JXB Advance Access originally published online on February 13, 2007
Journal of Experimental Botany 2007 58(6):1321-1332; doi:10.1093/jxb/erl297
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Characterization of ADP-glucose transport across the cereal endosperm amyloplast envelope
1Faculty of Life Sciences, 3.614 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK
2Dipartmento di Biologia Vegetale, University of Naples (Federico II), Via Foria 223, 80139, Naples, Italy
3Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
* To whom correspondence should be addressed. E-mail: itetlow{at}uoguelph.ca
Received 2 August 2006; Revised 7 December 2006 Accepted 11 December 2006
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Abstract |
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Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[
-32P]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid. Key words: ADP-glucose transport, amylopectin, amyloplasts, amylose, starch synthesis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Adenosine 5' diphosphate glucose pyrophosphorylase (AGPase, EC 2.7.7.27 [EC] ) catalyses an important step in starch biosynthesis of higher plants, being responsible for the synthesis of the nucleotide diphosphate sugar precursor ADP-glucose (ADP-Glc) which acts as a glucosyl donor in the reactions catalysed by starch synthases (EC 2.4.1.21 [EC] ). In the majority of plant cells the AGPase reaction takes place exclusively in plastids. However, the endosperms of cereals and other graminaceous plants also possess an extra-plastidial (presumably cytosolic) form of AGPase (Beckles et al., 2001). A number of studies have shown that the extra-plastidial form of AGPase is responsible for the majority of AGPase activity in maize, barley, rice, and wheat (Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikka et al., 2001; Tetlow et al., 2003b). Isolated amyloplasts of wheat, maize, and barley are able to import exogenous ADP-Glc in order to sustain physiological rates of starch biosynthesis (Tetlow et al., 1994; Möhlmann et al., 1997; Patron et al., 2004). This implies that, in cereal endosperms, much of the carbon used for the biosynthesis of storage starch is synthesized in the cytosol and transported as ADP-Glc across the amyloplast envelope membrane. By contrast, in non-photosynthetic dicotyledonous tissues, starch synthesis is dependent on a supply of hexose-phosphate and ATP, the substrates for ADP-Glc synthesis catalysed by AGPase. These are delivered to the amyloplast via separate transporters, the glucose 6-phosphate/phosphate translocator (Kammerer et al., 1998) and a plastidial nucleotide transporter (AATP) which imports ATP in exchange for ADP (Neuhaus et al., 1997), and is unable to transport ADP-Glc (Schünemann et al., 1993; Möhlmann et al., 1997; Tjaden et al., 1998b). Isolated wheat and maize endosperm amyloplasts are able to support in vitro starch biosynthesis through import of glucose 1-phosphate and ATP (Tetlow et al., 1994; Patron et al., 2004). Some dicotyledonous heterotrophic plastids and chloroplasts have the capacity to import exogenous ADP-Glc for starch biosynthesis (Pozueta-Romero et al., 1991; Baroja-Fernández et al., 2003; Muñoz et al., 2005) and, since these cells do not possess an extra-plastidial AGPase, it has been proposed that ADP-Glc is synthesized in the cytosol by an ADP-dependent sucrose synthase (EC 2.4.1.13 [EC] ) (Baroja-Fernández et al., 2001, 2004). Irrespective of the pathway of synthesis and delivery of ADP-Glc in starch-storing plastids, its utilization by starch synthases during the process of starch synthesis results in the formation of ADP, following the addition of the glucosyl moiety on the growing
-glucan chain. Presumably, ATP required for the synthesis of ADP-Glc must be regenerated from the ADP, either from within the plastid via the plastidial adenylate pool, or in the cytosol. In plastids which import ADP-Glc for starch synthesis, it is thought that ADP-Glc import is coupled to the export of adenylates, either as ADP or AMP (see below). Given the importance of cereals in the worldwide production of storage starches for human consumption and other non-food applications (Morell and Myers, 2005), the activity of the amyloplast ADP-Glc transporter is clearly a key component of the starch biosynthetic pathway, having the potential to influence plastidial ADP-Glc contents which, in turn, can influence starch content and quality (Tjaden et al., 1998a; Clarke et al., 1999). Studies of low-starch mutants of maize (Brittle1) and barley (lys5), which accumulate ADP-Glc in the endosperm, show mutations in one or more amyloplast inner envelope polypeptides. In maize, the Brittle1 mutant is deficient in four major amyloplast envelope polypeptides (39–44 kDa) which were detected in normal kernels by antibodies raised against the presumptive ADP-Glc transporter, termed BT1 (ZmBT1; Cao et al., 1995; Sullivan and Kaneko, 1995; Cao and Shannon, 1997). When compared with organelles from normal endosperm Brittle1 shows reduced rates of ADP-Glc uptake into amyloplasts (Liu et al., 1992; Shannon et al., 1996, 1998). Similarly, recent studies with the barley lys5 mutant, which has a defective amyloplast membrane protein showing homology to BT1 (HvNST1), show a reduced capacity for ADP-Glc uptake by isolated endosperm amyloplasts (Patron et al., 2004). Despite the strong circumstantial evidence which points to the BT1 gene product being a plastidial ADP-Glc transporter in cereal endosperms, there has been no direct evidence that the protein transports ADP-Glc. Analysis of the primary amino acid sequences of maize and barley BT1 proteins indicates both homologues have no clear sequence similarities to the nucleotide sugar transporters (NSTs) which are present in the endoplasmic reticulum and Golgi apparatus (Abeijon et al., 1997). Moreover, structural analysis of BT1 proteins suggests they are members of the mitochondrial carrier family (MCF) of transporters which transport a diverse array of metabolites, including ATP, but not nucleotide sugars (Sullivan et al., 1991; Picault et al., 2004). Non-graminaceous plants also possess BT1 homologues; AtBT1 of Arabidopsis thaliana and StBT1 of Solanum tuberosum share approximately 65% homology to ZmBT1 and HvBT1. Recently, functional characterization of StBT1 has provided an insight into the role of BT1 homologues in dicotyledonous plants, i.e StBT1 protein was found to be located in the plastid with ubiquitous expression in autotrophic and heterotrophic tissues. Import studies, using Escherichia coli for heterologous expression, showed that StBT1 is an adenine nucleotide uniporter with relatively narrow substrate specificity for AMP, ADP, and ATP (Leroch et al., 2005). Furthermore, StBT1 was unable to transport ADP-Glc, and it is speculated that its function is to provide the cytosol and other compartments with adenine nucleotides produced inside the plastid. Characterization of ADP-Glc transport by maize amyloplasts following reconstitution of envelope membranes into liposomes indicates the involvement of an antiport mechanism whereby ADP-Glc is imported in exchange for AMP or ADP (Möhlmann et al., 1997).
The aim of the work reported in this current paper was to gain an insight into the mode of action of the ADP-Glc transport system in wheat endosperm amyloplasts. The approach taken involved reconstitution of amyloplast envelope membrane proteins into liposomes in order to characterize the transport system with respect to substrate utilization and potential counter-exchange mechanisms. Furthermore, a photo-affinity analogue of ADP-Glc, 8-azido-[-32P]ADP-Glc, was employed to label covalently the polypeptide(s) responsible for substrate binding in in vitro experiments with intact amyloplasts. The results show that the ADP-Glc transporter of the amyloplast envelope imports ADP-Glc in exchange for adenine nucleotide phosphates, which in vivo is likely to be ADP, and that the polypeptide responsible for substrate binding is a 38 kDa envelope membrane protein.
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Materials
L-
-Phosphatidylcholine (PC) (soy type II S), L--dipalmitoyl phosphatidylcholine (DPPC), ADP-Glc, 8-azido-ATP, 8-azido-ADP, and 8-azido-AMP were purchased from Sigma Chemical Co., Poole, Dorset, UK. 5-(N-2, 3-Dihydroxypropylacetamido)-2, 4, 6-triiodo-N, N1-bis-(2, 3-dihydroxypropyl) isophthalamide (Nycodenz) was purchased from Nycomed (UK) Ltd, Birmingham, UK. The non-ionic surfactant polyethylglycol p-t-octylphenol (Triton X-100) was purchased from Calbiochem Corp., San Diego, CA, USA. [U-14C]ADP, ADP-[U-14C]Glc, [3H]AMP, and [3H]DPPC were purchased from Amersham International, Amersham, Bucks, UK. 8-Azido-[-32P]ATP was from ICN Pharmaceuticals Inc., Irvine, CA, USA. Anion exchange resin (Dowex AG-1X8, Cl–-form; 100–200 mesh) was purchased from Bio-Rad Laboratories Ltd, Hemel Hempstead, Herts, UK. Cellulose nitrate filters (0.2 µm pore size; 47 mm diameter) were purchased from Whatman International (Maidstone, Kent, UK). Silicone oil (AR200 grade) was purchased from Wacker Chemie, Munich, Germany. All other reagents were of analytical grade, and aqueous solutions were made up in double-distilled water which was routinely filtered through 0.22 µm disposable filters (Whatman).
Plant material and amyloplast isolation
Spring wheat (Triticum aestivum L. cv. Axona) was grown under conditions previously described by Tetlow et al. (1993) and the developing ears tagged at the onset of anthesis (the first appearance of anthers). Endosperm tissue was obtained from developing grains taken from the mid-ear region of the head at various stages of endosperm development between 12 d after pollination (DAP) and 20 DAP, and used to prepare amyloplasts. Between 500 and 800 grains (approximately 8–25 g fresh weight) were used to prepare amyloplasts mechanically according to the method described by Tetlow et al. (2003b). Amyloplasts prepared in this way were 60–75% intact as judged by the latency of the plastid marker enzyme alkaline inorganic pyrophosphatase (APPase, EC 3.6.1.1
[EC]
) (Tetlow et al., 1993). For the preparation of amyloplast envelope membranes, amyloplast pellets were lysed in rupturing buffer [100 mM N-TRIS(hydroxymethyl)methyl glycine (Tricine)KOH, pH 7.8, 1 mM Na2-ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 5 mM MgCl2, 10 µg cm–3 chymostatin, 100 µM each of antipain dihydrochloride, bestatin, leupeptin, pepstatin, E-64, and aprotinin, 10 µM phosphoramidon and 1, 10-phenanthroline, and 500 µM 3, 4 dichloroisocoumarin], frozen in liquid nitrogen and stored at –80 °C. The maximum mitochondrial contamination of the amyloplast preparations at each of the various stages of development was <0.2%, based on recoveries of the marker enzymes citrate synthase (EC 4.1.3.7
[EC]
) and cytochrome c oxidase (EC 1.9.3.1
[EC]
), and the maximum cytosolic contamination of amyloplast preparations was 0.4%, based on the recovery of uridine 5' diphosphate glucose (UDP-glucose) pyrophosphorylase (UGPase, EC 2.7.7.9
[EC]
) and pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90
[EC]
) (data not shown).
Enzyme assays
The following enzymes were assayed at 25 °C as described previously: APPase (EC 3.6.1.1
[EC]
), UGPase (EC 2.7.7.9
[EC]
), alcohol dehydrogenase (EC 1.1.1.1
[EC]
), citrate synthase (EC 4.1.3.7
[EC]
), pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1. 90) (Tetlow et al., 1993), and cytochrome c oxidase (EC 1.9.3.1
[EC]
) (MacDonald and ap Rees, 1983).
Isolation of amyloplast envelopes
Amyloplast lysates were centrifuged at 13 000 g for 5 min to remove starch grains in a micro-centrifuge precooled to 4 °C and the supernatant removed and centrifuged at 100 000 g [207 kPa. (30 psi)] for 20 min in a Beckman Airfuge (TLA 100). The resulting membrane pellet was rinsed three times in rupturing buffer and then resuspended in rupturing buffer and stored on ice before solubilization (see below). In some experiments envelope membranes were washed in rupturing buffer containing 1.5 M NaCl to remove extrinsic and loosely attached membrane-associated proteins.
Measurement of starch synthesis by isolated amyloplasts
Amyloplasts (prepared from endosperm 12–20 DAP) were incubated for 45 min at 25 °C with 5 mM ADP-[U-14C]Glc. Incorporation of isotope into material insoluble in methanol-KCl was determined, followed by digestion with amyloglucosidase and -amylase to release [14C]glucose according to methods described by Tetlow et al. (1994). Controls contained boiled plastid preparations.
Preparation and characterization of liposomes
Liposomes (vesicles) were prepared by bath-sonicating aqueous lipid mixtures essentially according to the methods described by Tetlow et al. (1996), except that the lipid mixtures were composed of soy PC and prepared in an aqueous buffer containing 100 mM Tricine-NaOH (pH 7.5) and 30 mM potassium gluconate (liposome buffer). For some experiments, 2 µCi [3H]DPPC was added to the lipid mixture to obtain radiolabelled liposomes in order to evaluate the effectiveness of the Dowex AG-1X8 columns and the filtration methods at removing non-transported radiolabelled substrates using the methods described by Hutchinson et al. (1989). For counter-exchange transport experiments substrates were preloaded during sonication; AMP, ADP, and ADP-Glc were preloaded at various final concentrations up to 20 mM (made up in liposome buffer) and unentrapped material removed by gel filtration chromatography as described by Tetlow et al. (1996). Liposome size distribution was monitored during the different stages of preparation by photon correlation spectroscopy using a Malvern autosizer (model RR146).
Reconstitution of amyloplast membrane proteins into liposomes
The methods used to reconstitute amyloplast membrane proteins into liposomes were modifications of methods described by Tetlow et al. (1996) and Möhlmann et al. (1997). Amyloplast envelope membrane protein [approximately 120–150 µg protein in a volume of 90 µl of liposome buffer containing 20% (v/v) glycerol] was solubilized by the addition of 10 µl Triton X-100 to a final concentration of 0.1% (v/v). The solubilized membrane proteins were rapidly (<10 s) combined with 900 µl liposomes, resulting in a lipid:detergent ratio of 100:1, and transferred to liquid nitrogen. After thawing at room temperature, the freeze–thaw step was repeated to allow incorporation of the solubilized membrane proteins into liposomes (Kasahara and Hinkle, 1977). Proteoliposomes were then sonicated for 30 s under a constant stream of nitrogen. Unincorporated material was removed by passing the mixture through an NAP-10 gel filtration column (Amersham International) equilibrated with a buffer containing 10 mM Tricine-NaOH (pH 7.5) and 250 mM potassium gluconate at 4 °C. Eluted proteoliposomes were stored on ice and used for transport experiments within 2 h.
Metabolite transport experiments
Transport experiments were initiated by the addition of 100 µl proteoliposomes, preincubated at 25 °C for 2–3 min, to 100 µl of liposome buffer containing radiolabelled nucleotides or nucleotide sugars (0.05 µCi per assay). In all cases, transport experiments were conducted at 25 °C. When 14C-labelled substrates were used, uptake was stopped at timed intervals by placing the assay mixture onto a 1.5 cm3 column of Dowex AG-1X8 resin pre-equilibrated with 200 mM Tricine-NaOH (pH 7.5) (equilibration buffer). Proteoliposomes were eluted from the column by washing with three volumes of 0.5 cm3 equilibration buffer and the radioactivity within the proteoliposomes measured using a Tricarb 2100TR liquid scintillation counter (Packard Bioscience C., Meriden, USA). When using 3H-labelled substrates, uptake was stopped by placing the assay mixture onto a pre-wetted cellulose nitrate filter on a fritted manifold under vacuum as previously described (Tetlow et al., 1996). A transport assay stopped at zero time was used as a control for all experiments. Further controls included the use of liposomes lacking membrane proteins in order to account for metabolite leakage or diffusion. All rates of metabolite transport presented are corrected for metabolite leakage (typically <1% per hour; data not shown) and radioisotope associated with proteoliposomes at zero time.
Measurement of ADP and AMP efflux from amyloplasts
Efflux of ADP and AMP from isolated amyloplasts was measured following incubation with ADP-Glc. Isolated amyloplasts were prepared as described above and carefully resuspended in amyloplast resuspension buffer containing 0.62 M sorbitol and used immediately for efflux experiments. Assays contained 180 µl amyloplasts (200–300 µg protein) plus 20 µl amyloplast resuspension buffer containing 5 mM ADP-Glc which was layered onto silicone oil (AR200 grade) in a 400 µl vertical-sided polypropylene tube (Alpha Laboratories, Eastleigh, Hants, UK) as described previously (Emes and Traska, 1987; Tetlow et al., 1998). Amyloplasts were incubated with ADP-Glc at 25 °C for up to 40 min and incubations stopped by centrifugation of the organelles through silicone oil. The supernatant was removed and efflux of AMP and ADP measured by the luciferin/luciferase technique as described by Mills (1986).
Kinetic studies
The Km values for the substrates of the ADP-Glc transporter were determined from double-reciprocal plots using near-saturating concentrations of the non-varied substrate. Ki values for AMP and ATP were determined by varying their concentrations at several fixed concentrations of ADP-Glc or ADP and measuring ADP-Glc/ADP exchange or ADP/ADP-Glc exchange. The Ki values for AMP and ATP were determined from Dixon plots (Segel, 1975).
Synthesis of 8-azido-[-32P]ADP-Glc
8-Azido-[-32P]ADP-Glc was synthesized from commercially available 8-azido-[-32P]ATP using purified E. coli ADP-Glc pyrophosphorylase (AGPase, EC 2.7.7.27
[EC]
, specific activity 0.034 µmol ADP-Glc formed min–1 mg–1 protein). The reaction mix contained 50 mM HEPES-KOH (pH 7.5), 5 mM glucose 1-phosphate, 2 mM fructose 1, 6-bisphosphate, 5 mM MgCl2, 0.5 mg cm–3 bovine serum albumen, 100 µM 8-azido-[-32P]ATP, 2 units inorganic pyrophosphatase (PPiase, EC 3.6.1.1
[EC]
; Sigma; from Baker's yeast), and 0.2 units AGPase, and was incubated at 37 °C for 45 min. Reactions were terminated by heating to 95 °C for 2 min and cooled on ice. AGPase was omitted from control reactions. Conversion of 8-azido-[-32P]ATP into 8-azido-[-32P]ADP-Glc was greater than 99%, as judged by scintillation counting of reaction products following separation by thin layer chromatography (TLC) (see below). Protein was removed from the stopped reaction by precipitation with ice-cold acetone, and the supernatant dried down in a vacuum centrifuge and resuspended in a minimal volume of distilled water. 8-Azido-[-32P]ADP-Glc was purified by TLC using PEI-cellulose plates (20x20 cm, 100 µm thick; Merck) in a mobile phase of Na-formate (adjusted to pH 3.4 with solid NaOH). Samples were run with 10 mM standards of ADP-Glc, and 8-azidoforms of ATP, ADP, and AMP made up in distilled water. Material on the plates was separated by immersing the lower 1 cm edge of the plate, sequentially, into 0.5 M Na-formate for 30 s, 2 M Na-formate for 2 min, and 4 M Na-formate for 1.5–2 h until the solvent front was within a few centimetres of the top of the plate. The plate was air-dried and the standards visualized under UV light. The sample of 8-azido-[-32P]ADP-Glc was covered to avoid photo-cross-linking of the sample, and located by its relative position to ADP-Glc standards (all nucleotides shared the same mobility as their 8-azido forms) and autoradiography. The region of the plate containing 8-azido-[-32P]ADP-Glc was excised and the material scraped off the backing of the plate and placed into a 1.5 cm reaction tube, and the 8-azido-[-32P]ADP-Glc was recovered by mixing overnight with 0.5 cm3 distilled water on a rotating table at room temperature. The sample was then centrifuged (13 000 g for 2 min) and the supernatant removed and dried in a vacuum centrifuge. The recovered 8-azido-[-32P]ADP-Glc was resuspended in amyloplast resuspension buffer and used in photo-affinity labelling experiments. Recovery of material from the TLC plates was approximately 75%. The identity of the 32P-labelled product recovered from the TLC plates was further confirmed as 8-azido-[-32P]ADP-Glc following its conversion back into 8-azido-[-32P]ATP by the reverse reaction of E. coli AGPase in which 5 mM tetrasodium pyrophosphate replaced glucose 1-phosphate, and PPiase and ATP were omitted (data not shown).
Photo-affinity labelling experiments
In vitro labelling of the ADP-Glc transporter of the amyloplast envelope membranes was performed with freshly prepared intact amyloplasts using the photo-affinity reagent 8-azido-[-32P]ADP-Glc. Reactions were initiated by the addition of 180 µl amyloplasts (approximately 200–300 µg protein) to unlabelled substrates plus 8-azido-[-32P]ADP-Glc (2 µCi per sample) made up in amyloplast resuspension buffer [50 mM N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid (HEPES)-NaOH, pH 7.5, 5 mM MgCl2, 1 mM Na2-EDTA, 1 mM KCl, 1 mM DTT, 0.8 M sorbitol]. Reactions, in a total volume of 200 µl, were incubated at 25 °C on the inside wells of inverted 1.5 cm3 reaction tube caps (Treff Laboratories, Switzerland) for 2 min followed by exposure for 4 min to a UV halogen reflector lamp (115 V/60 Hz, nominal intensity at 30 cm=1500 W cm–2, peak wavelength of 254 nm; XX-15S bench lamp, UVP; San Gabriel, CA, USA) set at a height of 30 cm from the sample. Following the photo-cross-linking reaction, the sample was mixed with 0.8 cm3 of ice-cold rupturing buffer, transferred to a reaction tube, and the amyloplast membranes isolated using the methods described above. The isolated membranes were washed three times (pellet resuspended and centrifuged) in rupturing buffer containing 1.5 M NaCl to remove loosely attached proteins and extrinsic membrane proteins. The NaCl-washed membrane pellet was rinsed with rupturing buffer and resuspended in sodium dodecyl sulphate (SDS)-sample buffer [containing 62.5 mM TRIS-HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) ß-mercaptoethanol, 0.001% (w/v) Bromophenol Blue] and heated to 100 °C for 3 min prior to separation of polypeptides by SDS-polyacrylamide gel electrophoresis (PAGE). Control samples were incubated in the dark.
Conditions for the photo-irradiation reactions were optimized using 8-azido-ATP. The formation of nitrene radicals from the photo-reactive azido group following exposure to UV light was monitored by measuring the absorbance of 100 µM solutions of 8-azido-ATP (made up in 40 mM TRIS-HCl, pH 7.5) at 282 nm in quartz cuvettes with a Cecil CE5501W spectrophotometer (Cecil Instruments) (Potter and Haley, 1982).
SDS-PAGE
Protein samples from isolated amyloplast envelopes were separated by SDS-PAGE using 10% (w/v) acrylamide gels following the method of Laemmli (1970). Following electrophoresis of samples from photo-irradiation experiments, gels were washed in 1.0 l of a solution of 10% (v/v) isopropanol and 5% (v/v) glacial acetic acid overnight, prior to drying and autoradiography.
Protein determination
The protein content of wheat endosperm plastid and plastid envelope preparations was determined using the Bio-Rad protein assay (Bio-Rad, Hemel Hempstead, Herts, UK) according to the manufacturer's instructions and using thyroglobulin as a standard (Bradford, 1976).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Reconstitution of the amyloplast ADP-Glc transporter
A detailed kinetic analysis of the ADP-Glc transporter from wheat endosperm amyloplasts was undertaken using reconstituted plastid envelope membranes. Time-dependent transport of both 0.05 and 0.5 mM ADP-[U-14C]Glc into proteoliposomes required the presence of a preloaded counter-exchange substrate inside the proteoliposome (provided at a concentration of 20 mM). This transport was linear for up to 2–3 min of incubation, as has been shown previously (Tetlow et al., 2003a). All transport rates for the time-course experiments were calculated after deducting the rate of transport of 0.5 mM ADP-[U-14C]Glc into proteoliposomes containing buffer (no counter-exchange substrate). The rate of ADP-Glc uniport was approximately 0.1 nmol min–1 mg–1 protein.
The concentration-dependence of ADP-[U-14C]Glc uptake was measured using proteoliposomes preloaded with 20 mM counter-exchange substrates (AMP, ADP, ATP) (Fig. 1A). The rate of ADP-[U-14C]Glc uptake increased with increasing concentrations of ADP-Glc up to 1 mM. An Eadie–Hofstee plot of these data indicated a Km for ADP-Glc of 0.43 mM (Fig. 1B; Table 1) independent of counter-exchange adenylate within the proteoliposomes. Rates of ADP-[U-14C]Glc transport were greatest with AMP as a counter-exchange substrate (Vmax=2.71±0. 32 nmol min–1 mg–1 protein, n=3), followed by ADP (Vmax=1.48±0. 05 nmol min–1 mg–1 protein, n=3) and ATP (Vmax=0.52±0. 02 nmol min–1 mg–1 protein, n=3). Proteoliposomes preloaded with 20 mM ADP-Glc were incubated with [U-14C]ADP or [3H]AMP at concentrations of up to 1 mM. Increasing the ADP concentration from 0 mM to 0.5 mM increased ADP uptake into the ADP-Glc-loaded proteoliposomes, and the rate of uptake was saturated at concentrations above 0.5 mM (Fig. 2A). Similarly, increasing concentrations of AMP, particularly between 0.01 mM and 0.2 mM, resulted in an increase in AMP uptake via counter-exchange for ADP-Glc. Eadie–Hofstee transformations of these data produced an apparent Km for both ADP and AMP of 0.2 mM (Table 1). Transport of AMP into proteoliposomes containing buffer without counter-exchange substrates was observed, and showed saturable kinetics with a Km of 0.18 mM (Fig. 2B; Table 1).
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A detailed kinetic study was carried out to determine the effects of the presence of various adenylates on [U-14C]ADP uptake into proteoliposomes in counter-exchange for ADP-Glc. All ATP and AMP concentrations examined reduced ADP uptake (Fig. 3), with a calculated Ki for a competitive inhibitor (Dixon plot) of 2 mM for ATP and 1.5 mM for AMP (Table 1). An alternative approach to determine the inhibitory effects of ATP and AMP on the ADP-Glc/ADP counter-exchange process was to measure their effect on the transport of ADP-[U-14C]Glc into proteoliposomes preloaded with ADP. Figure 4A shows that increasing the concentration of ATP had little effect on ADP-[U-14C]Glc uptake by proteoliposomes. A small decrease in ADP-Glc uptake was observed when the AMP concentration was increased from 0 mM to 10 mM (Fig. 4B), but there was no significant inhibition of ADP-Glc uptake by ATP or AMP (Table 1). To determine whether the ADP-Glc transport system interacted with UDP-Glc, ADP-[U-14C]Glc transport into proteoliposomes preloaded with either ADP or AMP was measured in the presence of varied concentrations of UDP-Glc. No effect of UDP-Glc was observed on ADP-Glc uptake (data not shown).
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Efflux of AMP and ADP from amyloplasts incubated with ADP-Glc
The results of the transport experiments with reconstituted amyloplast envelope membrane proteins indicated that ADP-Glc is transported via counter-exchange with either ADP and/or AMP. Despite the fact that during starch biosynthesis ADP will be produced as a by-product of the starch synthase reaction, plastidial adenylate pools may be subject to equilibration by the activity of adenylate kinase. The ADP-Glc transporter of maize amyloplasts was shown to counter-exchange either ADP or AMP for ADP-Glc (Möhlmann et al., 1997). Therefore, in order to determine the nature of the counter-exchange substrate during starch biosynthesis, efflux experiments were conducted whereby intact amyloplasts were incubated with ADP-Glc (under conditions previously shown to support physiological rates of starch synthesis) and the efflux of ADP and AMP measured. The efflux experiments shown in Fig. 5 show that little or no ADP or AMP is detected leaving the amyloplast when organelles are incubated in buffer with no ADP-Glc (Fig. 5A, B). However, efflux of ADP and AMP was detected following incubation of plastids with 5 mM ADP-Glc. Measurement of ADP efflux from intact amyloplasts incubated with ADP-Glc showed a time-dependent increase in ADP efflux which was also dependent upon plastid intactness (Fig. 5A). The calculated efflux of ADP from intact plastids incubated with ADP-Glc was 516 nmol mg–1 protein after 40 min. This value is in good agreement with the net rate of conversion of ADP-[U-14C]Glc into starch by intact plastids in separate experiments, which was calculated as 625±212 nmol mg–1 protein (n=5 replicate amyloplast preparations) over a 45 min incubation period at 25 °C.
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Efflux of AMP from intact plastids incubated with ADP-Glc was relatively low (Fig. 5B). Maximal efflux of AMP, after 20 min incubation of amyloplasts with ADP-Glc, was <100 nmol mg–1 protein and, when taking into account the AMP present at zero time, was undetectable at the 40 min incubation period.
Photo-affinity labelling amyloplast envelope proteins with 8-azido-[-32P]ADP-Glc
Photo-affinity labelling experiments were conducted with the substrate analogue 8-azido-[-32P]ADP-Glc in order to identify any polypeptide(s) on the amyloplast envelope responsible for substrate binding during the process of ADP-Glc uptake and the subsequent ADP-Glc-dependent starch synthesis. When intact amyloplasts were incubated with 0.1 mM 8-azido-[-32P]ADP-Glc, only a single 38 kDa envelope polypeptide was cross-linked in a UV light-dependent manner (Fig. 6). Treatment of amyloplast envelope membranes with 1.5 M NaCl removed approximately 30–40% of the loosely attached (extrinsic) proteins associated with the plastid envelope (data not shown). Experiments involving fractionation of amyloplast envelopes with NaCl following cross-linking with 8-azido-[-32P]ADP-Glc confirmed that the substrate analogue was only bound to an intrinsic envelope membrane protein associated with the NaCl-washed fraction (Fig. 6). Maize anti-Brittle1 antibodies showed no cross-reaction with the respective transporter proteins from wheat endosperm amyloplast envelope membrane proteins on immunoblots (data not shown).
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Two different approaches were used to characterize the ADP-Glc transport system which sustains ADP-Glc-dependent starch biosynthesis in cereal endosperms: (i) a proteoliposome-based transport assay using reconstituted amyloplast envelope proteins was employed; (ii) measurement of efflux of counter-exchangeable substrates in intact amyloplasts was made during ADP-Glc-dependent starch biosynthesis. Both approaches indicated that ADP-Glc transport into plastids operates via a counter-exchange mechanism. ADP-Glc transport experiments with reconstituted amyloplast membrane proteins in proteoliposomes showed that transport of this substrate was saturable, following Michaelis–Menten kinetics, and had a requirement for a counter-exchange substrate, as rates of ADP-Glc uniport were negligible. Uniport of AMP was measured in proteoliposomes containing reconstituted amyloplast envelope membranes (Fig. 2B). Since the proteoliposomes used in these experiments contained a mixture of reconstituted plastidial metabolite transporters, it is likely that the AMP uniport measured was due to the activity of a transporter other than the ADP-Glc transporter, for example, a wheat homologue of the adenine nucleotide transporter StBT1 described by Leroch et al. (2005). In addition to adenine nucleotide uniporters, other uniport transporters are known to be present in heterotrophic plastids; for example, an inorganic phosphate uniporter has been characterized from cauliflower bud amyloplasts (Neuhaus and Maass, 1996). UDP-Glc was not transported into proteoliposomes containing amyloplast envelope membrane proteins, which is consistent with previous reports showing that when UDP-Glc was provided as a precursor, starch biosynthesis by amyloplasts was not dependent on intactness (Tyson and ap Rees, 1988; Tetlow et al., 2003a).
Assuming that the ADP-Glc transported across the amyloplast envelope is utilized by the starch synthases for starch synthesis, then the immediate by-product of these reactions will be ADP. It follows, therefore, that the counter-exchange-dependent transport of the soluble substrate (ADP-Glc) and product (ADP) of starch synthesis would achieve balance of the cellular adenylate pools. Efficient rates of ADP-Glc transport could be achieved, in vitro, with either ADP or AMP in proteoliposomes. This has been previously observed for transport studies with reconstituted maize amyloplast envelope membrane proteins (Möhlmann et al., 1997). At present it is not possible to draw any firm conclusions as to the class of the translocator responsible for the transport of ADP-Glc across the amyloplast envelope as no amino acid sequence is available for this protein. Three structurally unrelated classes of intracellular adenine nucleotide transporters are known; the mitochondrial ADP/ATP carriers (AACs), which function as dimers with six transmembrane domains (Fiore et al., 1998), the plastidic AATP which exists in all higher and lower plants and exhibits an 11–12 transmembrane domain structure (Linka et al., 2003; Reiser et al., 2004), and the peroxisomal ATP/AMP carrier (Palmieri et al., 2002). The BT1 proteins are structurally related to the MCF class of mitochondrial metabolite transporters, which include the AACs (Patron et al., 2004), but have no obvious sequence similarities at the amino acid sequence level to the NSTs of the Golgi and endoplasmic reticulum. Functional reconstitution of the BT1 proteins would determine whether they are ADP-Glc transporters and, if so, to what class they belong. However, given the substrate specificities and mode of action determined for the transporter in this study, it is likely that the ADP-Glc transport system operating in plastid envelopes of cereal endosperm amyloplasts is, biochemically, more closely related to the NSTs operating in other eukaryotic organisms. Many of the NSTs characterized to date are Golgi-localized carriers which transport nucleotide sugars and phosphoadenosine-5'-phosphosulphate in stoichiometric exchange for the corresponding nucleotide monophosphate during glycoprotein processing and sulphation reactions within the lumen. Examples include Golgi-localized GDP-mannose/GMP transporters in yeast and a variety of UDP-sugar/UMP and UDP-GlcNAc/UMP transporters in rat liver (Waldman and Rudnick, 1990; Milla et al., 1992; Berninsone et al., 1994). The requirement of Golgi NSTs for nucleotide monophosphates in exchange for nucleotide sugars is underlined by the importance of nucleotide diphosphatases (NDPases) for efficient transport (Abeijon et al., 1989; Berninsone et al., 1994, 1995), and the fact that NDPases are inhibitors of some of the processing reactions, for example, mannosylation, taking place in the Golgi lumen. However, it could be argued that the transport of ADP-Glc into cereal endosperm amyloplasts may be different from the nucleotide-sugar import machinery operating in the Golgi apparatus.
Kinetic analysis of the reconstituted amyloplast ADP-Glc transporter revealed a similar affinity for both ADP and AMP. Under physiological conditions, it could be argued that ADP-Glc/ADP exchange is more likely than ADP-Glc/AMP exchange. Previous studies with amyloplasts estimate the plastidial ADP-Glc concentration to be in the region of 0.9 mM (Clarke et al., 1999), and in maize endosperm, ADP-Glc concentrations were in the same order as other adenylates (Shannon et al., 1996). Given the significance of the role of the ADP-Glc imported into the amyloplast, higher concentrations of ADP (a by-product of the starch synthase reaction) than AMP would be expected to be generated during starch biosynthesis. Although adenylate kinase activity in these plastids was not measured, this assumes little or no equilibration of adenylate pools by adenylate kinase. The results of the ADP and AMP efflux experiments support this idea, showing that during ADP-Glc-dependent starch biosynthesis ADP is the major adenylate exported from intact amyloplasts. The rates of ADP efflux (13 nmol min–1 mg–1 protein) correlate well with the measured rates of ADP-Glc-dependent starch synthesis in intact plastids (14 nmol min–1 mg–1 protein). In addition, unlike the situation with the glycosylation reactions in the Golgi, where the nucleotide diphosphates must be removed by NDPases (see above), there is no evidence to suggest that ADP is an inhibitor of the reactions catalysed by starch synthases. Although ADP-Glc exchange stoichiometry was not directly measured, electro-neutral exchange of dianionic species at physiological pH ranges is a reasonable assumption based on the close stoichiometry between ADP efflux and ADP-Glc-dependent starch biosynthesis in intact amyloplasts determined experimentally in the results reported here (although ADP-Glc/AMP exchange is not electro-neutral). Furthermore, a similar situation is observed in the Golgi NST systems mentioned above. Studies with a plant Golgi NST in pea revealed transport of UDP-Glc dependent on counter-exchange with nucleotide phosphates, although transport of UDP-Glc appeared to be equally efficient with UDP or UMP as counter-exchange substrates, a situation not dissimilar to that observed for the amyloplast ADP-Glc transporter reported here (Muñoz et al., 1996). Interestingly, transport-inhibition experiments with proteoliposomes showed that ATP was a strong inhibitor of ADP uptake, but not ADP-Glc uptake (Figs 3, 4; Table 1). This is consistent with there being at least two transporters present in the amyloplast envelope involved in ADP exchange. The plastidial AATP, which counter-exchanges ADP and ATP (Neuhaus et al., 1997), has no affinity for ADP-Glc or AMP, and transport of ADP is competitively inhibited by ATP. By contrast, the transport of ADP-Glc is not significantly inhibited by ATP, as shown here, suggesting it is not a primary substrate for the ADP-Glc transporter. Such kinetic properties would allow uptake of ADP-Glc to be independent of variation in cytosolic ATP content.
Cross-linking studies with the photo-reactive ADP-Glc analogue, 8-azido-[-32P]ADP-Glc clearly showed that a single intrinsic membrane polypeptide of 38 kDa is responsible for substrate binding during ADP-Glc-dependent starch synthesis by amyloplasts. The kinetic data for uptake of ADP-Glc also support earlier findings using isolated amyloplast membranes showing that the photo-affinity labelling of a 38 kDa membrane protein by 8-azido-[-32P]ADP-Glc could be reduced by the addition of non-radioactive ADP or AMP, but with little effect with ATP (Tetlow et al., 2003a). Immunoblotting studies with the putative ADP-Glc transporter from maize (BT1) indicated that it is composed of a number of membrane polypeptides between 39 kDa and 44 kDa (Cao and Shannon, 1997). The cross-linking data in the present study are not inconsistent with the possibility that the wheat homologue of BT1 involved in substrate binding is a 38 kDa polypeptide. However, until the function of the Brittle1 gene product is elucidated directly through functional reconstitution, the molecular identity of the cereal ADP-Glc transporter remains an open question.
In summary, the kinetic properties of the ADP-Glc transporter have been characterized from amyloplasts of wheat endosperm and it has been shown that uptake of extra-plastidial ADP-Glc proceeds via a counter-exchange mechanism. During starch biosynthesis, the ADP generated by the actions of starch synthases is the most likely intra-plastidic substrate for the transporter during the process of counter-exchange with extra-organellar ADP-Glc and the transport process has a 1:1 stoichiometry.
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Acknowledgements |
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We are grateful to Dr Hanping Guan (ExSeed Genetics L.L.C., IA, USA) for the gift of purified E. coli AGPase. We thank Dr Malcolm N Jones (retired) of the Faculty of Life Sciences, University of Manchester for helpful discussions and suggestions and for the use of his laboratory facilities, and to Dr Torsten Möhlmann and Dr Ekkehard Neuhaus (Technische Universität Kaiserslauten, Kaiserslautern, Germany) for assistance with liposome transport experiments in their laboratory and for the gift of Arabidopsis anti-ATP/ADP transporter antibodies. We thank Dr Tom Sullivan (University of Wisconsin, Madison) for maize anti-Brittle1 antibodies. Wheat was grown by Mr Thurston Heaton and Mr David Newton at the Firs Experimental Grounds, University of Manchester, UK. This research was supported by funding from the BBSRC.
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Abbreviations |
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AAC, mitochondrial ADP/ATP transporter; AATP, plastidial ATP/ADP transporter; ADP-Glc, ADP-glucose; AGPase, adenosine 5' diphosphate glucose pyrophosphorylase; APPase, alkaline inorganic pyrophosphatase; DAP, days after pollination; DPPC, L-
-dipalmitoyl phosphatiylcholine; DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetic acid; MCF, mitochondrial carrier family; NDPase, nucleotide diphosphatase; NST, nucleotide sugar transporter; PAGE, polyacrylamide gel electrophoresis; PC, L--phosphatidylcholine; PPiase, inorganic pyrophosphatase; SDS, sodium dodecyl sulphate; TLC, thin-layer chromatography; Tricine, N-TRIS(hydroxymethyl)methyl glycine; Triton X-100, polyethylglycol p-t-octylphenol; UDP-GlcNAc, uridine 5' diphosphate N-acetylglucosamine; UGPase, uridine 5' diphosphate glucose pyrophosphorylase. |
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