Plant Physiology and Biochemistry 83 (2014) 168e179

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Research article

Multimeric states of starch phosphorylase determine proteineprotein interactions with starch biosynthetic enzymes in amyloplasts Renuka M. Subasinghe a, Fushan Liu a, Ursula C. Polack a, Elizabeth A. Lee b, Michael J. Emes a, Ian J. Tetlow a, * a b

Department of Molecular and Cellular Biology, Science Complex, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada Department of Plant Agriculture, University of Guelph, Guelph, Ontario N1G 2W1, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 May 2014 Accepted 20 July 2014 Available online 6 August 2014

Proteineprotein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucansynthetic and phosphorolytic directions. Proteineprotein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, proteineprotein interactions were broken when amyloplast lysates were dephosphorylated in vitro, and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with [g-32P]-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Amylopectin Amyloplast Protein phosphorylation Proteineprotein interactions Starch branching enzyme Starch phosphorylase Starch synthesis

1. Introduction Starch is an important carbon reserve synthesized within the plastids of higher plants and green algal cells, allowing them to meet variable metabolic demands over a day/night cycle and in the

Abbreviations: ADP-Glc, ADP-glucose; ae
, amylose extender; AGPase, adenosine 50 diphosphate glucose pyrophosphorylase; APase, alkaline phosphatase; BSA, bovine serum albumen; Bt2, AGPase small subunit; DAP, days after pollination; DBE, debranching enzyme; D-enzyme, disproportionating enzyme; DP, degree of polymerization; DTT, dithiothreitol; GBSSI, granule-bound starch synthase I; Glc, glucose; Glc-1P, a-D-glucose 1-phosphate; Iso, isoamylase; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)-propanesulfonic acid; MOS, maltooligosaccharides; MS, mass spectrometry; NP-40, nonyl phenoxylpolyethoxyl ethanol; PBS, phosphate buffered saline; Pho1, plastidial starch phosphorylase; Pho2, cytosolic starch phosphorylase; Pi, inorganic orthophosphate; SBE, starch branching enzyme; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SP, starch phosphorylase (Pho1); SS, starch synthase; TRIS, tris-(hydroxymethyl)-aminomethane; Triton X-100, polyethylglycol p-t-octylphenol; U, units of enzyme activity (defined as 1 mmol per min); ZPU1, pullulanase. * Corresponding author. Fax: þ1 519 837 1802. E-mail address: [email protected] (I.J. Tetlow). http://dx.doi.org/10.1016/j.plaphy.2014.07.016 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.

case of plants also acts as a long-term carbon store in seeds and tuberous tissues. The major route of starch synthesis is via the nucleotide diphosphate sugar precursor ADP-glucose (ADP-Glc), synthesized by the highly regulated enzyme ADP-Glc pyrophosphorylase (AGPase, E.C. 2.7.7.27) (Greenberg and Preiss 1964). ADPGlc is in turn the substrate for a class of glucan-elongating enzymes called starch synthases (SS, E.C. 2.4.1.21), which, together with starch branching enzymes (SBE, E.C. 2.4.1.18) and debranching enzymes (DBEs, including isoamylases [Iso], E.C. 3.2.1.41 and pullulanase [ZPU1], E.C. 3.2.1.68) are the three major groups of enzymes responsible for the synthesis of water-insoluble semicrystalline starch granules (for recent reviews see HennenBierwagen et al., 2012; Tetlow, 2011). Amongst the many other enzymes involved in starch metabolism, starch phosphorylase (SP, E.C. 2.4.1.1) has long been implicated in the pathway of starch biosynthesis, although its precise role has yet to be defined (see review by Schupp and Ziegler, 2004). a-Glucan phosphorylases are found in many different organisms as dimers or tetramers (Nakano and Fukui 1986; Albrecht et al., 1998; Buchbinder et al., 2001; Higgins et al., 2013) and catalyze a

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reversible (equilibrium) reaction; (a-1,4-linked Glc)n þ Glc1P 4 (a-1,4-linked Glc)n þ 1 þ inorganic orthophosphate (Pi), meaning that subcellular Glc-1P and Pi levels will influence the direction of catalytic flux. Plants possess two forms of a-glucan phosphorylases, a plastidial form involved in starch synthesis/ metabolism called Pho1 (or PhoL, because of its low affinity for hydro-soluble glycogen as a glucan substrate), and a cytosolic form termed Pho2, or PhoH, which has a high affinity for glycogen and high-molecular-weight heteroglycan (Shimomura et al., 1982; Yang and Steup, 1990). Pho2 is closely related to bacterial and mammalian a-glucan phosphorylases and is involved in cytosolic polyglucan metabolism, including metabolism of a-glucans derived from the products of starch degradation (exported from the plastid € et al., 2004; Weise et al., 2004; Fettke et al., as maltose (Niittyla 2012)). Pho2 therefore plays no role in plastidial starch biosynthesis. Expression studies of the plastidial SP gene (Pho1, hereafter referred to as SP) in leaf and storage tissues correlate closely with periods of active starch biosynthesis (Tsai et al., 1970; Duwenig et al., 1997; Van Berkel et al., 1991; Yu et al., 2001), and genetic evidence from Chlamydomonas reinhardtii and rice (Oryza sativa e et al., L.) indicates a role for SP in starch accumulation (Dauville 2006; Satoh et al., 2008). In addition, there is no direct evidence for SP-mediated starch degradation; loss of SP in potato (Solanum tuberosum L.) and Arabidopsis (Arabidopsis thaliana L.) caused no measurable alteration to diurnal starch accumulation and turnover (Sonnewald et al., 1995; Zeeman et al., 2004), which is consistent with the prevailing view of a primarily amylolytic route of starch degradation in plants (Zeeman et al., 2010; Fettke et al., 2012). Further, amyloplasts possess high Pi/Glc-1P ratios which, it has been argued, would indicate that the SP reaction is favored in the direction of phosphorolysis (a-glucan degradation) (Wirtz et al., 1980; Tiessen et al., 2012). Studies of SP from maize amyloplasts showed that the phosphorolytic reaction was stimulated in the presence of malto-oligosaccharides (MOS), further supporting the notion of a predominantly phosphorolytic-acting SP in plastids. However, recent biochemical studies of rice endosperm SP clearly indicate that the SP reaction favors -glucan synthesis over degradation, even in the presence of high Pi levels (Hwang et al., 2010). Both SP reactions show high sensitivity to e et al., 2006; Burr and Nelson, inhibition by ADP-Glc (Dauville 1975; Matheson and Richardson, 1978), and it has been suggested by Hwang et al. (2010) that low ADP-Glc levels are maintained early in endosperm development (3e5 DAP) by an active ADP-Glc pyrophosphatase (E.C. 3.1.4, Rodriguez-Lopez et al., 2000) thereby stimulating extension of short MOS (DP 419) by SP. SP is thus envisaged as having a role in starch granule initiation by producing MOS primers for subsequent action by SSs. Research on rice endosperm SP also supports the notion that SP is involved in granule initiation through provision of precursor branched maltodextrins via functional interactions with SBEs (Nakamura et al., 2012). Recent work with potato tuber discs and Arabidopsis leaves show that [U-14C]-Glc from [U-14C]-Glc-1P can be efficiently incorporated into starch granules following transport into plastids, potentially via a SP-mediated route (Fettke et al., 2010, 2011). In addition to the possible role of SP in granule initiation, an alternative route of starch synthesis is also borne out from genetic studies in rice which indicates a predominantly SP-mediated route of starch synthesis at low temperatures (Satoh et al., 2008). There is increasing evidence that many of the enzymes of storage starch synthesis, including various SS and SBE isoforms, are found associated in multi-enzyme complexes which are assembled through the action of a phosphorylation-dependent regulatory pathway in amyloplasts (Tetlow et al., 2008; Hennen-Bierwagen

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et al., 2008, 2009; Emes and Tetlow 2012). In developing wheat endosperm amyloplasts, a proportion of SP is associated with SBEI and SBEIIb in a phosphorylation-dependent protein complex (Tetlow et al., 2004). SP has also been shown to associate in a multienzyme complex in amyloplasts of the maize amylose extender (ae
) mutant lacking the SBEIIb protein (Liu et al., 2009). Interestingly, the phosphorylation-dependent association of SP in the ae
protein complex caused SP, normally a stromal enzyme, to become granule-bound, and this phenomenon has also been shown in highamylose barley genotypes lacking SBEII (Ahmed et al., pers. comm.). The precise role of SP within the different protein complexes remains unclear. In sweet potato root, plastidial SP activity with different substrates is regulated by proteolytic cleavage mediated through the 20S proteasome, initiated by phosphorylation of serine residues on the protein (Chen et al., 2002; Young et al., 2006; Lin et al., 2012). The following study aimed to examine the posttranslational regulation of SP in developing maize endosperm amyloplasts. A functional recombinant maize SP was purified and used as an affinity ligand in proteineprotein interaction studies. Results show that SP is readily phosphorylated by a plastidial protein kinase, and that it interacts with different isoforms of SBE in a phosphorylation-dependent manner, depending upon its multimeric state. 2. Materials and methods 2.1. Plant material Wild-type CG102 maize (Zea mays) was grown in a field in Guelph, Ontario during the summer of 2011. The ae
allele was examined in the common maize inbred line background, CG102. The mutant ae
allele was ae1-ref (stock#517B) obtained from the Maize Genetics Cooperation Stock Center and back-crossed into CG102 for three generations. Pollinated kernels were collected at 9e12 days after pollination (DAP), 20e25 DAP, and 29e35 DAP and used to prepare endosperm amyloplasts. Plastid preparations were flash frozen in liquid nitrogen and stored at
80  C until future use. 2.2. Amyloplast isolation Maize endosperm amyloplasts were isolated using a modification of the methods described by Tetlow et al. (2008). Fresh endosperm tissue was washed and chopped with a razor blade in ice-cold amyloplast extraction buffer (50 mM N-(2-hydroxyethyl) piperazine-N0 -ethanesulfonic acid (HEPES)/KOH, pH 7.5, containing 0.8 M sorbitol, 1 mM KCl, 2 mM MgCl2, and 1 mM Na2-EDTA,). The resulting whole cell extract was then filtered through four layers of Miracloth (CalBiochem) wetted in the same buffer. Approximately 25 mL of the filtrate was then carefully layered onto 15 mL of 3% (w/ v) Histodenz (Sigma) in amyloplast extraction buffer followed by centrifugation at 100g at 4  C for 20 min and the supernatant was carefully decanted. Intact amyloplasts appeared as a yellow ring on top of the starch in the pellet and were lysed osmotically by the addition of ice-cold rupturing buffer containing 100 mM N-tris (hydroxymethyl) methyl glycine (Tricine)/KOH, pH 7.8, 1 mM Na2EDTA, 1 mM dithiothreitol (DTT), 5 mM MgCl2, and a protease inhibitor cocktail (ProteCEASE™ [G-Biosciences, USA] used at 10 mL per cm3). The plastid lysate was then centrifuged at 13,500g for 2 min at 4  C to remove starch granules followed by ultracentrifugation at 120,000g for 15 min in a Beckman Optima Max-XP Ultracentrifuge to separate plastidial envelope membranes. The supernatant from the ultracentrifugation step, termed plastid stroma (0.5e1.2 mg protein per mL), was flash frozen in liquid nitrogen and stored at
80  C until future use.

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2.3. Isolation and analysis of starch granule-bound proteins Isolation of starch granule-bound proteins (i.e. proteins trapped inside the granule matrix as opposed to proteins attached to the granule surface, which we term granule-associated proteins) was performed as described as previously (Tetlow et al., 2004; Liu et al., 2009). Starch granules from plastid lysates (see above) were resuspended in cold aqueous washing buffer (50 mM tris(hydroxymethyl) aminomethane (Tris)-acetate, pH 7.5, 1 mM Na2-EDTA, and 1 mM DTT) and centrifuged at 3000g for 1 min at 4  C. This washing step was repeated 5 times. The pellet was then washed 3 times with acetone followed by 3 washes with 2.0% (w/v) SDS. Starch granule-bound proteins were extracted by boiling the washed starch in SDS-loading buffer (62.5 mM TriseHCl, pH 6.8, 2% [w/v] SDS, 10% [w/v] glycerol, 5% [v/v] b-mercaptoethanol, 0.001% [w/v] bromophenol blue). Boiled samples were centrifuged at 13,000g for 5 min and the supernatant was used for SDS-PAGE and immunoblotting analysis of granule-bound proteins. 2.4. Phosphorylation of amyloplast proteins in vitro Plastid lysates (containing 0.5e1.2 mg protein per mL) prepared from maize amyloplasts isolated from endosperm at 20e25 DAP were incubated with 1 mM ATP (made up in rupturing buffer containing 25 mM tetra-sodium pyrophosphate as a protein phosphatase inhibitor) with between 600 and 1200 Ci/mmol [g-32P]-ATP (Perkin-Elmer, Boston, MA) in a total volume of 0.5 mL for 30 min at 25  C with gentle rocking. The phosphorylation reaction was terminated by the addition of 10 mM Na2eEDTA made up in 100 mM TricineeKOH (pH 7.8). Radiolabeled proteins were separated by SDS-PAGE, visualized by autoradiography and identified by immunoblotting and mass spectrometry. 2.5. Enzyme assays 2.5.1. Starch phosphorylase A quantitative assay for SP was employed measuring glucansynthesizing activity as described by Chang and Su (1986) with some modifications. SP activity was assayed in a final volume of 200 mL containing 50 mM 2-(N-morpholino) ethanesulfonic acid (MES)eNaOH buffer (pH 6), 1 mM DTT, 10 mM Glc-1P (Sigma), 2.5% (w/v) rabbit liver glycogen (type III, Sigma-Aldrich) (or other glucan substrates, where appropriate) and 50 mg of amyloplast stromal proteins. The assay system was pre-incubated at 37  C for 2 min. Protein extracts (60 mL) were added immediately before initiation of the reaction followed by the addition of 20 mL 50 mM [U-14C] a-D-glucose 1-phosphate (Glc-1P) (3.7e7.4 kBq per assay; Amersham Biosciences). The mixture was incubated at 37  C for 30 min, and terminated by heating at 95  C for 5 min. An 8% (w/v) aqueous solution of rabbit liver glycogen (type III, Sigma-Aldrich) was added to the mixture and precipitated together with newly elongated glucan by adding 1 mL of 75% (v/v) methanole1% (w/v) KCl (MeOH/KCl) followed by centrifugation at 13,500g for 5 min. The precipitated glucan was resuspended with 0.3 mL H2O on a disruptor for 5 min; glucan was again precipitated with 1 mL of MeOH/KCl and centrifuge at 13,500g for 5 min; the pellet was resuspended again with 0.5 mL of H2O and washed glucan added to a vial for counting 14C radioactivity in a Beckman LS6500 liquid scintillation counter. The phosphorolytic activity of SP was determined using a spectrophotometric-based assay according to the methods described by Tickle et al. (2009).

2.5.2. Starch branching enzyme SBE was assayed indirectly by stimulation of incorporation of 14C from [U-14C] Glc-1P into glucan by phosphorylase a according to methods previously described in Tetlow et al. (2008). 2.6. Zymograms and native-PAGE SP and SBE activities were also determined using native-PAGE zymograms. Zymograms for measuring SP activity were run according to methods modified previously (Tetlow et al., 2004, 2008). Zymograms were in-gel assays employing native 5% (w/ v) polyacrylamide gels in 375 mM TriseHCl, pH 8.8, and 10 mg of the a-amylase inhibitor Acarbose (“Prandase”, Bayer) containing 0.1% (w/v) rabbit liver glycogen (type III, Sigma-Aldrich) as primer in the gel (total volume of 7 mL) and incubated for 16 h at 28  C in a buffer containing 100 mM sodium citrate (pH 6.5) and 20 mM Glc-1P. Zymograms were developed with Lugol's (I2/KI) solution and visualized immediately, SP producing a dark blue colored band of glucan product (the position of this activity band aligned with an immuno-reactive band with anti-SP antibodies on corresponding native gels) and SBE isoforms producing brown colored bands. Native gels were prepared as for zymograms but with substrates and inhibitors omitted. Following electrophoresis, native gels were electroblotted onto nitrocellulose (see below). Protein samples used for zymograms and native-PAGE were mixed with a buffer containing 62.5 mM TriseHCl, pH 6.8, 10% (w/ v) glycerol, and 0.001% (w/v) bromophenol blue prior to loading into wells. 2.7. SDS-PAGE and immunoblotting Protein samples for SDS-PAGE and immunoblotting were mixed with SDS-loading buffer and boiled for 5 min. SDS gels used were either 12% (w/v) acrylamide gels or precast NUPAGE Novex 4e12% BisTris acrylamide gradient gels (Invitrogen Canada), following the manufacturer's instructions. Gradient gels were run at room temperature in a 3-(N-morpholino)-propanesulfonic acid (MOPS)based running buffer prepared according to the manufacturer's (Invitrogen) instructions. Immunoblotting procedures were as described by Liu et al. (2012a). For immunoblotting, separated proteins in gels were transblotted onto nitrocellulose membranes (Pall Life Science), and blocked in 1.5% (w/v) BSA for 15 min at room temperature with shaking. The purified maize SSI, SSII, GBSSI, SBEI, SBEIIa, SBEIIb, Bt2 and Iso-1 antibodies (mentioned above) were used at a dilution of 1:1000 times in 1.5% (w/v) BSA; the purified SP and ZPU1 antibodies were diluted 1:2000 in 1.5% (w/v) BSA. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) was used as a secondary antibody. 2.8. Expression and purification of recombinant proteins in Escherichia coli The SP and SBEIIb cDNA sequences from CG102 were ligated to a pET29a vector (Novagen) with restriction sites NcoI and XhoI after removing the sequence coding the transit peptide. The recombinant plasmid was transformed to ArcticExpress competent cells (Stratagene) and protein expressions were induced by 1 mM IPTG at 10  C and agitated (250 rpm) for 24 h. E. coli (ArcticExpress) cells were collected by centrifugation and lysed using “BugBuster Protein Extraction Reagent” (Novagen). Recombinant proteins were purified from inclusion bodies. A Protein Refolding Kit (Novagen catalog no. 70123-3) was employed for the purification of inclusion bodies and refolding of recombinant proteins following the manufacturer's manual. Soluble recombinant SBEIIb

or SP protein (from CG102) was precipitated by 35% ammonium sulfate followed by dialysis 4 h at 4  C. The dialyzate was centrifuged for 30 min at 27,000g at 4  C, and the supernatant applied to a 1 mL ResourceQ column (connected to an AKTA Explorer FPLC, Amersham Biosciences at 4  C) pre-equilibrated with 50 mM triseacetate buffer, pH 7.5 containing 0.05% (v/v) Triton X100. The columns were initially washed with the equilibration buffer, followed by elution with 0.6 M KCl in the equilibration buffer. The flow rates were 1 mL/min, and 0.5 mL/fraction were collected. Eluates were tested by SDS-PAGE and Coomassie Blue staining for recombinant SP or SBEIIb. Isolated protein was concentrated on a Centricon YM50 filter (Millipore) and then applied to a Superdex 200 10/300GL gel permeation column (see below). The biochemical functions of SP and SBEIIb were measured using 14C-labeled substrate assays and native gel zymogram assays (see above). Recombinant proteins were stored at
20  C in 40% (v/v) glycerol and catalytic activities were checked every 2e3 months. 2.9. Size exclusion chromatography Maize endosperm amyloplast stroma preparations were fractionated by size exclusion chromatography as described previously (Tetlow et al., 2008). Partially purified recombinant SP and SBEIIb samples were separated by size exclusion chromatography in order to separate monomeric and multimeric forms of recombinant SP, and monomeric forms of SBEIIb, from inactive aggregates prior to experiments. A Superdex 200 10/300GL gel permeation column was connected to an AKTA Explorer FPLC (Amersham Biosciences) at 4  C. The column was routinely calibrated using commercial protein standards from 13.7 kDa to 669 kDa (Amersham Biosciences). The column was pre-equilibrated with two column volumes of running buffer containing 10 mM HEPESeNaOH, pH 7.5, 100 mM NaCl, 1 mM DTT and 0.5 mM PMSF, at a flow rate of 0.25 mL/min. The protein samples (approximately 1 mg/mL) were loaded onto the column in a final volume of 0.5 mL and fractions of 0.5 mL were collected. 2.10. Preparation and analysis of polyclonal maize antibodies Polyclonal rabbit antisera targeted to maize AGPase small subunit (Bt2), SSI, SSIIa, GBSSI, SBEI, SBEIIa, SBEIIb, SP, SSIV, pullulanase (ZPU1), and Iso-1 were raised against synthetic peptides prepared commercially (http://www.anaspec.com/services/antibody.asp). The specific peptide sequence used for anti-maize Bt2 antibody was NADNVQEAARETDGYF corresponding to amino acid residues 482e497 of the full-length sequence (Gen-Bank accession no. AAZ82467.1). The specific peptide sequences used for anti-maize SSI, SSIIa, GBSSI, SBEI, SBEIIa, SBEIIb, SP, Iso-1 and Iso-2 antibodies were as described in Liu et al. (2009), for SSIV and pullulanase as described in Liu et al. (2012a), and for anti-maize Bt2 as described in Liu et al. (2012b). Crude antisera were further purified using peptide affinity columns. Respective synthetic peptides were individually coupled to sulfolink resin slurry (Pierce) and washed in TriseHCl, pH 8.5. The washed columns were then blocked with 50 mM cysteine in the same washing buffer. Antisera containing polyclonal maize antibody was applied to the column and washed with 10 mL RIPA (50 mM TriseHCl, pH 7.5, 150 mM NaCl, 1% (w/v) nonyl phenoxylpolyethoxyl ethanol (NP-40), 0.5% (w/v) Na-deoxycholate, 0.1% (w/v) sodium dodecyl sulfate [SDS]), 10 mL sarcosyl buffer [NETN (20 mM TriseHCl, pH 8.0, 1 M NaCl, 1mM Na2eEDTA and 0.5% (w/v) NP-40)] and 10 mL of 10 mM TriseHCl pH 7.8. Pure antibody bound to the column was eluted with 100 mM glycine pH 2.5 and neutralized by adding 10 mM TriseHCl pH 7.5 to the eluted

fractions. Pre-immune sera for each of the antibodies used above were employed as negative controls, and showed no cross-reaction with proteins from plastid lysates and co-immunoprecipitation experiments (data not shown). 2.11. Immunoprecipitation Immunoprecipitation and co-immunoprecipitation experiments were conducted using the methods described by Tetlow et al. (2004), with some modifications. Purified peptide-specific antibodies (each approximately 10 mg) were individually used for the co-immunoprecipitation experiments with maize amyloplast lysates (1 mL, between 0.5 and 1.2 mg/mL protein, using amyloplasts isolated 20e25 DAP). The mixture of antibody and amyloplast extract was incubated at room temperature on a rotator for 50 min and the immunoprecipitation of the antibody performed by adding 50 mL of Protein A-Sepharose (Sigma-Aldrich) made up as a 50% (w/v) slurry with phosphate buffered saline (PBS, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 M KH2PO4, pH7.4) at room temperature for 40 min. The Protein A-Sepharose/antibody/protein complex was centrifuged at 2000g for 5 min at 4  C in a refrigerated microfuge, and the supernatant discarded. The pellet was washed 5 times (1.3 mL each) with PBS, followed by washing 5 times with a buffer containing 10 mM HEPESeNaOH, pH 7.5, and 150 mM NaCl. Washed pellets were boiled in SDS-loading buffer and separated by SDS-PAGE, followed by immunoblot analysis (see above). In order to exclude the possibility that the co-immunoprecipitation of the

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2.13. Estimation of protein content Protein was determined using the Bio-Rad protein assay (BioRad Laboratories Canada) according to the manufacturer's instructions and using thyroglobulin as a standard.

with the possibility of one missed cleavage at a MS tolerance of 100 ppm and a MS/MS tolerance of 0.1 Da, and included the following variable modifications: carbamidomethyl-Cys, and oxidized Met and biotinylation.

3. Results 2.14. Mass spectrometry 3.1. Localization of SP in amyloplasts In-gel digestion with trypsin and preparation of peptides for mass spectrometry (MS) were as described previously (Tetlow et al., 2008). UPLC-MS/MS analyses were performed using a nanoAcquity™ UPLC System (Waters, Milford, MA) in combination with a QTOF micro mass spectrometer (Waters Micromass, Manchester, U.K.). The column used was a 75 mm  150 mm Atlantis™ dC18 column packed with 3 mm particles with an initial Symmetry™ C18 trapping column of 180 mm  20 mm with 5 mm particles. The LC was coupled with the mass spectrometer using a Universal Nanoflow Sprayer (Waters, Manchester, U.K.) operated with a PicoTip Emitter (New Objective, Woburn, MA) with an inner diameter of 10 mm. UPLC-MS/MS analyses were carried out at a flow rate of 400 nL/min and a column temperature of 35  C. Samples were loaded onto the trapping column and washed for 3 min with 2% solution B (acetonitrile with 0.1% formic acid). Peptides were separated using a linear gradient from 90% solution A (water with 0.1% formic acid), 2% Be60% A, 40% B in 40 min. The mass spectrometer was operated in positive ion mode with a capillary voltage of 3600 V and a cone voltage of 35 V. Data were acquired in data dependent acquisition mode, one survey scan of 2 s was carried out followed by up to three MS/MS scans (of 2 s) of each of the three most intense precursor ions. MS/MS spectra were processed using Peaks Studio 4.5 (Bioinformatics Solutions Inc., Waterloo, ON, Canada) and searched against the protein database NCBI (Ma et al., 2003). These searches were performed using trypsin specificity

Fig. 1. Localization of SP in maize amyloplast lysates. Amyloplasts (approximately 0.8 mg per mL) were prepared from developing endosperms at 20e25 DAP (individual kernel fresh weight approximately 300 mg) and fractionated as described in Materials and Methods. 12e20 mg of soluble (stromal) proteins, envelope membrane protein and starch granule proteins were loaded onto each gel lane and separated on 4e12% gradient polyacrylamide gels, electroblotted onto nitrocellulose membranes, and developed with peptide-specific anti-maize SP antibodies. Arrow indicates crossreactions of the anti-SP antibodies with the SP polypeptide (approximately 112 kDa). MW, molecular mass markers.

The sub-organellar location of SP in maize endosperm amyloplasts was determined using western blotting following fractionation of isolated amyloplasts from endosperm extracted 20e25 DAP. Fig. 1 shows that SP is exclusively localized to the plastid stroma and is visible as a single 112 kDa polypeptide. Immunoblot

Fig. 2. Fractionation of SP from maize amyloplast stroma by size exclusion chromatography. A. Amyloplast extracts were prepared from endosperms at 15e35 DAP, and 0.5 mL of soluble proteins from these extracts (approximately 1.5e2 mg protein per mL) was applied to a Superdex 200 10/300GL gel permeation column. Protein fractions eluting from the column were separated by 12% SDS-PAGE and then subjected to immunoblot analysis using peptide-specific anti-maize SP antibodies. B. Amyloplast extracts pre-treated with either 1 mM ATP or 30 U APase prior to separation by size exclusion chromatography. Numbers on the top of the blots indicate the column fraction number (early elution and greater molecular mass on the left) and numbers in boxes refer to the elution position (marked by a vertical arrow) of molecular mass markers (in kDa; Amersham Biosciences), as determined in independent column runs under identical conditions. The positions of molecular mass markers for SDS-PAGE are indicated on the left side of each immunoblot. The position of the SP cross-reacting with the respective anti-maize antibody is marked as a horizontal arrow on the blots.

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analysis of maize endosperm whole cell extracts using anti-maize plastidial SP antibodies revealed a single immuno-reactive polypeptide (Fig. 1) of the same mass (112 kDa) as found in amyloplast lysates. No SP protein was detected in plastid envelope membrane fractions or in washed starch granules.

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Interestingly, undenatured SP from amyloplast lysates and whole cell extracts could not be immunoprecipitated using the anti-SP antibodies, although it could be detected following western blotting of undenatured proteins with these antibodies (see Fig. 6A and later discussion).

Fig. 3. Analysis of recombinant SP. 3A shows catalytic activity of recombinant SP in both glucan-synthetic and phosphorolytic directions using zymogram analysis (30 ug protein per lane) prior to separation by size exclusion chromatography and MS analysis. Western blots in 3A show that recombinant SP cross-reacts with peptide-specific anti-SP antibodies and anti-S-tag antibodies. 3B shows the fractionation of multimeric forms of recombinant SP by size exclusion chromatography detected by western blotting, and in 3C analysis of the catalytic activity of corresponding fractions containing SP by zymogram analysis in the glucan-synthetic direction. Numbers on the top of the blots indicate the column fraction number (early elution and greater molecular mass on the left) and numbers in boxes refer to the elution position (marked by a vertical arrow) of molecular mass markers (in kDa; Amersham Biosciences), as determined in independent column runs under identical conditions. The positions of molecular mass markers for SDS-PAGE are indicated on the left side of each immunoblot (MW).

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3.2. Size exclusion chromatography of maize amyloplast stromal proteins Extracts of soluble proteins from maize amyloplasts were separated and eluted through a Superdex 200 gel permeation column in order to determine the aggregation state of SP. Elution profiles for native SP were similar to those observed previously (Liu et al., 2009). SP eluted as a broad peak at apparent molecular masses greater than that which would be predicted if SP was eluting as a 112 kDa monomer. The observed elution profile (Fig. 2A) corresponds to SP existing predominantly as a high-molecular-weight multimer of >440 kDa at all developmental stages analyzed (15e35 DAP), which may include tetrameric forms of SP, or possibly SP-containing heteromeric protein complex assemblies. Elution of SP from the gel permeation column was unaffected by pre-treatment of amyloplasts with 1 mM ATP or 30 U alkaline phosphatase (APase) (Fig. 2B). Immuno-reactive bands migrating less than the size of the SP monomer (112 kDa) on SDS-PAGE were detected in ATP- and APase-treated samples eluting from the gel permeation column (Fig. 2B) and are presumed to be products of proteolytic degradation, but were not analyzed further. 3.3. Using recombinant SP to study proteineprotein interactions in maize endosperm amyloplasts Recombinant maize SP-containing an S-protein fusion tag at the N-terminus was produced in E. coli and used to study proteineprotein interactions either through co-immunoprecipitation, or attached to a solid S-agarose support for affinity binding studies. The recombinant SP isolated from bacterial lysates was catalytically

active (in both glucan-synthetic and phosphorolytic directions) and could be detected using anti-SP and anti-S-tag antibodies (Fig. 3A, zymograms). The recombinant protein was separated by SDS-PAGE and stained with Coomassie Blue (gel not shown); the excised gel band was identified as SP following in-gel trypsin digestion and analysis of peptides using MS (Fig. 3A, MS data). Given the tendency of recombinant proteins to form aggregates and inclusion bodies, the recombinant maize SP was subjected to gel permeation chromatography and zymogram analysis to separate the various multimeric forms from artificially produced aggregates (Fig. 3B and C). The results show various aggregation states of SP including large, catalytically inactive aggregates of >440 kDa and two peaks of protein corresponding to tetramers and monomers. Gel permeation chromatography analysis of SP from plastid lysates (Fig. 2) suggests native SP exists as a multimeric enzyme. Recombinant SP fractions corresponding to tetrameric and monomeric forms of SP (tetramers derived from fractions 21 to 23 from the Superdex 200 column and monomers derived from fractions 28 to 30) were used in subsequent proteineprotein interaction experiments with amyloplast lysates. The partially purified forms of recombinant SP (tetramer and monomer fractions from the Superdex 200 column) were attached to a solid S-agarose support for affinity binding studies to determine proteineprotein interactions involving SP in maize amyloplasts. Fig. 4 shows the results obtained with recombinant SP forms immobilized to S-agarose and incubated with amyloplast stromal preparations (isolated 20e25 DAP). After removal of the plastid extracts and washing the beads, proteineprotein interactions were detected using western blotting with various peptide-specific antisera (see Materials and Methods). Many proteins tested

Fig. 4. Use of recombinant maize SP multimers as affinity ligands to study proteineprotein interactions in amyloplast lysates. 120 mg of catalytically active recombinant maize SP tetramer or monomer (S-tagged at the N-terminus) were immobilized onto S-protein agarose and incubated with 1 mL of amyloplast lysates (0.8e1 mg protein per mL, isolated 20e25 DAP) for 1 h at 25  C. After removal of the plastidial lysates, the beads containing the recombinant protein and interacting proteins were washed and then boiled in SDSloading buffer, proteins separated by SDS-PAGE using 4e12% acrylamide gradient gels, blotted onto nitrocellulose, and probed with various peptide-specific anti-maize antibodies. Lanes 1e3 are amyloplast extracts mixed with the immobilized recombinant protein and show, respectively, untreated amyloplasts, amyloplasts treated with 1 mM ATP or 30 U APase. Lanes 4e6 are the same treatments using “empty” S-protein agarose beads, lacking recombinant proteins. Lane 7, amyloplast stroma. Horizontal arrows indicate crossreactions with the various antibodies used. MW indicates molecular mass markers with their molecular masses shown on the left of the blot.

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Fig. 6. Functional interactions between SP and SBEIIb. (A) Activity gel (zymogram) and SDS-PAGE analysis of SBE isoforms and SP from amyloplasts of wild-type (wt) and ae
endosperms (20e25 DAP). Approximately 100 mg of soluble proteins per lane were separated on a 7-cm native 5% acrylamide gel containing 0.2% (w/v) maltoheptaose, and then developed for 3 h at 28  C. SBE and SP (glucan-synthetic) activities were visualized by staining with I2/KI and are labeled. Identical gels were electroblotted onto nitrocellulose membranes and probed with peptide-specific anti-maize SP antisera, allowing the identification of SP activity from the zymogram. Western blots of SDS gels probed with antimaize SP antisera showed equal amounts of SP in wild-type and ae
plastid lysates. (B) Analysis of SP (glucan-synthetic direction) activities from amyloplasts of ae
endosperms in which increasing amounts of recombinant maize SBEIIb were added and incubated for 1 h at room temperature prior to separation by native-PAGE and zymogram analysis as in A, or (C) assayed for SP activity using a 14C-based assay with glycogen as the glucan primer. The zymogram also detects the activities of SBE isoforms.

remained in the supernatant irrespective of pre-treatment of the amyloplasts. However, zymogram analysis of the supernatant showed loss of most of the SP activity when amyloplasts had been treated with ATP (Fig. 7C), and most of the SBEIIb was removed due to its association in the protein complex with SSIIa and SSI (Fig. 7B). Activities of SBEI and SBEIIa were unaffected (Fig. 7C). Treatment of recombinant SP with ATP caused no change in glucan-synthetic or phosphorolytic activity, indicating that ATP was not having any direct effect on SP, independent of proteineprotein interactions (data not shown). 4. Discussion This study has shown that SP is a soluble protein found in the plastid stroma in a multimeric state throughout the major kernelfilling stages of maize endosperm development and supports previous studies (Mu et al., 2001). Gel permeation chromatography analysis of native maize SP allowed us to estimate the size of SP during endosperm development, showing that throughout the developmental stages analyzed (15e35 DAP, corresponding to the major period of starch deposition in the endosperm) SP exists as one or more multimeric complexes which could represent a population of homo-tetrameric and/or heteromeric forms. The predominant form of SP eluted in the region corresponding to a molecular mass of approximately 440 kDa, consistent with

previous studies (Liu et al., 2009). Interestingly, the 112 kDa SP monomer was not detected in amyloplast preparations, reinforcing the view that the enzyme exists in a multimeric state in vivo. Given the various multimeric states of SP, and its known ability to form protein complexes with other starch biosynthetic enzymes (Tetlow et al., 2004; Liu et al., 2009), this study was directed at understanding the role played by the different multimeric states of SP in forming protein complexes. The available anti-maize SP antibodies used in this study were raised against a synthetic peptide (YSYDELMGSLEGNEGYGRADYFLV, corresponding to residues 900e923 of the full-length amino acid sequence of SP) and showed a high degree of specificity to the denatured SP polypeptide. Surprisingly, native SP in solution was not precipitated by the antibodies, although the protein was detected following non-denaturing PAGE (Fig. 7C). Consequently, the anti-maize SP antibodies could not be employed for immunoprecipitation studies and instead, an alternate strategy was adopted in which recombinant maize SP was expressed in E. coli and used as an affinity ligand for proteineprotein interaction experiments. It was necessary to fractionate the catalytically active monomers and tetramers of recombinant SP from artifacts (highmolecular mass aggregates of >440 kDa which were catalytically inactive) in order to study proteineprotein interactions. The tendency of many recombinant proteins to form aggregates that are not seen in vivo is common (Liu et al., 2012a). Gel permeation

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Fig. 7. Co-immunoprecipitation experiment using anti-maize SSIIa antibodies and wild-type maize amyloplasts. (A, B) The trimeric protein complex consisting of SSI, SSIIa and SBEIIb was immunoprecipitated with anti-SSIIa antibodies following pre-treatment of amyloplast lysates with 1 mM ATP. Western blots of the co-immunoprecipitated proteins and resulting supernatant following SDS-PAGE were probed with anti-SSI, SBEIIb and SP antibodies. (C) Zymogram of SP activity (glucan-synthetic) in the supernatant following SSIIa immunoprecipitation. MW indicates the positions of molecular mass markers (shown as bands) with their corresponding masses in kDa on the blots. AP, amyloplast soluble protein extract; APase, treatment with 30 U alkaline phosphatase; ATP, treatment with 1 mM ATP.

chromatographic analysis of native SP indicates that monomeric or dimeric forms are not present in amyloplasts in vivo. Since much of the enzyme in amyloplasts exists as an aggregate of approximately 440 kDa (Fig. 2), it must be concluded that when SP forms protein complexes with other proteins such as SBEI and SBEIIb (see below), it is in a lower-order multimer (monomer, dimer or trimer). Previous experimental evidence supports the existence of homomeric and heteromeric SBE complexes (Tetlow et al., 2004, 2008). Treatment with APase (a non-specific phosphatase) caused disassociation of SP/SBE interactions (Fig. 4), but no alteration in the elution profile of SP following gel permeation chromatography (Fig. 2B). This suggests that when SBE isoforms dissociate from a complex with SP, SP forms a homo-tetrameric structure of similar size to an SP/SBE complex. Therefore SP probably exists as a homo-multimer (tetramer) and a hetero-multimer complex with associated SBE isoforms. Direct proteineprotein interaction studies using the recombinant maize SP with maize amyloplast lysates indicate that SP can form ATP-dependent heteromeric protein complexes with SBEI and SBEIIb, and that these associations can be broken following treatment with APase (Fig. 4). This is consistent with results of earlier protein interaction studies in amyloplasts of wheat endosperm describing the ATP-dependent formation of a protein complex in which SP, SBEI and SBEIIb are associated (and broken with APase treatment) (Tetlow et al., 2004). A number of enzymes involved in amylopectin biosynthesis form ATP-dependent protein complexes (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008; Liu et al., 2009) and are also found as granule-bound proteins. It has recently been proposed that catalytically active protein complexes involved in amylopectin cluster biosynthesis become granule-bound through

their physical association with SSIIa (Liu et al., 2012b). Neither SP nor SBEI are found as granule-bound proteins in wild-type starch granules, and are not known to interact with SSIIa. We therefore propose that the SP/BE protein complexes either operate at the surface of the nascent granule, in some way modifying a-glucans, or that they are involved in MOS modification in the plastid stroma (see below). An interesting observation made in this study was the stimulatory effect of SBEIIb on the catalytic activity of SP (see Fig. 6B). Loss of SBEIIb protein in the maize ae
mutant caused a reduction in SP activity (Fig. 6A, and Liu et al., 2009), despite equivalent levels of SP protein being present in both wild-type and ae
mutant plastid extracts. SP activity in plastid extracts from the ae
mutant was stimulated upon addition of increasing amounts of recombinant SBEIIb (Fig. 6B). The mechanism for SP stimulation by SBEIIb is unclear. It is possible that the stimulatory effect of SBE isoforms arises from the creation of non-reducing ends for a-glucan elongation by SP in a manner analogous to the phosphorylase a-stimulation assay (Tetlow, 2012). Evidence from this study suggests that stimulation of SP activity could also be through direct association with SBEIIb. It is noteworthy that SBEIIa did not interact with SP (data not shown). The SP/SBEIIb interaction is ATP-dependent (Fig. 4), and labeling experiments with [g-32P]-ATP show that SP is readily phosphorylated (Fig. 5). The formation of the SP/SBE protein complex in wheat endosperm amyloplasts was also ATP-dependent, where SP (interacting with SBEIIb) was phosphorylated on a serine residue(s) (Tetlow et al., 2004). A recent, high throughput proteomic analysis of developing maize seeds also detected phosphorylation of maize SP (Walley et al., 2013). SP appears to have multiple

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phosphorylation sites with four phosphoserines and one phosphothreonine detected in a short motif between residues 536 and 565, as well as a phosphoserine at residue 68. Bioinformatic analysis predicted several functional protein interaction partners including SBEs (Liu et al., pers comm.), consistent with the experimental data reported here and elsewhere (Tetlow et al., 2004). Recent in vitro experiments by Nakamura et al. (2012) show that rice SP can synthesize maltodextrins in a primer-independent fashion, and that their synthesis is accelerated by the presence of SBEs, notably SBEI and SBEIIb. SBE activity was also enhanced in the presence of SP, and the authors suggest, but provided no evidence, that this is due to a physical association between SP and SBE. The branched maltodextrins formed by SP and SBE are resistant to isoamylase treatment, and it was suggested by Nakamura et al. (2012) that these structures act as primers for the biosynthesis of amylopectin, possibly initiating the synthesis of individual clusters, as proposed by Jeon et al. (2010). Studies with Arabidopsis mutants suggest amylopectin priming can be achieved with SP and SBE and/ or SBE interacting with specific SS isoforms (SSIII and SSIV) (D'Hulst rida, 2010). The data in this paper give support to this and Me model, showing direct proteineprotein interactions involving SP and SBEI and SBEIIb; SBEIIb stimulating the catalytic activity of SP through this association. If the SP/SBE complex is involved in creating isoamylase-resistant branched maltodextrins for the initiation of amylopectin clusters or higher order structures such as blocklets, presumably they need to be loosely associated with the glucan in order for the cluster-synthesizing SS/SBE complexes to continue work on the structure, and therefore do not become granule-bound. Funding This work was supported by an Ontario Ministry of Agriculture Food and Rural Affairs (OMAFRA) BioCar grant (project number 048572) awarded to IJT and to Natural Sciences and Engineering Research Council (NSERC) Discovery Grants awarded to MJE (project no. 262209) and IJT (project no. 341722). Acknowledgments The authors acknowledge Dr. Dyanne Brewer (University of Guelph Advanced Analysis Center) for MS analysis and data interpretation. References Albrecht, T., Greve, B., Pusch, K., Kossmann, J., Buchner, P., Wobus, U., Steup, M., 1998. Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies. Eur. J. Biochem. 251, 343e352. Buchbinder, J.L., Rath, V.L., Fletterick, R.J., 2001. Structural relationships among regulated and unregulated phosphorylases. Annu. Rev. Biophys. Biomol. Struct. 30, 191e209. Burr, B., Nelson, O.E., 1975. Maize a-glucan phosphorylase. Eur. J. Biochem. 56, 539e546. Chang, T., Su, J.C., 1986. Starch phosphorylase inhibitor from sweet potato. Plant Physiol. 80, 534e538. Chen, H.-M., Chang, S.-C., Wu, C.-C., Cuo, T.-S., Wu, J.-S., Juang, R.-H., 2002. Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis. Physiol. Plant. 114, 506e515. e, D., Chochois, V., Steup, M., Haebel, S., Eckermann, N., Ritte, G., et al., 2006. Dauville Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii. Plant J. 48, 274e285. rida, A., 2010. The priming of storage glucan synthesis from bacteria D'Hulst, C., Me to plants: current knowledge and new developments. New Phytol. 188, 13e21. Duwenig, E., Steup, M., Kossmann, J., 1997. Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs. Planta 203, 111e120.

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