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Direct Transport of Adpglucose by an Adenylate Translocator Is Linked to Starch Biosynthesis in Amyloplasts

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Direct Transport of Adpglucose by an Adenylate Translocator Is Linked to Starch Biosynthesis in Amyloplasts Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5769-5773, July 1991 Plant Biology Direct transport of ADPglucose by an adenylate translocator is linked to starch biosynthesis in amyloplasts (carboxyatractyloside/double silicone oil layer centrifugation/starch synthase/sucrose synthase/sycamore) JAVIER POZUETA-ROMERO, MARCO FREHNER*, ALEJANDRO M. VIALEt, AND TAKASHI AKAZAWA Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464-01, Japan Communicated by Andre T. Jagendorf, February 19, 1991 ABSTRACT Starch biosynthesis has been studied by using Previously, we reported the detection, by immunoanalysis, amyloplasts isolated from cultured cells ofsycamore trees (Acer of a putative adenylate (ATP/ADP) translocator in the amy- pseudoplatanus L.). Highly purified intact amyloplasts, free loplast envelopes isolated from cultured sycamore cells (6). from mitochondria and starch granules derived from broken We have now examined the kinetic properties of this trans- amyloplasts, were isolated from a Percoll step gradient. Sub- porter by using intact amyloplast preparations and have sequently, the double silicone oil layer centrifugation technique found evidence for the direct transport ofADP-Glc across the was used to study adenylate transport in the amyloplasts. An amyloplast envelope, which is tightly linked to the formation adenylate-specific carrier was found to be active in the uptake of starch. of ATP, ADP, AMP, and, most importantly, ADPglucose (ADP-Glc). Kinetic analyses showed that the uptake of these MATERIALS AND METHODS adenylates was mutually competitive with each other. In con- Cell Culture and Amyloplast Isolation. Amyloplasts from trast to the mitochondrial adenylate carrier, in amyloplasts nonstarved suspension culture cells of sycamore trees (Acer only ATP and ADP-Glc uptake were inhibited by carboxya- pseudoplatanus L.) were isolated according to the Percoll tractyloside. Evidence is presented that the ADP-Glc trans- step-gradient method (7, 8) with some modifications; the final ported into the amyloplast stroma can be used in starch gradient consisted of 2 ml each of Percoll layers ofp = 1.057, synthesis catalyzed by starch synthase (ADP-Glc:1,4-a-D- 1.084 (including protoplast lysate), 1.098, and 1.210 g/ml. To glucan 4-a-D-glucosyltransferase, EC 2.4.1.21). We propose eliminate contamination by starch granules derived from that starch biosynthesis in amyloplasts is tightly coupled with broken amyloplasts, an extra dense layer of Percoll (p = the direct transport of ADP-Glc synthesized in the cytosol by 1.210 g/ml), obtained after evaporation of the commercial sucrose synthase (ADP-Glc:D-fructose 2-a-D-glucosyltrans- product, was placed in the bottom of the gradient. After ferase, EC 2.4.1.13). centrifugation at 2000 x g in a swinging-bucket type rotor, amyloplasts were recovered from the interface between the The amyloplast is a specifically differentiated plastid that Percoll layers with p = 1.098 and 1.210 g/ml, whereas free synthesizes and accumulates reserve in starch granules pelleted down to the bottom (see Fig. la). starch the stroma. Collected amyloplasts were sedimented at 1000 x g after Although it is frequently hypothesized that chloroplasts and dilution in MEM buffer (10 mM Mops, pH 7.5/1 mM EDTA/ amyloplasts are ontogenically and evolutionarily related to 0.5 M mannitol) and resuspended in sampling buffer (25 mM each other, they differ in their carbon and adenylate flow (1). Hepes, pH 7.5/20 mM KCl/1 mM EDTA/0.45 M mannitol) Chloroplasts produce ATP and NADPH necessary for pho- [final 6-phosphogluconate dehydrogenase (6PGDH) specific tosynthetic CO2 fixation (Benson-Calvin cycle). Although it activity = 460 ± 90 units/liter]. The intactness of the amy- is established that fixed products are exported to the cytosol loplasts was determined as reported (1). by means of the phosphate translocator (2), some ofthem are Vesicles from amyloplast envelopes were prepared accord- utilized for the formation of starch in the chloroplast. ing to the method described by Liedvogel and Kleinig (9) for ADPglucose pyrophosphorylase (glucose-i-phosphate ade- obtaining vesicles from daffodil chromoplasts. Vesicles were nylyltransferase, EC 2.7.7.27) utilizes the photochemically resuspended in sampling buffer but without mannitol (hypo- generated ATP and glucose 1-phosphate (Glc-1-P) for the tonic buffer; 1 mg of protein/ml). synthesis of ADPglucose (ADP-Glc), which serves as the Adenylate Transport. Unless indicated otherwise, transport glucosyl donor in starch formation. This enzyme is believed experiments were performed with Bio-Rik 0.4-ml plastic tubes to play a key role in the overall gluconeogenic mechanism in from Bio Plastics (Osaka) at 4°C in a refrigerated TOMY the chloroplast (3). On the other hand, amyloplasts possess (Tokyo) MRX-151 centrifuge. A conventional single silicone neither ATP-generating nor C02-fixing capabilities. Provided oil layer centrifugation technique (10) was used for assaying an analogous mechanism of starch biosynthesis operates in the time course of uptake of adenylates in intact amyloplasts. both organelles (3), one must postulate the presence of In experiments examining the inhibitory effect of carboxya- adenylate and sugar-phosphate shuttles in the amyloplast tractyloside (CAT) on the adenylate transport, amyloplast envelope membranes. It can be envisioned that imported preparations were preincubated at 4°C with the reagent (100 ATP and carbon compounds from the cytosol would be jM) for 30 min. Subsequent kinetic studies of adenylate utilized eventually for the synthesis of ADP-Glc by ADP-Glc pyrophosphorylase. However, the extreme physical fragility Abbreviations: ADP-Glc, ADPglucose; CAT, carboxyatractyloside; of this starch-filled organelle has made it difficult to isolate Glc-1-P (or Glc-6-P), glucose 1-phosphate (or glucose 6-phosphate); structurally and functionally competent amyloplasts and to 6PGDH, 6-phosphogluconate dehydrogenase; UDP-Glc, UDPglu- examine their transport capabilities linked to the mechanism cose. of gluconeogenesis (4, 5). *Present address: Department of Plant Science, Swiss Federal Institute of Technology, Eidgenossiche Technische Hochschule- Zentrum, CH-8092 Zurich, Switzerland. The publication costs of this article were defrayed in part by page charge tPresent address: Department ofMicrobiology, Facultad de Ciencias payment. This article must therefore be hereby marked "advertisement" Bioquimicas y Farmaceuticas, Universidad de Rosario, 2000 Ro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. sario, Argentina. 5769 Downloaded by guest on September 25, 2021 5770 Plant Biology: Pozueta-Romero et al. Proc. Nati. Acad. Sci. USA 88 (1991) uptake were carried out by using the double silicone oil layer and mitochondria, respectively (7). The sucrose synthase centrifugation technique. The precise details of this experi- (ADP-Glc:D-fructose 2-a-D-glucosyltransferase, EC 2.4.1.13) mental technique will be described elsewhere (J.P.-R., M.F., activity in the protoplast lysates (7) was assayed as described and T.A., unpublished results). A summary of the procedure (12), replacing ATP and UDP with UTP and ADP, respec- is as follows: amyloplasts resuspended in sampling buffer (40 tively. Starch content was estimated as reported (7). ,Al, p = 1.031 g/ml) are separated by a layer oflight silicone oil To examine the covalent a( -l 4) linkage formed between (75 ,ul, p = 1.003 g/ml) from the labeled metabolite-containing [14C]glucose and the terminal end of the amylose molecules, incubation layer (30 mM Hepes, pH 7.5/1 mM EDTA/25 mM TLC separation of the /8-amylolytic products of the labeled KCl/0.4 M sucrose; 60 Aul, p = 1.059 g/ml). The incubation of starch granules isolated from the amyloplasts incubated with amyloplasts with metabolite in the incubation layer begins ADP-['4C]Glc was performed. The identification of radiola- immediately after inversion of the light silicone layer during beled adenylates transported in amyloplasts was carried out centrifugation. The incubation time varies as a function of the by using ascending cellulose TLC [isobutyric acid/water/ centrifugal force (g); we determined empirically that centrif- ammonia/EDTA (37 g/liter) (500:280:21:8, (vol/vol)] fol- ugation at 750 x g corresponds to a 37-sec incubation period, lowed by radioautography. which was employed throughout the present kinetic analyses. Reagents. The radiochemicals {[3H]ATP, [3H]ADP, [3H]- The reaction stops when amyloplasts traverse the incubation AMP, ADP-[14C]Glc, [3H]UTP, UDP[14C]glucose (UDP- layer to the heavy silicone oil layer (75 ,ul, p = 1.066 g/ml), [14C]Glc), [14C]Glc, 3H20, and [14C]sorbitol} were purchased which is located below the incubation medium. Finally, the from Amersham. /3-Amylase, hexokinase (no. H4502), glu- amyloplasts sediment to the pelleting layer (100 ,.d of 0.55 M cose-6-phosphate dehydrogenase (no. G5885), and phospho- sucrose containing 10% HCI04, p = 1.075 g/ml) located under glucoisomerase (no. P5381) were purchased from Sigma. the lower silicone layer. Wackersilicone AR 20 Pharmas from Wacker-Chemie Since the sorbitol-permeable space (intermembrane space) (Munich) and SH 704 from Toray Silicone (Osaka) were used is accessible to every metabolite tested, real values of me- in the transport experiments. tabolite transport mediated by carrier proteins located in the amyloplast inner membranes are obtained after determining RESULTS the sorbitol-impermeable space (3H20-permeable space
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