53 (t961) Mazelis, M., Miflin, B.J., Pratt, H.M.: A chloroplast-localized diaminopimelate decarboxylase in higher plants. FEBS Lett. 64, 197-200 (1976) McLaren, J.S., Barber, D.J. : Evidence for carrier-mediated transport of L-leucine into isolated pea (Pisum sativum L.) chloroplasts. Planta 136, i4%i51 (I977)
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W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts
Miflin, B.J. : The inhibitory effects of various amino acids on the growth of barley seedlings. J. Exp. Bot. 20, 810 819 (1969) Miflin, B.J. : The location of nitrite reductase and other enzymes related to amino acid biosynthesis in the plastids of roots and leaves. Plant Physiol. 54, 550-555 (1974) Miflin, B.J., Beevers, H. : Isolation of intact plastids from a range of plant tissues. Plant Physiol. 53, 870-874 (1974) Miflin, B.J., Lea, P.J.: Amino acid metabolism. Ann. Rev. Plant Physiol. 28, 299 329 (1977) Miflin, B,J., Bright, S.W.J., Davies, H.M., Shewry, P.R., Lea, P.J. : Amino acids derived from aspartate: Their biosynthesis and its regulation. Proc. VI Long Ashton Symp., "Nitrogen Assimi, lation of Plants," (in press) Mills, W.R. : Studies on amino acid biosynthesis and its regulation in isolated chloroplasts of the Vicieae. Diss. Abstr. No. 78 01951. Ph.D. thesis, Miami Univ., Oxford, O., USA (1977) Nobel, P.S. : Chloroplast shrinkage and increased photophosphorylation in vitro upon illuminating intact plants of Pisum sativum. Biochim. Biophys. Acta 153, 170 182 (1968) Nobel, P.S., Cheung, Y.S. : Two amino-acid carriers in pea chloroplasts. Nature New Biol. 237, 207-208 (1972)
Parenti, F., Margulies, M.: In vitro protein synthesis by plastids of Phaseolus vulgaris. I. Localization of activity in the chloroplasts of a chloroplast containing fraction from developing leaves. Plant Physiol. 42, 1179-1182 (1967) Ramirez, J.M., Del Campo, E.F., Arnon, D.I.: Photosynthetic phosphorylation as energy source for protein synthesis and carbon dioxide assimilation by chloroplasts. Proc. Natl. Acad. Sci. USA 59, 606-611 (1968) Shah, S.P.J., Cossins, E.A. : Pteroylglutamates and methionine biosynthesis in isolated chloroplasts. FEBS Lett. 7, 267-270 (1970) Spencer, D., Unt., H. : Biochemical and structural correlation in isolated spinach chloroplasts under isotonic and hypotonic conditions. Aust. J. Biol. Sci. 18, 197510 (1965) Wolosiuk, R.A., Buchanan, B.B.: Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature 266, 565-567 (1977) Wong, K.F., Cossins, E.A.: Control of methionine synthesis by lysine in Lemna minor. Phytochem. 15, 921-925 (1976)
Received 6 February; accepted 26 May 1978
Planta 142, 153-160 (1978)
Effects of Lysine, Threonine and Methionine on Light-driven Protein Synthesis in Isolated Pea (Pisum sativum L.) Chloroplasts * W. Ronald Mills** and Kenneth G. Wilson Department of Botany, Miami University, Oxford, OH 45056, USA
Abstract. Light-driven incorporation of [14C]leucine (LEU) into protein in isolated pea chloroplasts was inhibited by 0.1 and 1 m M lysine (LYS), 0.1-10 m M threonine (THR) and 1 mM methionine (MET). Equimolar combinations of LYS plus T H R were inhibitory at both 0.1 and 0.5 mM. Incorporation of [I4C] aspartic acid (ASP) and [3H]tyrosine (TYR) was also reduced by 1 mM LYS or THR. In the cases tested, LYS and/or T H R inhibitions were partially or fully reversed by 0.1 m M MET. [35S]MET incorporation was unaffected or stimulated by LYS and THR. These data are consistent with the hypothesis that MET is biosynthesized in isolated chloroplasts and that its synthesis is regulated by LYS and/or THR. Of 16 other amino acids tested at 1 mM, isoleucine, phenylalanine, tryptophan, tyrosine and valine inhibited protein synthesis. Key words: Amino acids - Biosynthetic regulation - Chloroplasts (protein synthesis) - P i s u m Protein synthesis.
Introduction Increasing evidence indicates that chloroplasts are a major site of amino-acid biosynthesis as well as sulfur and nitrogen reduction (see Miflin and Lea, 1977). Recent studies have shown several enzymes involved in the biosynthesis of the nutritionally important aspartate-family amino acids to be associated with plas* A portion of this work was included in the Doctoral Dissertation of W.R.M. (Mills, 1977) ** Present address: Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ, U.K. Abbreviations." DCMU= 3-(3,4-dichlorophenyl)-1,1-dimethylurea; ASP=aspartic acid; HS=homoserine; ILE=isoleucine; LEU= leucine; LYS =lysine; MET=methionine; THR=threonine; TYR = tyrosine
tids. These include: aspartate kinase (Miflin et al., in press), homoserine dehydrogenase (Bryan et al., 1977; Burdge et al., unpublished), diaminopimelate decarboxylase (Mazelis et al., 1976) and acetolactate synthetase (Miflin, 1974). Furthermore, Shah and Cossins (1970) reported that enzyme extracts from pea chloroplasts could catalyze the synthesis of methionine from homocysteine. Isolated intact chloroplasts incubated in light also appear capable of incorporating radio-labeled CO2 or aspartate into some or all of the aspartate-family amino acids (Kirk and Leech, 1972; Mills, 1977). Biosynthetic regulation of the aspartate-derived amino acids in higher plants has generally been investigated in two ways. One approach has been to study the properties of enzymes involved in the biosynthesis of these amino acids in vitro. Often these enzymes have been found to be affected in an allosteric manner by certain pathway end-products (see Miflin et al., in press). A second method has been to observe the influence of aspartate-derived amino acids on the growth of plants or plant cultures (see Miflin et al., in press). In some cases the effects of aspartate-family amino acids on protein synthesis and metabolism of labeled precursors have also been examined. For example, LYS and T H R have been shown to inhibit growth and protein synthesis in M a r c h a n t i a gemmalings (Dunham and Bryan, 1969, 1972) and M i m u l u s seedlings (Henke etal., 1974; Henke and Wilson, 1974). These effects have often been interpreted in terms of feedback control of enzymes occurring early in the pathway for the biosynthesis of LYS, MET, T H R and ILE from ASP, such as aspartate kinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) (see Fig. 1). Henke and Wilson (1974) have suggested that MET biosynthesis in M i m u l u s seedlings treated with LYS and/or T H R is insufficient to support normal rates of protein synthesis and growth. Presumably these compounds inhibit en-
0032-0935/78/0153/$01.60
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W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts
ASPARTATE ~-ASPARTYLPHOSPHATE ~'ASPARTYL~iMIALDEHYDE
~)
~>1
HOMOSERINE
O-PHOSPHOHOMOSERI )'1 NE~ THREONINE 4, ,b + +
ISOLEUCINE
METHIONINE
LYS|NE
Fig. 1. s c h e m e for the biosynthesis of LYS, T H R , M E T and ILE from ASP. The following numbers indicate enzymes which have been shown to be feedback-controlled in plants: 1 aspartate kinase ; 2 dihydrodipicolinic acid synthetase; 3 homoserine dehydrogenase; 4 threonine synthetase; 5 acetolactate synthetase. (Taken from Bryan, 1976; Miflin and Lea, 1977)
with an ISCO Model SR Spectroradiometer, Lincoln, Neb. U S A ; ca. 10,000 lx) for 11 14 d at 21 + 3 ~ C. Plants were treated on alternate days with water or Hoagland's version 2 nutrient solution (Arditti and D u n n , 1969). Prior to harvest, seedlings were placed in darkness 16 24 h to reduce starch in the chloroplasts. This was followed by 30 min in light to reduce starch further and enhance subsequent photophosphorylation in vitro (Nobel, 1968; Chrang et al., 1975; Anderson and Avron, 1976; Wolosiuk and Buchanan, 1977). Generally, 5 15 g of young leaves and stems were harvested, and washed 3 times with ice-cold, sterile distilled water. Chloroplasts were isolated essentially by the method of Bottomley et al. (1974). The leaves and stems were placed in a 50-ml homogenization vessel with 45 ml of ice-cold grinding medium. They were homogenized for 4 s with a Sorvall Omni-mixer (DuPont Instruments, Newton, Conn., USA) set at 7.5. The brei was filtered through two layers of cheesecloth and two layers of Miracloth (Calbiochem, San Diego, Cal., USA) and centrifuged in a refrigerated centrifuge with a swing-out rotor at 2,000 x g for 50 s. The supernatant was decanted, and the pellet washed with 45 ml of the isolating medium and centrifuged as above. The grinding medium contained 3 3 0 m M sorbitol, 5 0 m M N-tris(hydroxyethyl)methylglycine(Tricine)-KOH (ph 8.4), 2 m M E D T A (ethylendiaminetetraacetic acid), t m M MgCI~ and 4 m M 2-mercaptoethanol. All glassware and utensils were oven-sterilized while solutions were filter-sterilized. The intactness of chloroplast preparations was judged with phase-contrast microscopy (Spencer and Unt, 1965) as well as with transmission electron microscopy. In the latter, chloroplasts with an electron-dense stroma and with visibly unbroken envelopes were considered to be intact. Chlorophyll was determined by the method of A r n o n (1949).
Incubation of Chloroplasts
zymes common to the biosynthesis of aspartate-derived amino acids, ultimately blocking the flow of carbon from ASP to MET. Isolated, intact pea chloroplasts have been shown to incorporate labeled amino acids into protein when light is the sole energy source (Blair and Ellis, 1973). Neither exogenous amino acids nor cofactors are required for high rates of synthesis. If amino acids are biosynthesized in isolated chloroplasts they should be available for incorporation into protein. As in whole plants, LYS and/or T H R could regulate MET production. Consequently, MET would be reduced below the level required for high rates of protein synthesis. In this paper we report findings from experiments in which the effects of exogenous amino acids on light-driven protein synthesis in isolated pea chloroplasts were examined.
Material and Methods
Isolated choroplasts were resuspended in the KC1 incubation medium of Ramirez et al. (1968). This medium contained 200 m M KC1, 66 m M Tricine-KOH (pH 8.3) and 6.6 m M MgCI2. The chloroplast suspensions were placed in 10 m m diameter x 75 m m test tubes and radiolabeled LEU, ASP, M E T or T Y R was added to make the final volume. The tubes were incubated in a water bath at 20 _+ 1~ C. The partially submerged tubes (1 cm from the surface at the level of the sample suspension) were positioned at a 30~ angle to the water surface and shaken reciprocally at 80 cycles/rain. White light from two 500-W photoflood lamps (Photoflood E B U No. 2; General Electric, Cleveland, O., USA) provided an energy fluence rate at the level of the sample of ca. l0 s ~tW cm 2. Tubes for dark treatments were wrapped in aluminium foil. Samples of resuspended chloroplasts were periodically checked for bacterial contamination by plating aliquots on nutrient agar and counting colonies after incubation for 36 h at 37 ~ C. The nutrient agar was prepared by adding Difco (Difco Laboratories, Detroit, Mich., USA) Bacto Nutrient Broth (8 gfl) to 1%' Difco Bacto-Agar. The contamination of the chloroplast preparation by other organelles was estimated by utilizing marker enzymes. The mitochondrial marker, cytochrome oxidase, was assayed according to Hackett (1964) as modified by Miflin (1974), while catalase, the microbody marker, was assayed by the method of Aebi (1974).
Growth of Seedlings and Isolation of Chloroplasts
Determination of Radioactivity Incorporated into Protein
Plants of Pisum sativum L., cv. Alaska (W. Atlee Burpee Co., Warminster, Pa. USA), were grown in vermiculite in a growth room under continuous illumination (ca. 5 • 104 p c m - 2 ; measured
Following incubation, the chloroplast suspensions were rapidly cooled on ice, then spotted and dried on W h a t m a n (Whatman, Clifton, N.J., USA) 3-mm filter papers. The discs or squares
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts (2.5 cm diameter or 2.5 cm square) were prepared for liquid scintillation counting by the procedure of Mans and Novelli (1961). The processed discs or squares were placed in scintillation vials containing 5 ml of BBOT-toluene (8 g of 2,5-Bis[5'-tert-butyl-2benzoxazolyl]thiophene/1 toluene). The discs were counted in a Packard (Packard Instrument Co., Downers Grove, Ill., USA) TriCarb 3310 Liquid Scintillation Spectrometer at ca. 70% counting efficiency.
Chemicals All amino acids, EDTA, Triton X-100, 2-mercaptoethanol, Tricine, ribonuclease A, chloramphenicol and cycloheximide were obtained from Sigma Chemical Co., St. Louis, Mo., USA; actinomycin D from Merck, Sharp & Dohme, Rahway, N.J., USA; DCMU from ICN Pharmaceuticals, Plainview, N.Y., USA; and BBOT from Eastman Kodak, Rochester, N.Y., USA. Other unlabeled chemicals were obtained from Matheson, Coleman & Bell, Norwood, O., USA. The following radioactively labeled chemicals were acquired from New England Nuclear Corp., Boston, Mass., USA: L-[U14Claspartic acid, 203 mCi/mmol; L-[U-l*C]leucine, 325mCi/ mmot; L-l~hS]methionine, 511.9 Ci/mmot; L-[3,5-3H]tyrosine, 60.3 Ci/mmol.
155
Table 1. Characteristics of light-driven protein synthesis in isolated pea chloroplasts. A final volume of 0.1 ml contained 20 gg chlorophyll and 0.5 laCi of [~C]LEU; incubation was for t5 rain. The mean light-control value was 7570 cpm Treatment
Incorporation (% of light control)
Zero time Lysed Dark ATP (2 mM) Dark • ATP (2 mM) NH4C1 (1 mM) DCMU (2 ~tg/ml) D-Threo-Chloramphenicol (100 gg/ml) Cycloheximide (100 Fg/ml) Actinomycin D (10 lag/ml) Ribonuclease A (15 gg/ml)
8 9 27 93 38 19 36 37 93 126 99
9 Dark 9 Light
7.5
Results 7
Characteristics of Light-driven Protein Synthesis in Isolated Chloroplasts L i g h t a n d i n t a c t c h l o r o p l a s t s a r e r e q u i r e d for h i g h r a t e s o f s y n t h e s i s ( T a b l e 1). M a x i m a l r a t e s n e a r 6.0, 1.0, 0.1 a n d 0 . 1 n m o l m g 1 chlorophyll h-1 were o b s e r v e d w i t h [ 1 4 C ] L E U , [14C]ASP, [ 3 H ] T Y R , a n d [ 3 S S ] M E T , in this o r d e r . I n c o r p o r a t i o n o f [ I ~ C ] L E U i n t o p r o t e i n is r e d u c e d b y t h e u n c o u p l e r a m m o n i u m c h l o r i d e , by t h e e l e c t r o n - f l o w i n h i b i t o r D C M U , a n d the 70S-ribosome inhibitor chloramphenicol. However, n e i t h e r t h e 8 0 S - r i b o s o m e i n h i b i t o r c y c l o h e x i m ide, n o r t h e R N A - s y n t h e s i s i n h i b i t o r a c t i n o m y c i n D , n o r r i b o n u c l e a s e A r e d u c e d s y n t h e s i s at t h e c o n c e n t r a t i o n s tested, A T P at 2 m M was o n l y slightly s t i m u l a t o r y in t h e d a r k ( T a b l e 1). As noted by Jagendorf and colleagues, early studies on protein synthesis in isolated chloroplasts were complicated by bacterial contamination (App and Jagendorf, 1964; Bamji and Jagendorf, 1966). Therefore, the preparations of isolated chloroplasts were examined in a variety of ways to determine if contaminating bacteria affected the observed protein synthesis. Figure 2 illustrates a time course for the incorporation of radiolabeled LEU into protein. Incorporation is markedly light-dependent and the rate approaches zero after ca. 15-20 min. Incorporation by bacteria should be linear over this time interval (Bamji and Jagendorf, 1966). Furthermore, neither light dependence nor the sensitivity to photophosphorylation inhibitors evident in Figure 2 should be exhibited by bacteria. Treatment of the plastid suspensions with 2% Triton X-100 detergent, which solubilizes chloroplasts but not whole cells, bacteria or nuclei (Parenti and Margulies, 1967), left less than 1% of the radioactivity in the 10,000 x g pellet. Addition-
to 5.c
2.5
TIME (MIN)
Fig. 2. Time course for light-driven incorporation of [14C]LEU into protein by isolated pea chloroplasts. The final volume was 0.3 ml. Each tube contained 27.1 gg of chlorophyll and lgCi of [14C]LEU. At specified times, 0.05-ml aliquots were removed from each tube and processed for liquid scintillation counting
ally, neither plating on nutrient agar nor examination by transmission electron microscopy indicated bacterial contamination. Cytochrome oxidase and catalase activities have been used as markers for mitochondria and microbodies, respectively (Miflin and Beevers, 1974). After the isolation of the chloroplasts, the initial brei and the chloroplast fraction were checked with these markers for the presence of contaminating mitochondria and microbodies (Table 2). Less than 0.1% of the total catalase and cytochrome-oxidase activity was found in the chloroplast fraction, while 3% of the total chlorophyll was in this fraction. Thus, it is unlikely that results on light-driven protein synthesis in isolated pea chloroplasts have been affected by bacterial, mitochondrial or microbody contamination. T h e c h l o r o p l a s t p r e p a r a t i o n s w e r e f o u n d to be 45 8 5 % i n t a c t as j u d g e d by p h a s e - c o n t r a s t m i c r o s c o p y ( S p e n c e r a n d U n t , 1965) a n d t r a n s m i s s i o n electron microscopy.
156
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts
TaMe 2. Marker enzyme and chlorophyll distribution in chloroplast preparation. Chloroplasts were isolated as described in Materials and Methods. Aliquots were taken from the filtered brei and the resuspended chloroplast pellet and analyzed for chlorophyll content or catalase and cytochrome oxidase activity
100
LYS
Amount or activity
Organelle marker
Chlorophyll (gg/fraction) Catalase (gmol fraction 1 min- 1) Cytochrome oxidase (nmol fraction- l min + l)
Chloroplast pellet
Crude homogenate (cpm)
cpm
% of crude homogenate
7600 7600
200 5
3.0 0.07
2400
2
0.08
i 100 .~
.+,sI
oi ..I
Table 3.
Effect of aspartate-family amino acids on light-driven protein synthesis in isolated pea chloroplasts. In experiments with [i4C]LEU and [I+C]ASP label, the final volumes were 0.1 ml containing 5-20 gg chlorophyll and 0.5 gCi. Samples were incubated for 30 rain. With [3H]TYR, the final volume was 0.4 ml containing 16 lxg chlorophyll and 1.0 gCi. Incubation was for 15 min. The mean light control values were ll,099cpm with [a4C]LEU, 1039 cpm with [14CLASP and 1813 cpm with [3H]TYR Amino acid (raM)
Incorporation (% of light control) [i4C]LEU
None, dark LYS 1.0 THR 1.0 MET 0.1 MET 1.0 LYS 1.0+MET 0.1 THR 1.0+MET 0.l Amino acid mixture ~
21 58 69 103 . 70 92 95 95
[I+C]ASP
[3H]TYR
45 48 62 95 82 -
30 73 89 86 92 -
@ 0.
MET
75
Fig. 3. Effect of varying concentrations of LYS, THR and MET on light-driven incorporation of 1+C into protein by isolated pea chloroplasts. Incubation time was 15 rain. A final volume of 0,4 ml contained 0.1 gCi of [i4C]LEU
The amino acid mixture was composed of the following L-isomers, at 10 p.M each: alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glutamic acid, histidine, isoleucine, phenylalanine, proline, serine, tryptophan, tyrosine and valine
Effects of Aspartate-family Amino Acids on Light-driven Protein Synthesis in Isolated Chloroplasts In preliminary experiments, LYS, T H R or MET at 1 mM inhibited [I+C]LEU incorporation into protein, while 0.1 m M MET had no effect (Table 3). Addition of 0.1 m M MET reversed the LYS and T H R inhibition, while a mixture of the protein amino acids, excluding LYS, T H R , MET and LEU, had no effect. Similar experiments were conducted using [I+C]ASP and [3H]TYR as labels (Table 3). LYS, T H R and MET at 1 m M inhibited It+ClASP incorporation in a manner similar to that observed with labeled LEU. With [3H]TYR, 1 mM LYS or T H R reduced synthesis; however, the inhibition was less than that
observed with the other labeled amino acids. Again 0.1 mM MET partially reversed the inhibition. When the time course of inhibition of [t4C]LEU incorporation into protein by 1 m M LYS and 1 m M T H R was examined, the inhibition was found to be similar at 2.5, 5 and 15 min (data not shown). Therefore, a 15-rain incubation period was used in subsequent experiments since this is approximately the time when incorporation becomes asymptotic. A separate group of studies was initiated to determine the concentration of LYS, T H R and MET having the greatest effect on the incorporation of [14C]LEU into protein (Figure 3). Protein synthesis was reduced about 20% by 0,1 m M and 30% by 1 mM LYS. Lower concentrations had no effect. One and 10 raM T H R inhibited synthesis, while lower
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts Table 4. MET reversal of L Y S + T H R inhibition of light-driven protein synthesis in isolated pea chloroplasts Amino acid (raM) [14C]LEU~ incorporation cpm/ lag chl
% of light co,trot
[3H]TYRb incorporation cpm/ lag chl
% of light centre!
151+ 1-
-
None
774_+ 59 -
None, dark
116_+
7 15
46_+11
30
LYSO1 + T H R 0.1
574_+103 74
103_+18
68
LYS0.1 + T H R 0.1 + M E T 0.1
757+_ 21 98
149+_22
99
LYS 05 + T H R 0.5
158_+ 62 20
LYS 0.5 + T H R 0.5 + M E T 0.1
667+ 56 86
A final volume of 0.2 ml contained 5 lag chlorophyll and 0.5 gCi of [14C]LEU. Incubation time was 15 min b A final volume of 0.4 ml contained 12 ~tg chlorophyll and 1.0 gCi [3H]TYR. Incubation time was 15 min
Table 5. Effect of various combinations of LYS, THR and MET on the incorporation of [35S]MET into protein in isolated pea chloroplasts. A final volume of 0. l ml contained 7.9 lag chlorophyll and 1/aCi MET; incubation time was 15 rain. The mean lightcontrol value was 18,905 cpm Amino acid
Incorporation (% of light control)
LYS 0.1 mM LYS 1.0 mM THR 0.l mM THR 1.0raM MET 0.1 mM MET 1.0raM LYS 10laM+THR 10gJM LYS 0.1 m M + THR 0.1 mM LYS 0.1 m M + T H R 0.1 mM + MET 0.1 laM LYS 0.1 mM + T H R 0.1 m M + MET 0.1 ~tm LYS 0.1 m M + T H R 0.1 m M + MET 10 ~tM
89 103 104 158 22 26 131 115 116 96 37
Table 6. Effect of various amino acids on light-driven protein synthesis in isolated pea chloroplasts. A final volume of 0.4 ml contained 22.1 lag chlorophyll and 0.1 laCi of [Ir or the final volume of 0.5 ml contained 13 1 gg chlorophyll and 1 laCi of [3H]TYR. Incubation was for 15 rain. The mean light-control values were 2423 cpm with [14C]LEU and 1266 cpm with [3H]TYR Amino acid (1 raM)
c o n c e n t r a t i o n s h a d little effect. S i m i l a r l y , p r o t e i n synthesis was i n h i b i t e d by 1 m M M E T a n d u n a f f e c t e d by l o w e r c o n c e n t r a t i o n s .
Effect of Equimolar L Y S + THR on Light-driven Protein Synthesis in Isolated Pea Chloroplasts C o m b i n a t i o n s o f L Y S + T H R at 0.1 a n d 0.5 m M e a c h r e d u c e d l i g h t - d r i v e n p r o t e i n s y n t h e s i s in i s o l a t e d p e a chloroplasts when [~4C]LEU or [3H]TYR were the l a b e l s ( T a b l e 4). T h e d e g r e e o f i n h i b i t i o n was as g r e a t or g r e a t e r t h a n w i t h L Y S o r T H R a l o n e at a n y concentration tested. Furthermore, addition of 0.1 m M M E T p a r t i a l l y o r fully r e v e r s e d t h e i n h i b i t i o n in all cases. L Y S + T H R at 1.0 m M e a c h or g r e a t e r p r o d u c e d v a r i a b l e results, b u t g e n e r a l l y w e r e less inhibitory (data not shown).
Effect of L YS, THR and M E T on Light-driven Incorporation of [35S]ME T into Protein T a b l e 5 s h o w s d a t a f r o m a series o f e x p e r i m e n t s in w h i c h t h e effects o f L Y S a n d / o r T H R o n [ 3 5 S ] M E T incorporation into isolated pea chloroplast protein was o b s e r v e d . A l l c o n c e n t r a t i o n s o f L Y S , T H R a n d L Y S + T H R t e s t e d r e s u l t e d in levels o f [ 3 5 S ] M E T incorporation approaching or exceeding the control.
157
Incorporation (% of light control) [14C]LEU
Alanine Arginine Asparagine Aspartic acid Cystine Glutamic acid Glutamine Glycine Histidine Isoleucine Phenylalanine Proline Serine Tryptophan Tyrosine Valine
98 93 103 99 96 95 94 95 96 56 69 100 96 43 61 88
[3H]TYR 80 -
Effect of Other Protein Amino Acids on Light-driven Protein Synthesis in Isolated Pea Chloroplasts T h e effect o f 16 p r o t e i n a m i n o acids, o t h e r t h a n L Y S , T H R , M E T a n d L E U , o n l i g h t - d r i v e n p r o t e i n synthesis in i s o l a t e d p e a c h l o r o p l a s t s a r e s h o w n in T a b l e 6. A l a n i n e , a r g i n i n e , a s p a r a g i n e , a s p a r t i c acid, cystine, g l u t a m i n e , g l u t a m i c acid, glycine, h i s t i d i n e , p r o line a n d serine h a d little o r n o effect. V a l i n e was slightly i n h i b i t o r y , w h i l e I L E was m o r e effective. I L E inhibited incorporation of both [~C]LEU and
158
W.R. Millsand K.G. Wilson: AminoAcidsand Protein Synthesisin Pea Chloroplasts
[3H]TYR; however, the effect was more pronounced with [14C]LEU as the label. The aromatic amino acids, phenylalanine, tyrosine and tryptophan, all markedly inhibited [a4C]LEU assimilation into protein.
Discussion
Although several enzymes of the biosynthetic pathway(s) of the aspartate-famity amino-acids have been found in plastids (Miflin, 1974; Mazelis et al., 1976; Bryan et al., 1977; Miflin et al., in press, the question of LYS, THR, MET and ILE biosynthesis and its regulation in isolated chloroplasts has received little attention. One way to investigate the localization of amino acid biosynthetic capacity in isolated organelles is to examine the effect of potential feedback inhibitors on protein synthesis i n these organelles. LYS and/or THR inhibited protein synthesis in this system and these effects were reversed by MET. This is the expected result if MET is biosynthesized in isolated pea chloroplasts and its synthesis regulated b y LYS and/or THR. Similar inhibition of growth (see Miflin et al., in press) and protein synthesis (Dunham and Bryan, 1971; Henke and Wilson, 1974) by LYS and/or THR and reversal by MET have been observed in several plants. It is unlikely that competition for uptake between LYS and/or THR and the labeled amino acid caused the inhibition. First, similar results were obtained when the structurally different amino acids, ASP, LEU and TYR, were used as labels, and they have been reported to be transported by different carriers in isolated chloroplasts (Heldt and Rapley, 1970; Nobel and Cheung, 1972; McLaren and Barber, 1977). Second, in other experiments we found little difference in levels of free [I~C]LEU taken up by isolated pea chloroplasts in the presence of LYS or THR, even though typical inhibition of protein synthesis was observed (Mills, 1977). Aarnes and Rognes (1974) isolated one aspartate kinase and two homoserine dehydrogenases from 3-dold etiolated pea seedlings. The activity of the aspartate kinase and one of the homoserine dehydrogenases was inhibited in vitro by THR. These enzymes were unaffected by LYS. Therefore, the inhibition of protein synthesis in isolated pea chloroplasts by LYS was undxpected (Table 3). However, the reversal of LYS inhibition by MET indicates that some enzyme(s) i n the)pathway from ASP to MET may be inhibited by L~S. Davies (1977) isolated two aspartate kinases fromcarrot cell SUSlSensioncultures. One enzyme was inhibited by LYS and the other by THR. The relative concentrations o f these enzymes varied with culture
age. Thus, one possibility is that two aspartate-kinase isozymes exist in pea chloroplasts. If the LYS-sensitive enzyme is bound to plastid membranes, it could have been removed during enzyme isolation. Alternatively, if LYS and THR sensitive forms vary with tissue age, as in cultured carrot cells, 3-d-old etiolated pea seedlings may only have a THR-sensitive form, while older, light-grown plants could have both. Obviously, LYS may exert its effect on other enzymes of MET biosynthesis (see e.g. Wong and Cossins, 1976). Inhibition of light-driven protein synthesis by equimolar combinations (0.1-0.5 mM) of L Y S + T H R were observed in several experiments (Table 4). The effects were reversed by MET. L Y S + T H R at these concentrations often reduced rates of protein synthesis to the level of the dark control. However, higher levels of L Y S + T H R (1.0raM) were often less inhibitory than lower concentrations, although this effect was variable. These results are difficult to understand and are being studied further. The unusual inhibition pattern may be related to the unique accumulation of HS during the first few weeks of pea seedling growth (Larson and Beevers, 1965). As a further test of the hypothesis that MET biosynthesis occurs in isolated pea chloroplasts and that LYS + T H R regulates the synthesis, incorporation of [3~S]MET into protein was examined (Table5). If isolated pea chloroplasts are incubated with labeled MET, the exogenous [3SS]MET and the endogenous MET should compete for incorporation into protein. Under inhibitory conditions, i.e. in the presence of LYS and/or THR, lowered MET biosynthesis should reduce isotope dilution. Hence, although overall protein synthesis should be reduced, a concomitant decrease in measurable [35S]MET incorporation should not be observed. Indeed, with all LYS and/or THR treatments, [35S]MET incorporation approaching or exceeding the control was observed. Miflin (1969) studied the effect of exogenous amino acids on the growth of barley seedlings. He noted that the most inhibitory ones, LYS, THR, MET, HS, LEU, TYR and valine, are all produced by branched pathways from common precursors. Similarly, with isolated pea chloroplasts, the amino. acids inhibiting protein synthesis the most are all biosynthesized in branched pathways. It is unclear if these effects result from reduced amino-acid biosynthesis because of feedback inhibition. However, it is interesting that similar groups of amino acids inhibit both light-driven protein synthesis in isolated pea chloroplasts and growth of barley seedlings. The use of isolated pea chloroplasts capable of high rates of protein synthesis with light as the only
W.R. Mills and K.G. Wilson: Amino Acids and Protein Synthesis in Pea Chloroplasts energy source has several a d v a n t a g e s . First, the p r e p a r a t i o n s c o n t a i n high p e r c e n t a g e s o f intact plastids, therefore s t r o m a l enzymes are n o t lost. Second, inc o r p o r a t i o n o f a m i n o acids into p r o t e i n s h o u l d facilitate c a r b o n flux t h r o u g h a m i n o - a c i d b i o s y n t h e t i c p a t h w a y s , as well as reduce the c o n c e n t r a t i o n o f end o g e n o u s a m i n o acids which c o u l d p o t e n t i a l l y act as f e e d b a c k inhibitors. T h i r d , when light is used to drive the reactions, it n o t only reduces the l i k e l i h o o d o f s p u r i o u s results c a u s e d by b a c t e r i a or o t h e r cont a m i n a n t s , b u t also eliminates the need for e x o g e n o u s A T P or r e d u c e d p y r i d i n e nucleotide. The d a t a p r e s e n t e d here are c o n s i s t e n t with the h y p o t h e s i s t h a t M E T is b i o s y n t h e s i z e d in i s o l a t e d p e a c h l o r o p l a s t s a n d t h a t this synthesis is r e g u l a t e d b y LYS and/or THR. First, LYS and THR alone and in specific c o m b i n a t i o n s inhibit l i g h t - d r i v e n p r o t e i n synthesis in p e a c h l o r o p l a s t s a n d this effect is r e v e r s e d b y M E T . S i m i l a r effects have been o b s e r v e d in whole p l a n t s a n d have been a t t r i b u t e d to i n h i b i t i o n o f M E T biosynthesis b y L Y S a n d / o r T H R . Second, i n c o r p o r a tion o f laSS]MET into p r o t e i n by i s o l a t e d p e a c h l o r o plasts is u n a f f e c t e d or s t i m u l a t e d by L Y S a n d / o r T H R while these c o m p o u n d s r e d u c e i n c o r p o r a t i o n o f o t h e r l a b e l e d a m i n o acids. This is e x p e c t e d if M E T p r o d u c tion occurs in i s o l a t e d c h l o r o p l a s t s a n d these c o m p o u n d s i n h i b i t M E T b i o s y n t h e s i s a n d thus reduce i s o t o p e dilution. Third, in p r e l i m i n a r y e x p e r i m e n t s we f o u n d t h a t r a d i o l a b e l e d M E T is p r o d u c e d f r o m [ l a C ] A S P a n d [35S]SO42-b y i s o l a t e d p e a c h l o r o p l a s t s i n c u b a t e d u n d e r c o n d i t i o n s for l i g h t - d r i v e n p r o t e i n synthesis (Mills, 1977). F u r t h e r m o r e , M E T b i o s y n thesis f r o m b o t h p r e c u r s o r s was i n h i b i t e d by LYS+THR at 0 . 5 r a M each. These d a t a , c o u p l e d with recent evidence showing t h a t a s p a r t a t e kinase, homoserine dehydrogenase, diaminopimelate decarbo x y l a t e a n d a c e t o l a c t a t e s y n t h e t a s e are f o u n d in plastids, i n d i c a t e t h a t LYS, T H R , M E T a n d I L E are all b i o s y n t h e s i z e d in c h l o r o p l a s t s . We wish to thank S.W.J. Bright and P.J. Lea (Rothamsted Experimental Station, Harpenden, Herts, U.K.), M.J. Muhitch and E.L. Burdge, III (Miami University), for critically reading the manuscript, and M.J. Powell (Miami University) for assistance with the electron microscopy. Financial assistance was provided in part by the Miami University Faculty Research Committee.
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Received 6 February; accepted 26 May 1978
