Planta 136, 147

Planta

151 (1977)

9 by Springer-Verlag 1977

Evidence for Carrier-Mediated Transport of L-Leucine into Isolated Pea (Pisum sativum L.) Chloroplasts J.S. McLaren and D.J. Barber* Department of Physiology and Environmental Studies, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, LE12 5RD, U.K.

Abstract. The uptake of leucine into isolated, intact,

pea chloroplasts was investigated using the silicone oil centrifugation technique. The internal: external ratio of leucine exceeded unity at low external leucine concentrations. Uptake of leucine at different e~ternal concentrations showed passive diffusion and carriermediated transport components. Competition for uptake was shown between leucine and isoleucine but not between leucine and glycine. Rates of diffusion of leucine were found to be low compared with glycine, however, fast carrier-mediated transport of leucine assumed more importance at physiological concentrations. Key words: Carrier -- mediated transport -- Chloroplast -- Leucine -- Pisum.

Contrary to the evidence of Nobel, Gimmler et al. (1974) reported low rates of net uptake of neutral amino acids, into isolated chloroplasts, and based on both osmotic volume changes and uptake of labelled amino acids found no evidence of carriermediated transport. In preliminary experiments it was found that isolated chloroplasts were capable of accumulating leucine in excess of the leucine concentration in the external medium. In view of this, and since Gimmler et al. (1974) did not report data for leucine uptake, the uptake of labelled leucine into isolated intact chloroplasts was investigated and the results reported here.

Materials and Methods Plant Material and Chloroplast Isolation

Introduction

In recent years, specific translocators, in the chloroplast membrane, have been established for ATP (Heldt, 1969), dicarboxylic acids and triose-phosphates (Heldt and Rapley, 1970). However, some controversy exists over the permeability of chloroplasts to amino acids. Based on reflection coefficients for amino acids, determined from osmotic responses of isolated pea chloroplasts, it was reported that (a) isolated chloroplasts are freely permeable to amino acids (Nobel and Wang, 1970) and (b) the chloroplast has two amino acid carriers (Nobel and Cheung, 1970). * Present address." Division of Biochemistry, North-East London Polytechnic, R o m f o r d Road, Stratford, L o n d o n Abbreviations." S I S = S u c r o s e impermeable space; T W S = T r i t i a t e d water space; SPS = Sucrose permeable space ; P G A = 3-phosphoglyceric acid; T C A = Trichloroacetic acid; T L C = Thin layer chromatography

Pisum sativum " M e t e o r " were grown in compost at 25 ~ C under low irradiance (10 W m -2) produced by warm white fluorescent tubes, and a daylength of 16 h. After 10-12 days, the leaves were harvested (one hour into the day cycle) and homogenised in extraction buffer (0.3 M sucrose, 25 m M Hepes, 2 m M EDTA, 2 m M Na ascorbate, pH 7.5) at top speed for 4 s using a polytron, type PT-20 with a PCU-2 power control. The homogenate was filtered through 4 then 8 layers of muslin and centrifuged at 3000 g for 60 s. The pellet was washed and resuspended in extraction buffer plus 1 m M Mg Clz and 1 m M Mn CI z. The isolation procedure was carried out at 0 ~ C and completed within 120 s. Chlorophyll content was determined using 80% acetone (MacKinney, 1941).

Physiological status o f isolated chloroplasts Isolation of chloroplasts by the method described above resulted in a heterogenous population of chloroplasts. The integrity of the chloroplasts in these routine preparations was determined as follows :(a) W h e n examined by phase contrast microscopy the preparations were estimated to contain between 60-80% intact chloroplasts. (b) Using ferricyanide as a Hill oxidant, and an oxygen elec~ trode to measure light dependent 02 evolution (Gimmler, et al.,

148

J.S. McLaren and D.J. Barber: Carrier-Mediated Transport of L-Leucine

1974; Heber and Santarius, 1970), chloroplast suspensions were found to contain 65% intact chloroplasts. (c) The chloroplast preparations fixed CO 2 at a rate of 70 gmol mg- 1 chlorophyll h- 1 and demonstrated concomitant 02 evolution with HCO3 or PGA as substrate. This corresponds to isolated chloroplasts of type A, as defined by Hall (1972).

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Movement of labelled compounds into isolated chloroplasts was measured using a modified silicone oil centrifugation technique (Heldt and Sauer, 1970). To micro-centrifuge tubes containing 25 ~al 2 M KOH and a 100 ill layer of inert silicone oil (Type AR100, from Wacker, Munich) was added 100 ~tl chloroplasts suspension (approx. 150 pg chlorophyll/ml) plus radioactive and non-labelled amino acid. All experiments were carried out at 20~ C in darkness. Incubations were terminated by centrifugation using a micro-centrifuge (Jobling Laboratories, model 320). The micro-centrifuge tubes were frozen and sawn across the oil layer, the sedimented chloroplast pellets were then solubilised by heating at 60~ C for 30 rain. Supernatants and dissolved pellets were counted in a Packard TriCarb Liquid Scintillation Spectrometer in conjunction with an automatic activity analyser for quench correction. Tritiated-water and [14C] sucrose treatments were run in parallel with each amino acid investigated. The osmotic volume of intact chloroplasts (sucrose impermeable space, SIS) was calculated from the difference between the tritiated water space (TWS) and the sucrose permeable space (SPS).

Results I n p r e l i m i n a r y e x p e r i m e n t s investigating leucine upt a k e into i s o l a t e d c h l o r o p l a s t s it was f o u n d that, after 180 s i n c u b a t i o n , the c o n c e n t r a t i o n o f leucine in the SIS was 10 times higher t h a n in the external m e d i u m . F i g u r e 1 shows the increase in i n t e r n a l leucine concent r a t i o n with time. Leucine c o n c e n t r a t i o n in the extern a l m e d i u m was 1 r a M . T h e internal to external r a t i o r a p i d l y exceeded u n i t y a n d 4 possible e x p l a n a t i o n s o f this were c o n s i d e r e d : (a) m e t a b o l i c c o n v e r s i o n o f leucine, (b) the presence o f an electrical p o t e n t i a l gradient, (c) a d s o r p t i o n a n d (d) c a r r i e r - m e d i a t e d t r a n s p o r t . (a) C o n v e r s i o n o f l e u c i n e , in the i s o l a t e d c h l o r o p l a s t , to a less p e r m e able p r o d u c t w o u l d result in a " m e t a b o l i c s i n k " leading to a n a p p a r e n t a c c u m u l a t i o n a g a i n s t its gradient. I s o l a t e d c h l o r o p l a s t s were i n c u b a t e d with [14C] leucine at 20 ~ C. T h e i n c u b a t i o n was t e r m i n a t e d , after 40 s, b y the a d d i t i o n o f 10% t r i c h l o r o a e t i c acid (TCA). I n c o r p o r a t i o n o f [14C]leucine into T C A - p r e c i p i t a t e d m a t e r i a l was only 8 % o f [14C]leucine u p t a k e into the chloroplasts. A l i q u o t s o f the s u p e r n a t a n t (from the T C A p r e c i p i t a t i o n ) c h r o m a t o g r a p h e d b y T L C ( M c L a r e n , 1976) resulted in a single p e a k o f radiactivity, which c o r r e s p o n d e d to leucine. These d a t a i n d i c a t e d t h a t there was n o m e t a b o l i c c o n v e r s i o n o f leucine, in isolated c h l o r o p l a s t s , u n d e r these experim e n t a l conditions.

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(b) D u e to the electrical p o t e n t i a l g r a d i e n t which exists across the c h l o r o p l a s t e n v e l o p e ( V r e n d e n b e r g et al., 1973 ; Bulycheva et al., 1972) c h a r g e d molecules m a y m o v e across the e n v e l o p e a g a i n s t their concent r a t i o n gradients. T h e o b s e r v e d a c c u m u l a t i o n o f leucine m a y have arisen since, at p H 7.5, a p p r o x i m a t e l y 1% o f the m o l e c u l e s are p r e s e n t as the anion. A t p H 5.9 leucine b e a r s no net charge, t h e r e f o r e a c c u m u l a t i o n a g a i n s t a c o n c e n t r a t i o n g r a d i e n t c o u l d n o t be driven b y a n electrical p o t e n t i a l gradient. T a b l e 1 shows t h a t at p H 5.9 the internal c o n c e n t r a t i o n s o f leucine was five times the external c o n c e n t r a t i o n . (c) A d s o r p t i o n o f leucine o n t o c h a r g e d sites in the c h l o r o p l a s t s was n o t c o n s i d e r e d to be significant because o f the following reasons. T h e r a t i o o f [12C] :[l~C]leucine used in these experiments was large (at least 330:1) a n d the internal c o n c e n t r a t i o n o f [14C]leucine did n o t decrease as the [12C]:[14C] r a t i o in the external m e d i u m was increased. This can be e x p l a i n e d in t e r m s o f a d s o r p t i o n only if large n u m b e r s o f b i n d i n g sites are available. This seems u n l i k e l y since o t h e r a m i n o acids such as glycine a n d m e t h i o n i n e , which are also zwitterionic at p H 7.6, fail to s h o w i n t e r n a l : e x t e r n a l r a t i o s in excess o f unity, even after p r o l o n g e d i n c u b a t i o n s (Barber, 1976). These d a t a indicate t h a t leucine accuTable 1. Internal concentration of leucine, in isolated chloroplasts, after 120 s incubation at 2 pH values. Each value is the mean of 3 replicates pHexternalmedium

Internalleucineconc. (mM)

Int/Extratio leucine

7.5 5.9

11.5• 4.8•

11 5

J.S. McLaren and D.J. Barber: Carrier-Mediated Transport of L-Leucine

mulated in the SIS not because of adsorption, which any similar molecule might be expected to demonstrate, but due to some characteristic of the chloroplast, specific to leucine. (d) Accumulation of compounds against their concentration gradients has been shown to be a property of carrier-mediated transport (Rosenberg and Wilbrandt, 1957; Wilbrandt and Rosenberg, 1961). The possible existence of a carrier for leucine was investigated in the follwing experiments:

149

Table 2. Volume of the sucrose impermeable space, after 30 s, with various concentrations of leucine in the chloroplast suspension medium Leucine conc. in external medium (raM)

Sucrose impermeable space Itl/mg chlorophyll

0.5 1.0 5.0 10.0 20.0

17_+2 I8_+2 18_+2 25_+3 21 _+4

1. Rates of Uptake 160

Figure 2 shows the rate of uptake of leucine at various concentrations ofleucine in the medium. Up to 5 mM leucine, in the medium, there was a rapid increase in the rate of uptake, followed by a slower linear increase with external concentration. These data are not consistent with transport of leucine by passive diffusion alone. Transformation of the data to a Hofstee plot revealed two separate components o f uptake (Fig. 3). On a Hofstee plot, passive diffusion is represented by a vertical line; linear regressions other than vertical represent carrier-mediated transport (Neame and Richards, 1972). The vertical component in Figure 3 was taken as passive diffusion while the second component was considered to represent carrier-mediated transport with a K m = 0 . 5 mM and V .... --48 nmol (mg chlorophyll)- 1 30 s 1. Internal leucine concentrations were determined by dividing the total uptake of leucine into the SIS, by the SIS volume at various concentrations of leucine in the medium. The internal:external ratio exceeded unity only at external concentrations of less than 5 mM (Fig. 4).

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Fig. 2. Rate of uptake of leucine, over 30 s, into intact chloroplasts at various external concentrations of leucine. Each point is the mean of 3 replicates. The broken line represents an estimate of passive diffusion

160

2. Competition Experiments On the basis of reflection coefficient measurements two different carriers for amino acids, in pea chloroplasts, were proposed, and competition predicted between glycine, alanine, leucine, isoleucine and valine; and between serine, threonine and methionine (Nobel and Cheung, 1972). Uptake of [14C]leucine into intact chloroplasts was measured in the presence of isoleucinc or glycine. Table 3 shows that 10 mM isoleucine decreased leucine uptake by 98%, while 10 mM glycine decreased the rate of uptake leucine by only 29%. This indicates competition between isoleucine and leucine for a common carrier whereas glycine shows little evidence of competition. Since the two carriers proposed by Nobel and Cheung (1972) were investigated using high molarities of amino acid, furt-

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J.S. McLaren and D.J. Barber: Carrier-Mediated Transport of L-Leucine

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Fig. 4. Internal:external ratio of leucine concentration, after 30 s, with isolated chloroplasts at various external leucine concentrations. Each point is the mean of 3 replicates

Table 3. Rate of uptake of leucine into isolated chloroplasts, over 30 s, in the presence of isoleucine or glycine. Each value is the mean of 3 replicates Amino acid in external medium

1 mM 5 mM 1 mM 1 mM

Rate of leucine uptake nmol (rag chlorophyll) ' 30 s 1

leucine 34 leucine 41 leucine+ 10 m M isoleucine 0.5 leucine+10 m M glycine 24

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Table 4. Uptake of [14C]glycine into isolated chloroplasts in the presence of other amino acids

A m i n o acid in external medium (60 raM)

Uptake of [14C]glycine [nmol (mg Chl)- a 20 s 1]

Alanine Serine Methionine Leucine Glycine

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her competition experiments measuring uptake of p4C]glycine were carried out here, in the presence of 60 mM amino acids. Contrary to previous findings (Nobel and Cheung, 1972), Table 4 shows that no competition was found between [14C]glycine and any of the amino acids tested. Measurements of the rate of uptake of glycine at various concentrations of glycine in the medium showed a linear response characteristic of passive diffusion (Fig. 5). Also, unlike leucine, glycine was never found to accumulate against its concentration gradient. From these results it would appear that movement of leucine and glycine into isolated chloroplasts occurs by diffusion. However, in the case of leucine, the evidence suggests that carrier-mediated transport assumes more importance at physiological concentrations.

Discussion

The rapid penetration of amino acids, into isolated chloroplasts, indicated by their low reflection coefficients (Nobel and Wang, 1970; Nobel and Cheung, 1972) has not been confirmed by measurement of rates of uptake of labelled amino acids (Gimmler et al., 1974). The rates of uptake of leucine {35 nmol (mg chlorophyll)- 1 30 s- 1 from 1 mM leucine} and glycine {18 nmol (mg chlorophyll) -1 30 s -~ from 1 mM glycine} reported here do not indicate that chloroplasts are freely permeable to amino acids. Gimmler et al. (1974) reported comparable rates of amino acid uptake and found no evidence for the existence of a carrier for any neutral amino acids. However, they did not publish any findings concerning leucine uptake. The data presented in this paper show that leucine could accumulate, in the chloroplast, against its own concentration gradient. These results cannot be explained by diffusion alone. It was shown that the leucine accumulation was not due to: 1. metabolic conversion inside the chloroplast, which could have effectively maintained a concentration gradient across the chloroplast envelope; 2. the presence of an electrical potential gradient; 3. adsorption of leucine onto charged sites in the chloroplast. The evidence from uptake kinetics and competitor experiments indicated that uptake of leucine was by carrier-mediated transport. Experiments were not carried out to determine the nature or driving force (e.g. Counter exchange) of the carrier. However, there appeared to be specificity for leucine and isoleucine. Nobel and Wang (1970) postulated the existence of a carrier specific for glycine and the aliphatic amino acids. The results presented here indicate cartier-mediated transport of leucine/isoleucine but not

J.S. McLaren and D.J. Barber: Carrier-Mediated Transport of L-Leucine

glycine. Since Nobel and co-workers used high concentrations of amino acids (> 100 raM) it appears unlikely that the aliphatic amino acid carrier corresponds to the carrier proposed from our data, which operates nearer physiological concentrations and saturates around 5 mM leucine. One possible reason for the existence of carriermediated transport of leucine may be that rates of diffusion are low compared with other amino acids. The estimated rate of diffusion of leucine (Fig. 2) is 75% lower than that of glycine (Fig. 5). However, at external concentration of 1 mM, the total leucine uptake was three times higher than that of glycine. This suggests that, at physiological concentrations, carrier-mediated transport of leucine ensures that movement of leucine across the chloroplast envelope does not become a rate limiting step. A second factor concerns the ability of chloroplast to synthesise amino acids. Isolated chloroplasts of Vicia faba were found to be able to synthesise most protein amino acids, but no synthesis of leucine by aminotransferase reactions could be detected (Kirk and Leech, 1972). The proposed leucine carrier could be important in maintaining the leucine pool in the chloroplasts. References Barber D.J. : Ph.D. Thesis, University of Liverpool (1976). Bulycheva A., Andrianov V.K., Kurella G.A., Litvin F.F. : Microelectrode measurements of the transmembrane potential of Pereromia metallica. Nature (Lond.) 236, 175-177 (1972) Gimmler H., Schafer G., Kraminer H., Heber U.: Amino acid permeability of the chloroplast envelope as measured by light

151

scattering, volumetry and amino acid uptake. Planta (Berl.) 120, 47-61 (1974) Hall D.O.: Nomenclature for isolated chloroplasts. Nature New Biol. 235, 125-126 (1972) Heber U., Santarius K,A.: Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z. Naturforsch. 25b, 718-728 (1970) Heldt H.W. : Adenine nucleotide translocation in spinach chloroplasts. FEBS Letters 5, 11 14 (1969) Heldt H.W., Rapley L. : Unspecific permeation and specific uptake of substances in spinach chloroplasts. FEBS Letters 7, 139 142 (1970) Heldt H.W., Sauer F.: The inner membrane of the chloroplast envelope as the site of specific metabolite transport. Biochim. Biophys. Acta 234~ 83-91 (1970) Kirk M., Leech R.M. : Amino acid biosynthesis by isolated chloroplasts during photosynthesis. Plant Physiol. 50, 228-234 (1972) MacKinney G.: Absorption of light by chlorophyll solutions. J. Biol. Chem. 140, 315 (194i) McLaren J.S.: P h . D . Thesis, University of Nottingham (1976) Neame K.D., Richards T.G.: Elementary kinetics of membralae carrier transport. Oxford: Blackwell Scientific Publications 1972 Nobel P.S., Cheung Y.N.S. : Two amino acid carriers in pea chloroplasts. Nature New Biol. 237, 207-208 (1972) Nobel P.S., Wang C.T.: Amino acid permeability of pea chloroplasts as measured by osmotically determined reflection coefficients. Biochim. Biophys. Acta 211, 79 87 (1970) Rosenberg T., Wilbrandt W. : Uphill transport induced by counterflow, J. Gen. Physiol. 41,289 296 (1957) Vredenberg W.J., Homann P.H., Tonk W.J.M.: Light-induced potential changes across the chloroplast enclosing membrane and expressions of primary events at the thylakoid membrane. Biochim. Biophys. Acta 314, 261-265 (1973) Wilbrandt W., Rosenberg T.: The concept of carrier transport and its corrollaries in pharmacology. Pharmacol. Rev. 13, 109-183 (1961)

Received 1 March ; accepted 10 M a y 1977

Evidence for carrier-mediated transport of L-leucine into isolated pea (Pisum sativum L.) chloroplasts.

The uptake of leucine into isolated, intact, pea chloroplasts was investigated using the silicone oil centrifugation technique. The internal: external...
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