TY - JOUR
AU - Tetlow, Ian J.
AB - Abstract The amylose extender (ae–) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein–protein interactions in amyloplasts of ae– mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein–protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae– amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae– endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae–), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis. Amylopectin, amyloplasts, amylose extender, high-amylose, protein phosphorylation, protein–protein interactions, starch branching enzyme, starch granule associated proteins, starch phosphorylase, starch synthase, starch synthesis Introduction Starch is a biologically and commercially important polymer of glucose (Glc) synthesized as discrete granules inside the plastids of higher plants and green algae. The starch granule is water-insoluble (and hence, osmotically inert), making it a suitable long-term storage carbohydrate for seeds and tubers of many plant species. This unique physical property is a result of the highly organized manner in which the glucan chains are packed together in the amylopectin component of starch, and the same level of molecular organization of storage starches is also apparent in leaf starch (Zeeman et al., 2002). Currently accepted models for starch structure involve the periodic clustering of branch points in amylopectin (French, 1984; Hizukuri, 1986; Waigh et al., 1999; Bertoft, 2004). Branch point distribution and frequency appears to be a critical factor in determining many of the physico-chemical properties of starches (Thompson, 2000). Low branch point frequency and close clustering of branch linkages are found in water-insoluble starch granules, as opposed to the more open, highly branched water-soluble polyglucans such as glycogen with longer inter-branch distances (Meléndez-Hevia et al., 2000). Three groups of enzymes are known to be involved in starch biosynthesis following the formation of the soluble precursor ADP-glucose (ADP-Glc) by ADP-Glc pyrophosphorylase (AGPase, EC 2.7.7.27). Starch synthases (SSs, EC 2.4.1.21) begin the process of amylopectin synthesis by catalysing the formation of linear α-(1→4)-linked glucan chains from ADP-Glc, which, in turn, are the substrates for starch branching enzymes (SBEs, EC 2.4.1.18) which introduce branch points into the glucan chain by cleaving internal α-(1→4) linkages and transferring the released reducing end to a C6 hydroxyl to create α-(1→6) linkages. These two interdependent activities (SS and SBE) are a key feature of polyglucan biosynthesis. The third group of enzymes, termed debranching enzymes [DBEs, including isoamylases (Iso), EC 3.2.1.41 and pullulanase, EC 3.2.1.68] appear to be important for the formation of semi-crystalline amylopectin (Morris and Morris, 1939; James et al., 1995; Mouille et al., 1996; Zeeman et al., 1998; Wattebled et al., 2005, 2008; Fujita et al., 2009). Higher plants possess multiple soluble forms of SSs (SSs I–IV), SBEs (SBE I and II), and DBEs (Iso 1–3, and one pullulanase-type DBE), each with varied affinity for different glucan substrates, suggesting specific and unique roles for each during amylopectin biosynthesis (Jespersen et al., 1993; Ball and Morell, 2003; Patron and Keeling, 2005). It is becoming clear that the complicated process of starch granule formation involves more than the co-ordinate expression of these three groups of core biosynthetic proteins. Recent work with cereal endosperms suggests that key enzyme activities involved in amylopectin biosynthesis, the SSs and SBEs, are assembled into functional units (protein complexes) to perform the task of granule formation (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008, 2009). In addition to functional protein complexes containing glucan-extending (SS) and branching (SBE) capabilities, previous work with wheat (Triticum aestivum L.) endosperm amyloplasts has also identified α-glucan (starch) phosphorylase (SP, EC 2.4.1.1) in a protein complex together with SBEI and SBEIIb (Tetlow et al., 2004), suggesting a biosynthetic role for SP in amylopectin synthesis. The first genetic evidence demonstrating a role for SP in amylopectin biosynthesis came from studies with plastidial SP-deficient (sta4) mutants of Chlamydomonas reinhardtii (Dauvillée et al., 2006). Analysis of the phenotype of the sta4 mutant in Chlamydomonas also implied interactions between SBEs and SP (Dauvillée et al., 2006). Recent work by Satoh et al. (2008) also points to a role for the plastidial form of SP (Pho-1) in starch synthesis in rice (Oryza sativa L.) endosperm. The precise role played by each of the protein complexes in starch granule assembly is, as yet, unclear. Protein phosphorylation is emerging as a potentially important mechanism for the control of starch biosynthesis in plants. All isoforms of SBE and at least one form of SS (SSIIa) have been shown to be phosphorylated by plastidial protein kinase activity and, in the case of SBEs, phosphorylation status modulated the catalytic activities of SBEII forms in both amyloplasts and chloroplasts (Tetlow et al., 2004). Significantly, all of the protein complexes between starch biosynthetic enzymes in endosperm amyloplasts and leaf chloroplasts studied to date are phosphorylation-dependent, indicating an important role for this post-translational control mechanism in controlling specific aspects of starch biosynthesis. In maize (Zea mays L.), many well-characterized mutations in the starch biosynthetic pathway show altered starch phenotypes, providing valuable insights into the biochemistry of the pathway (Lee, 2004). Analysis of these genetic resources indicated co-ordination and interdependence of starch biosynthetic activities (Boyer and Preiss, 1981; Singletary et al., 1997; Gao et al., 1998), which has recently been confirmed by the discovery that key components of amylopectin synthesis operate within protein complexes (Tetlow et al., 2004, 2008). Given the importance of α-glucan branching in amylopectin formation, and the involvement of SBE forms in protein complexes in cereal endosperm amyloplasts, a mutant of maize lacking the major form of SBEII (SBEIIb), termed amylose extender (ae–), was used to examine the effects of loss of branching enzyme activity on the ability to form protein complexes, and how this may influence starch structure in this mutant. In maize, SBEIIb is the most abundant protein in the amyloplast stroma (Mu et al., 2001) and therefore the predominant isoform of SBEII expressed in the endosperm, whereas SBEIIa (the product of a separate gene) is detected in almost all tissues (Fisher et al., 1996; Gao et al., 1997). Maize ae– starches are characterized by longer internal chain lengths in amylopectin compared with normal starches and less frequently branched outer chains (Hilbert and MacMasters, 1946; Banks et al., 1974; Klucinec and Thompson, 2002). Such starches are termed ‘high-amylose’ starches, due to the higher proportion of long-chain branch points in the amylopectin. Analysis of ae–.wx double mutants of rice and maize (lacking amylose) clearly show that the ae– mutation causes alterations in amylopectin structure (Nishi et al., 2001; Yao et al., 2004). The nature of the branching pattern of amylopectin probably has more important bearing on the physico-chemical properties of starch, than the chain length profile (Klucinec and Thompson, 2002). In addition to differences in granule ultrastructure between normal and ae– starch granules, the gross morphology of ae– granules differs markedly from normal or waxy granules, often appearing elongated (rather than ellipsoid), and smaller than normal granules (Boyer et al., 1976b; Fogher and Lorenzoni, 1981; Shannon and Garwood, 1984; Fannon et al., 1992). Earlier analysis of the ae– mutant by Colleoni et al. (2003) revealed interesting pleiotropic effects on other enzymes in the starch biosynthetic pathway including loss of measurable SBEI activity and altered properties of an isoamylase-type DBE in the mutant endosperm. It has been suggested that a lower SBE activity relative to SS activity in ae–-containing starches leads to more of the longer B chains (which may link clusters; Thompson, 2000) relative to shorter B chains, thus reducing the number of chains per cluster, and reducing the number of branch points present within periodic clusters (Boyer et al., 1976a; Klucinec and Thompson, 2002). The results of the present study show that a phosphorylation-dependent protein complex in normal endosperm containing SSI, SSIIa, SBEIIb, is lost in the ae– mutant and is replaced by novel protein–protein interactions consisting of SSI, SSIIa, SBEI, SBEIIa, and SP. These various interactions are reflected in the complement of starch synthesizing enzymes detected in starch granules as granule-associated proteins. The consequences of these novel protein–protein interactions on starch granule structure in ae– maize will be discussed. Materials and methods Plant material The ae– allele was examined in a common maize inbred line background, CG102. The mutant ae– allele was ae1-ref (stock 517B) obtained from the Maize Genetics Cooperation Stock Centre and backcrossed into CG102 for three generations. Wild-type Zea mays plants and mutant maize plants were grown either in the field or at 25–27 °C in the greenhouse at the University of Guelph under conditions previously described for growing wheat (Tetlow et al., 2008). Self-pollinated kernels, obtained through controlled pollinations, were collected at 9–12 d after pollination (DAP), 20–25 DAP, and 29–35 DAP and used to prepare endosperm amyloplasts, starch and whole cell soluble extracts. Plant materials were flash frozen in liquid nitrogen and stored in –80 °C until future use. Preparation of whole cell extracts from developing endosperm Whole cell extracts were prepared as described previously (Tetlow et al., 2003) with some modification. Approximately 10 g endosperm tissue was quickly frozen in liquid nitrogen and immediately ground using a chilled mortar and pestle under liquid nitrogen into a fine powder. The frozen powder was mixed with ice-cold rupturing buffer containing 100 mM N-tris (hydroxymethyl) methyl glycine (Tricine)/KOH, pH 7.8, 1 mM Na2-EDTA, 1 mM dithiothreitol (DTT), 5 mM MgCl2, and a protease inhibitor cocktail (Sigma-Aldrich, catalogue no. P 9599, used at 10 μl ml−1). The mixture was further gently ground and allowed to stand on ice for 5 min followed by centrifugation at 13 500 g for 5 min at 4 °C. The supernatant was subjected to ultracentrifugation at 120 000 g for 15 min in a Beckman Airfuge (at 25 psi) to remove membranes and particulate material. The supernatant obtained following ultracentrifugation was used for experiments. Amyloplast isolation Maize endosperm amyloplasts were isolated using a modification of the methods described by Tetlow et al. (2008). Fresh endosperm tissue (90–100g) was washed and chopped with a razor blade in ice-cold amyloplast extraction buffer (50 mM N-(2-hydroxyethyl) piperazine-N′-ethanesulphonic 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, catalogue no. 475855) wetted in the same buffer. Approximately 25 ml of the filtrate was then carefully layered onto 15 ml of 3% (w/v) Histodenz (Sigma, catalogue no. D2158) in amyloplast extraction buffer followed by centrifugation at 100 g 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 (see above). The plastid lysate was then centrifuged at 13 500 g for 2 min at 4 °C to remove starch granules followed by ultracentrifugation at 120 000 g for 15 min to remove plastidial membranes. The ultracentrifugation supernatant termed plastid stroma (0.5–1.1 mg protein ml−1) was flash frozen in liquid nitrogen and stored at –80 °C until future use. Isolation of starch granule bound proteins Isolation of starch granule bound proteins was performed as described as previously (Denyer et al., 1995; Tetlow et al., 2004). 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 3000 g for 1 min at 4 °C. This washing step was repeated five times. The pellet was then washed three times with acetone followed by three washes with 2% (w/v) SDS. Starch granule bound proteins were extracted by boiling the washed starch in SDS loading buffer [62.5 mM TRIS-HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.001% (w/v) bromophenol blue]. Boiled samples were centrifuged at 13 000 g for 5 min and the supernatant was used for SDS-PAGE and immunoblotting analysis of granule-associated proteins. Phosphorylation of amyloplast proteins in vitro Plastid lysates (containing 0.5–1.1 mg protein ml−1) prepared from wild type and ae– amyloplasts isolated from endosperm at 20–25 DAP were incubated with 1 mM ATP with between 600 and 1200 Ci mmol−1 γ-32P-ATP (Perkin-Elmer, Boston, MA) in a total volume of 0.5 ml for 30 min at 25 °C with gentle rocking. Phosphorylated stromal proteins were used in immunoprecipitation experiments (see below). Radiolabelled proteins were visualized by autoradiography. Expression of recombinant maize SS and SBE in Escherichia coli Plasmid vectors (pET 29, Novagen) containing maize SSI, SBEI, and SBEIIa sequences were kindly provided by Drs Alan Myers and Martha James (Iowa State University, USA). Maize SSI was constructed in pET-29a, this plasmid contains the complete coding sequence of mature SSI, specifically amino acids 39–640 according to the full-length cDNA sequence (GenBank accession no. AAB99957). Amino acid 39 was directly identified as the mature N terminus by amino acid sequence of the purified protein (Knight et al., 1998). The recombinant SSI protein was fused to the S-tag sequence at its amino terminus. Maize SBEI plasmid was constructed in pET-29b, amino acids 65–823 referring to its full-length cDNA sequence (GenBank accession no. AAC36471); maize SBEIIa plasmid was constructed in pET-29a, amino acids 21–814 according to the full-length cDNA sequence (GenBank accession no. AAB67316). Similar to SSI, recombinant SBEI, and SBEIIa were fused to the S-tag sequence at their amino termini. The recombinant plasmids of SSI, SBEI, and SBEIIa were individually transformed to Escherichia coli strain BL21-CodonPlus (DE3)-RP. A single clone picked up from each of an overnight plate was individually inoculated to Luria-Bertani media and grown at 30 °C overnight. Overnight media were diluted 20 times in the morning, and were grown at 37 °C for approximately 2 h until the density was 0.6–0.8 at A600. The expression of each recombinant protein was induced by adding IPTG to a final concentration of 1 mM and the cultures were grown for 3 h at 37 °C. E. coli cells were collected by centrifugation and lysed using ‘BugBuster Protein Extraction Reagent’ (Novagen catalogue no. 70584). Recombinant SSI, SBEI, and SBEIIa were purified from inclusion bodies. A Protein Refolding Kit (Novagen catalogue no. 70123-3) was employed for the purification of inclusion bodies and refolding of recombinant proteins following the manufacturer's manual. The biochemical functions of SSI, SBEI, and SBEIIa were measured using 14C-labelled substrate assays, and native gel zymogram assays (see below). Recombinant proteins were stored at –20 °C in 40% (v/v) glycerol and catalytic activities were checked every 2–3 months. Enzyme assays Starch synthases: SS activity was measured using ADP-[U-14C]Glc as described previously (Tetlow et al., 2008) with minor modifications. The activity of soluble SS was assayed by following the incorporation of 14C from ADP-[U-14C]Glc into glucan in a total assay volume of 200 μl using 60 μl extract. Reaction mixture contained 200 mM N, N-bis-(2-hydroxyethyl) glycine (Bicine)-KOH, pH 8.5, 50 mM potassium acetate, 200 mM sodium citrate, 20 mM DTT, 1 mM Na2-EDTA, and 8% (w/v) of rabbit liver glycogen (type III, Sigma-Aldrich). Extracts were added immediately prior to initiating reactions with 20 μl 20 mM ADP-[U-14C]Glc (0.67 kBq per assay; Amersham Biosciences) and were incubated at 25 °C for 20 min. Reactions were terminated by heating the mixture at 95 °C for 5 min followed by passing the mixture through 1 ml anion-exchange resin columns (AG1-X8 resin, Bio-Rad Laboratories) as described by Jenner et al. (1994). Starch branching enzymes: SBE was assayed indirectly by stimulation of incorporation of 14C from [U-14C] Glc1P into glucan by phosphorylase a according to methods previously described (Smith, 1990), and modified by Tetlow et al. (2008). The reaction contained, in a total volume of 200 μl, 100 mM sodium citrate, pH 7.0, 1 mM Na2-EDTA, 1 mM DTT, 2.5 mM AMP, and 0.2 unit rabbit muscle phosphorylase a (from rabbit muscle; product no. P-1261, Sigma-Aldrich). Protein extract (60 μl) was added immediately before initiation of the reaction with 20 μl 50 mM [U-14C] α-D-glucose 1-phosphate (Glc 1-P) (3.7–7.4 kBq per assay; Amersham Biosciences), incubated at 25 °C for 90 min, and terminated by heating at 95 °C for 5 min. Prior to washing with 75% (v/v) methanol–1% (w/v) KCl, an 8% (w/v) aqueous solution of rabbit liver glycogen (type III, Sigma-Aldrich) was added to precipitate the newly synthesized 14C-labelled glucan. The modified SS and SBE assays for maize were each optimized with respect to substrate concentration and glucan primer used, and reactions were linear with respect to protein concentration and reaction time prior to experimentation. Zymograms Zymograms for measuring SS and SBE 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 TRIS-HCl, pH 8.8, and 10 mg of the α-amylase inhibitor Acarbose (Bayer, ‘Prandase’). SS zymograms contained 0.3% (w/v) rabbit liver glycogen (type III, Sigma-Aldrich) as primer in the gel and were incubated for 48–72 h in a buffer containing 50 mM glycylglycine, pH 9.0, 100 mM (NH4)2SO4, 20 mM DTT, 5 mM MgCl2, 0.5 mg ml−1 bovine serum albumen (BSA), and 4 mM ADP-Glc. The native gel for SBE zymogram contained 0.2% (w/v) maltoheptaose (Sigma-M7755), 1.4 units phosphorylase a (from rabbit muscle; Sigma-Aldrich, catalogue no. P-1261) and was incubated in a buffer containing 20 mM 2-(N-morpholino) ethanesulphonic acid (MES)-NaOH, pH 6.6, 100 mM Na-citrate, 45 mM Glc-1-P, 2.5 mM AMP, 1 mM DTT, 1 mM Na2-EDTA for 2–3 h at 28 °C. SS and SBE zymograms were developed with Lugol's solution and visualized immediately. Size exclusion chromatography Maize endosperm amyloplast preparations or whole cell extracts were fractionated by size exclusion chromatography as described previously (Tetlow et al., 2008). 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 HEPES-NaOH, pH 7.5, 100 mM NaCl, 1 mM DTT, and 0.5 mM PMSF, at a flow rate of 0.25 ml min−1. The protein samples (about 1 mg ml−1) were loaded onto the column in a final volume of 0.5 ml and fractions of 0.5 ml were collected. Preparation and analysis of polyclonal maize antibodies Polyclonal rabbit antisera targeted to maize SSI, SSIIa, SBEI, SBEIIa, SBEIIb, SP, Iso-1, and Iso-2 were raised against synthetic peptides prepared commercially (http://www.anaspec.com/services/antibody.asp). The specific peptide sequences used were as follows: SSI, AEPTGEPASTPPPVPD, corresponding to residues 72–87 of the full-length sequence (GenBank accession no. AAB99957); SSIIa, GKDAPPERSGDAARLPRARRN, corresponding to residues 69–89 of the full-length sequence (GenBank accession no. AAD13341); SBEI, KGWKFARQPSDQDTK, corresponding to residues 809–823 of the full-length protein (GenBank accession no. AAC36471); SBEIIa, FRGHLDYRYSEYKRLR, corresponding to residues 142–157 of the full-length sequence (GenBank accession no. AAB67316); SBEIIb, PRGPQRLPSGKFIPGN, corresponding to residues 641–656 of the full-length sequence (GenBank accession no. AAC33764); SP, YSYDELMGSLEGNEGYGRADYFLV, corresponding to residues 900–923 of the full-length sequence (GenBank accession no. AAS33176), Iso-1, FTKHNSSKTKHPGTYIAC-NH2, corresponding to residues 269–286 of the full-length sequence (GenBank accession no. AAA91298), Iso-2, ARSYRYRFRTDDDGVV, corresponding to residues 37–52 of the full-length sequence (GenBank accession no. NP001105666), and granule-bound starch synthase I (GBSSI), QDLSWKGPAKNWENV, corresponding to residues 442–456 of the full-length sequence (GenBank accession no. ABW95928). Crude antisera were further purified using peptide affinity columns. Respective synthetic peptides were individually coupled to sulpholink resin slurry (Pierce) and washed in TRIS-HCl, pH 8.5. The washed columns were then blocked with 50 mM cysteine in the same washing buffer. Antisera containing the polyclonal maize antibody was applied to the column and washed with 10 ml RIPA [50 mM TRIS-HCl, 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 sulphate (SDS)], 10 ml sarcosyl buffer [NETN (20 mM TRIS-HCl, pH 8.0, 1 M NaCl, 1 mM Na2-EDTA, and 0.5% (w/v) NP-40)] and 10 ml of 10 mM TRIS-HCl pH 7.8. Pure antibody bound to the column was eluted with 100 mM glycine pH 2.5 and neutralized by adding 10 mM TRIS-HCl 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). Co-immunoprecipitation Co-immunoprecipitation experiments were conducted using the methods described by Tetlow et al. (2004), with some modifications. Purified SSI, SSIIa, SBEI, and SBEIIb antibodies (each approximately 10 μg) were individually used for the co-immunoprecipitation experiments with both wild-type and ae– mutant amyloplast lysates (1 ml, between 0.5–1 mg ml−1 proteins). The mixture of antibody and amyloplasts was incubated at room temperature on a rotator for 50 min and precipitation of the antibody performed by adding 50 μl 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 mM KH2PO4, pH 7.4) at room temperature for 40 min. The Protein A-Sepharose/antibody/protein complex was centrifuged at 2000 g for 5 min at 4 °C in a refrigerated microfuge, and the supernatant discarded. The pellet was washed five times (1.3 ml each) with PBS, followed by washing five times with a buffer containing 10 mM HEPES-NaOH, 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 below). In order to exclude the possibility that the co-immunoprecipitation of the proteins observed in the immunoprecipitation pellet was a result of SSs, SBEs or SP binding to the same glucan chain, plastid lysates used for immunoprecipitation were preincubated with glucan-degrading enzymes as follows. Wild-type and ae– plastid lysates were incubated with five units each of amyloglucosidase (EC 3.2.1.3, Sigma product number A7255, from Rhizopus) and α-amylase (EC 3.2.1.1, Sigma product number A2643, from porcine pancreas) for 20 min at 25 °C. Glucose released as a result of amyloglucosidase/α-amylase digestion of glucans was measured spectrophotometrically using a hexokinase/glucose 6-phosphate dehydrogenase-linked assay as described previously (Tetlow et al., 1994). Control experiments indicated that 0.1 mg glucan (glycogen, starch or amylopectin) could be completely digested under these conditions (data not shown). Immobilized S-tagged recombinant SS and SBE pull-down assay S-tagged recombinant SSI, SBEI, and SBEIIa were purified as described above. Approximately 120 μg of S-tagged recombinant protein, either SS or SBE, was immobilized to 120 μl of S-protein agarose (Novagen, catalogue no. 69704-4) according to the manufacturer's manual. The S-tagged (at the N-terminus) immobilized recombinant proteins and a parallel control (S-protein agarose without binding protein) were incubated with 1.5% (w/v) BSA at room temperature for 20–30 min in order to reduce non-specific binding of plastidial proteins to the column matrix. Agarose (with or without immobilized protein) was collected by low-speed centrifugation (500 g in a refrigerated microfuge) and then incubated with wild-type or ae– mutant amyloplast lysates individually at room temperature for 60 min. Each mixture was loaded onto a Bio-Rad Polyprep chromatography column (Bio-Rad, catalogue no. 731-1550) and washed with 250 ml washing buffer [20 mM TRIS-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) polyethylglycol p-t-octylphenol (Triton X-100)] to remove non-specifically bound proteins. The washed agarose bead pellet was then boiled in SDS-PAGE loading buffer, and the eluted proteins analysed by SDS-PAGE and immunoblotting. SDS-PAGE and immunoblotting Protein samples for SDS-PAGE and immnoblotting 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 4% to 12% BIS TRIS acrylamide gradient gels (Invitrogen Canada, catalogue no. NP0335BOX), following the manufacturer's instructions. Gradient gels were run at room temperature in a 3-(N-morpholino)-propanesulphonic acid (MOPS)-based running buffer prepared according to the manufacturer's (Invitrogen) instructions. 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 GBSSI, SSI, SSII, SBEI, SBEIIa, SBEIIb, Iso-1, and Iso-2 antibodies (mentioned above) were used at a dilution of 1:1000 times in 1.5% (w/v) BSA; the purified SP antibody was diluted 1:2000 in 1.5% (w/v) BSA. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) was used as a secondary antibody. Protein determination Protein was determined using the Bio-Rad protein assay (Bio-Rad Laboratories Canada) according to the manufacturer's instructions and using thyroglobulin as a standard. Mass spectrometry In-gel digestion with trypsin and preparation of peptides for MS were as described previously (Tetlow et al., 2008). Tandem electrospray mass spectra were recorded using a hybrid Q-TOF spectrometer (Micromass) interfaced to a Micromass CapLC capillary chromatograph as previously described (Tetlow et al., 2004). Results Immunological and enzymological characterization of plastid lysates from ae- maize mutants Immunoblots of amyloplast lysates probed with peptide-specific anti-maize SBEIIb antibodies showed an immunoreactive polypeptide of approximately 85 kDa in normal maize (Fig. 1A). Subsequent Q-TOF-MS analysis of the corresponding immunoreactive polypeptide identified the 85 kDa polypeptide as SBEIIb (data not shown), also confirming the specificity of these antibodies. Analysis of amyloplast lysates from ae– endosperm with anti-SBEIIb antibodies indicated that the SBEIIb protein is absent in this mutant (Fig. 1A). Analysis of immunoblots of amyloplast lysates probed with available antibodies raised against enzymes of starch synthesis showed that SSI, SSIIa, SBEI, SBEIIa, plastidial SP, Iso-1, Iso-2, and GBSSI were all present in the ae– mutant at levels comparable with that found in wild-type maize (Fig. 1A and Fig. 6C for GBSSI analysis). In addition, zymogram analyses were performed in order to determine the relative activities of the SBE isoforms in normal and ae– endosperm. Zymogram analysis of branching enzyme activity, coupled with immunodetection of isoforms in native polyacrylamide gels, clearly demonstrates the loss of catalytic activity in a region corresponding to SBEIIb in the ae– mutant (Fig. 1B). Further, the catalytic activity of SBEI in ae– amyloplasts was apparently reduced when compared with wild-type extracts (Fig. 1B). Analysis of the SBE zymogram in Fig. 1B also showed a reduction in SP activity (measured in the glucan-synthesizing direction) in ae– amyloplast extracts, despite equal amounts of SP protein present in wild-type and ae– amyloplasts as judged by immunoblot analysis (see also Fig. 1A). Fig. 1. Open in new tabDownload slide Immunological characterization of amyloplast lysates from wild-type and ae– maize. Amyloplast lysates (approximately 0.8 mg ml−1) were prepared from developing wild-type (wt) and ae– endosperms at 20–25 DAP (individual kernel fresh weight approximately 300 mg). 12 μg of soluble (stromal) proteins were loaded onto each gel lane and separated on 12% acrylamide gels, electroblotted onto nitrocellulose membranes, and developed with various peptide-specific anti-maize antibodies as shown in (A). Arrows indicate cross-reactions of each of the antibodies with its corresponding target protein; SBEIIb (approximately 85 kDa), SSI (approximately 74 kDa), SSIIa (approximately 76 kDa, but with a predicted mass of 85 kDa), SBEI (approximately 80 kDa), SBEIIa (approximately 90 kDa), SP (approximately 112 kDa), Iso-1 (approximately 80 kDa, but with a predicted mass of 90 kDa), and Iso-2 (approximately 90 kDa). MW, molecular mass markers. (B) Activity gel (zymogram) analysis of SBE isoforms and SP from whole cell extracts of wild-type and ae– endosperms (20–25 DAP). Approximately 100 μg 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 activities were visualized by staining with I2/KI. Identical gels were electroblotted onto nitrocellulose membranes and probed with peptide-specific anti-maize SBE and SP antisera, allowing the identification of specific SBE and SP activities from the zymogram. (C) Changes in catalytic activities of key amylopectin-synthesizing enzymes in ae– endosperm. The maximum catalytic activities of SS, SBE, and SP isoforms were measured in amyloplast lysates from wild-type (wt) and ae– endosperms at 20–25 DAP. Approximately 20 μg stromal proteins were used in each assay to measure total soluble SS, SBE, and SP activities (see Materials and methods). Results are the mean ±sem of 3–5 independent amyloplast preparations. The maximum catalytic activities of SS, SBE, and SP were measured in amyloplasts from ae-, and compared with amyloplast lysates from wild-type maize endosperm at equivalent stages of development (20–25 DAP) (Fig. 1C). Data in Fig. 1C compare total recoverable activities of SBE, SS, and SP in wild-type maize endosperm amyloplasts with ae– amyloplasts. Total branching enzyme activities in the ae– mutant was approximately half that of wild-type maize (P value <0.001), whereas measurable soluble starch synthase activity was significantly higher than in the wild type (P value <0.001). SP activity (measured in the glucan-synthesizing direction, as the incorporation of [U-14C]-glucose 1-phosphate into glycogen) was reduced in ae– amyloplasts (P value <0.001), consistent with the zymogram data shown in Fig. 1B. Gel permeation chromatography of starch-synthesizing enzymes from wild type and ae– mutants of maize Extracts of soluble proteins from maize amyloplasts were eluted through a Superdex 200 column. In wild-type extracts, the elution profiles for SSI and SSIIa were similar to those observed previously (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008). For each isozyme, two peaks of immunodetectable protein were found corresponding approximately to the native molecular sizes of the monomeric forms of SSI and SSIIa (75 kDa and 85 kDa, respectively), and also a high molecular weight (HMW) form of approximately 300 kDa which we have previously shown in maize corresponds to a complex between SSI, SSIIa, and SBEIIb (Fig. 2A, B; Hennen-Bierwagen et al., 2008). By contrast only a single peak of SBEI protein could be detected in wild-type maize, corresponding to the monomer of approximately 80 kDa (Fig. 2C). SP also eluted as a single HMW weight peak which probably corresponds to a tetrameric configuration of the 112 kDa subunit in wild-type maize (Mu et al., 2001) (Fig. 2D). In the ae– mutant, the elution profiles were similar to wild-type maize, although both SSI and SSIIa eluted earlier from the column than was the case with wild-type extracts, indicating that these proteins are components of larger complexes than those found in wild-type amyloplasts. Importantly, in ae– (but not in the wild-type) plastid lysates, SBEI eluted in a high molecular weight form, approximately coincident with the HMW forms of SSI and SSIIa (Fig. 2C). Fig. 2. Open in new tabDownload slide Fractionation of SSs, SBEs, and SP by size exclusion chromatography. Amyloplast extracts were prepared from wild-type (wt) and ae– endosperms at 20–25 DAP, and 0.5 ml of soluble proteins from these extracts (approximately 1.5–2 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 various peptide-specific anti-maize antibodies; anti-SSI (A), anti-SSIIa (B), anti-SBEI (C), and anti-SP (D). 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 (MW) for SDS-PAGE are indicated on the left side of each immunoblot. The position of each cross-reacting protein with the respective anti-maize antibody is marked as a horizontal arrow on the blots. (This figure is available in colour at JXB online.) Soluble SSs co-immunoprecipitate with different isozymes of SBE in wild-type and mutant plants Peptide-specific antibodies were used in reciprocal co-immunoprecipitation experiments to analyse protein–protein interactions among starch synthesizing enzymes in wild-type and ae– maize at 20–25 DAP. All of the antibodies used in co-immunoprecipitation experiments (anti-SSI, anti-SSIIa, and anti-SBEI) were able to recognize, and precipitate, their respective target protein (Fig. 3A–C). Figure 3 shows that antibodies to either SSI or SSIIa co-precipitated each other in all cases. In wild-type maize, SBEIIb was clearly co-immunoprecipitated by either SSI or SSIIa antibodies whereas neither SBEIIa, SBEI, nor SP were detected (Fig. 3A, B). Significantly, in the ae– mutant antibodies to either SSI or SSIIa also co-precipitated SBEI, SBEIIa, and SP (Fig. 3A, B). It is also notable that, in wild-type and ae– amyloplast lysates, no interactions were observed between SSs, SBEs, and Iso-1 or Iso-2 (Fig. 3A-C). Anti-SBEI co-immunoprecipitation experiments demonstrated the reciprocal associations predicted from the above (Fig. 3C). In wild-type maize amyloplast extracts, neither SSI, SSIIa, SBEIIa, nor SP was immunoprecipitated with antibodies to SBEI. Interestingly, SBEIIb was associated with SBEI in wild-type maize. In the case of the ae– mutant, antibodies to SBEI were clearly able to co-precipitate SSI and SSIIa. Co-precipitation of SBEIIa by SBEI antibodies was not observed in wild-type lysates, and only slightly in the ae– mutant. No co-immunoprecipitation of SBEIIa with SBEIIb antibodies was observed in wild-type amyloplast lysates (Fig. 5B). Preincubation of wild-type and ae– plastid lysates with glucan-degrading enzymes (amyloglucosidase and α-amylase) did not prevent co-immunoprecipitation of SS, SBE, and SP isoforms, which indicates that their association in each of the genotypes tested is due to specific protein–protein interactions, and not a result of SSs, SBEs or SP binding to a common glucan chain. Fig. 3. Open in new tabDownload slide Co-immunoprecipitation of stromal proteins from wild-type and ae– amyloplasts. 0.5–1 ml amyloplast lysates (0.8–1 mg protein ml−1) prepared from wild type and ae– endosperm 20–25 DAP were incubated with peptide-specific anti-SS and anti-SBE antibodies at 25 °C for 50 min, and then immunoprecipitated with Protein-A-Sepharose. The washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μl SDS-loading buffer and 30 μl loaded onto 4–12% pre-cast acrylamide gradient gels, electroblotted onto nitrocellulose, and developed with various anti-maize antisera as shown. (A) Co-immunoprecipitation using anti-SSI antibodies, (B) co-immunoprecipitation using anti-SSIIa antibodies, and (C) co-immunoprecipitation using anti-SBEI antibodies. Horizontal arrows indicate cross-reactions with the various antisera used: SSI at 74 kDa, SSIIa at 76 kDa, SBEI at 80 kDa, SBEIIa at 90 kDa, SBEIIb at 85 kDa, SP at 112 kDa, Iso-1 at 80 kDa, and Iso-2 at 90 kDa. The large band observed at approximately 50 kDa in all lanes is due to autorecognition of the IgG heavy chain. MW, indicates prestained molecular mass markers with their molecular masses shown on the left of the immunoblot. (This figure is available in colour at JXB online.) All of the protein–protein interactions observed between amyloplast proteins from wild-type and ae– endosperm at 20–25 DAP (see above) were also observed in endosperm at later stages of endosperm development (29–35 DAP). However, when co-immunoprecipitation studies were performed using earlier stages of development (9–12 DAP), no protein–protein interactions were detected in wild-type or ae– material (data not shown). Using recombinant proteins to study protein–protein interactions in wild-type and ae– amyloplasts Recombinant maize proteins were produced in Escherichia coli and used to study protein–protein interactions either through co-immunoprecipitation, or attached to a solid S-agarose support for affinity binding studies. Purified, catalytically active recombinant forms of maize SSI, SBEI, and SBEIIa were used in interaction experiments with wild-type and ae– amyloplast lysates (Fig. 4). Figure 4 shows the results obtained with recombinant proteins immobilized to S-agarose. All of the recombinant proteins used in the study retained their catalytic activity whilst attached to S-agarose beads, as determined by 14C-based SS and SBE assays described in the Materials and methods (data not shown). Anti-SSI, anti-SBEI, and anti-SBEIIa antibodies all recognized their respective recombinant proteins (Fig. 4A–C). For each of the recombinant proteins used in the affinity chromatography experiments, no corresponding native protein was observed in the washed beads, indicating that, in these experiments, no homomeric protein interactions occurred. Fig. 4. Open in new tabDownload slide Use of recombinant maize SSs and SBEs as affinity ligands to study protein–protein interactions in amyloplast lysates. 120 μg of catalytically active recombinant maize enzymes (S-tagged at the N-terminus of each protein) were immobilized onto S-protein agarose and incubated with 1 ml of either wild-type or ae– amyloplast lysates (0.8–1 mg protein ml−1) 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 SDS-loading buffer, proteins separated by SDS-PAGE using 4–12% acrylamide gradient gels, blotted onto nitrocellulose, and probed with peptide-specific anti-maize antibodies. (A) Recombinant maize SSI immobilized to S-protein agarose. (B) Recombinant maize SBEI immobilized to S-protein agarose. (C) Recombinant maize SBEIIa immobilized to S-protein agarose. Horizontal arrows indicate cross-reactions with the various antibodies used. MW, indicates molecular mass markers with their molecular masses shown on the left of the blot. (This figure is available in colour at JXB online.) Fig. 5. Open in new tabDownload slide Phosphorylation-dependent protein–protein interactions in maize endosperm amyloplasts. Wild-type and ae– amyloplasts (20–25 DAP) were preincubated with either 1 mM ATP or 25 units E. coli alkaline phosphatase (APase) for 30 min at 25 °C. Non-treated samples were incubated in an equivalent volume of rupturing buffer. Following preincubation, samples were then incubated with peptide-specific anti-SSIIa (A) and anti-SBEIIb (B) antibodies at 25 °C for 50 min, and then immunoprecipitated with Protein-A-Sepharose. The washed Protein-A-Sepharose antibody–antigen complexes were boiled in 200 μl SDS-loading buffer and 30 μl loaded onto 4–12% precast acrylamide gradient gels, electroblotted onto nitrocellulose, and developed with various anti-maize antisera as shown. Horizontal arrows indicate cross-reactions with the various antisera used: SSI at 74 kDa, SSIIa at 76 kDa, SBEI at 80 kDa, SBEIIa at 90 kDa, SBEIIb at 85 kDa, and SP at 112 kDa. The large band observed at approximately 50 kDa in all lanes is due to autorecognition of the IgG heavy chain. MW, indicates molecular mass markers with their molecular masses shown on the left of the immunoblot. (C) Autoradiography of 32P-labelled phosphoproteins immunoprecipitated with anti-maize SSIIa antibodies from wild-type and ae– amyloplast stroma. Amyloplasts were incubated with 1 mM [γ-32P]-ATP for 30 min at 25 °C, and then incubated with anti-maize SSIIa antibodies as described in the Materials and methods. Arrows shown on the autoradiograph indicate phosphoproteins co-precipitating with SSIIa which were identified by immunoblot analysis with various maize antisera: a, SBEI; b, SP; and c, SBEIIb. MW indicates the positions of molecular mass markers (shown as bands) with their corresponding masses in kDa. (This figure is available in colour at JXB online.) Fig. 6. Open in new tabDownload slide Analysis of starch granule bound proteins from wild-type and ae– amyloplasts. Starch granules were isolated from amyloplasts at 20–25 DAP and washed extensively to remove proteins loosely bound to the granule surface. 40 mg of purified starch was boiled in 600 μl SDS-loading buffer and 35 μl of the supernatant from the boiled sample loaded onto gels. (A) Granule bound proteins were separated by SDS-PAGE using 4–12% acrylamide gradient gels and visualized by silver staining. Horizontal arrows indicate polypeptides which were excised and identified by Q-TOF-MS analysis. MW, indicates molecular mass markers with their molecular masses shown on the left of the gel. (B) Silver-stained polypeptides were excised from the polyacrylamide gels in (A) and digested with trypsin, and the recovered peptides sequenced using Q-TOF-MS to identify polypeptides from wild-type and ae– starch granules. The identity of presumptive proteins is shown in bold. The peptide sequences presented for each polypeptide analysed and identified were acquired from a single representative in-gel digest. (C) Starch granule proteins separated by SDS-PAGE were also subjected to immunoblot analysis using various peptide-specific anti-maize antibodies. Horizontal arrows indicate cross-reactions with the various antibodies used. MW, indicates molecular mass markers with their molecular masses shown on the left of the blot. (This figure is available in colour at JXB online.) SSIIa and SBEIIb from wild-type amyloplast lysates bound to recombinant maize SSI attached to S-agarose beads (Fig. 4A), showing the same interaction observed in the co-immunoprecipitation experiments (Fig. 3). No interaction was observed between recombinant SSI and SBEI, SBEIIa, or SP from wild-type amyloplast lysates (Fig. 4A). By contrast, when amyloplast lysates from ae– maize were incubated with the recombinant maize SSI, in addition to interacting with SSIIa, the recombinant SSI also interacted with SBEI, SBEIIa, and SP (Fig. 4A). It can be noted from the immunoblot shown in Fig. 4A that when ae– lysates were incubated with recombinant SSI, two proteins showing cross-reactivity with the anti-SBEIIa antisera, and which were close in molecular mass to the expected size of SBEIIa, were present in the washed beads containing the recombinant SSI. MS analysis of the lower of these protein bands (approximately 90 kDa, and marked with an arrow on Fig. 4A) showed that the protein was SBEIIa (data not shown). Similar experiments using recombinant maize SBEI attached to S-agarose beads indicated interaction only with SBEIIb in wild-type maize plastid lysates (Fig. 4B). However, when using amyloplast lysates from the ae– mutant, SSI, SSIIa, SBEIIa, and SP interacted with recombinant maize SBEI (Fig. 4B). When wild-type amyloplast lysates were incubated with recombinant maize SBEIIa no interactions were observed with other enzymes of starch synthesis (Fig. 4C). Using ae– plastid lysates, SSI, SSIIa, SBEI, and SP all interacted with the recombinant maize SBEIIa (Fig. 4C). Results obtained by using unbound recombinant proteins were identical to those in which the protein was immobilized to S-agarose (data not shown). The effects of protein phosphorylation on protein–protein interactions ATP and alkaline phosphatase (APase) were employed in co-immunoprecipitation experiments similar to those described in Fig. 3 to test whether the formation of the protein complexes was influenced by protein phosphorylation. In all cases, treatment with ATP or APase did not affect the ability of any of the antibodies used to bind to, and precipitate, their respective target protein (Fig. 5A, B). In wild-type amyloplast lysates, interactions between SSIIa and SSI, and SSIIa and SBEIIb (as determined by immunoprecipitation with anti-SSIIa antibodies) were strongly enhanced by pre-incubation of lysates with 1 mM ATP, and reduced to nearly undetectable levels following dephosphorylation with APase (Fig. 5A, B). Addition of a protein phosphatase inhibitor cocktail (Sigma) to ATP-treated plastid lysates caused no increase in co-immunoprecipitation of the interacting proteins (data not shown). Importantly, the phosphorylation-controlled formation of protein complexes in wild-type amyloplast lysates was shown to be reversible. An insoluble form of APase attached to agarose beads was used to dephosphorylate proteins in amyloplast lysates. Following removal of the insoluble APase, protein complexes were re-formed by the addition of 1 mM ATP to the plastid lysates following further incubation for 30 min at 25 °C (Fig. 5A, lane marked APase, +ATP). In wild-type maize, no interactions were observed between SSIIa and SBEI (Fig. 5A) or SSIIa and SP (data not shown), irrespective of pretreatment with ATP or APase. When antibodies to SBEIIb were employed as the precipitating agent, reciprocal, phosphorylation-dependent interactions were observed between SBEIIb and SSIIa, and SSI (Fig. 5B). In addition, SBEIIb was shown to interact with SBEI. As with the other protein–protein interactions observed in wild-type amyloplast lysates, this interaction was enhanced by treatment with ATP and eliminated following incubation with APase (Fig. 5B). No interaction was observed between SBEIIb and SP. Surprisingly, when ae– amyloplast lysates were used in the co-immunoprecipitation experiments described in Fig. 5, the interactions observed between SSI, SSIIa, and SBEI were unaffected by pretreatment with either ATP or APase (Fig. 5A). Conceivably, the lack of a response comparable to the wild type could be due to the experimental conditions and the novel complexes observed in the ae– mutant may be less accessible to the different pretreatments in vitro. In order to determine whether the proteins within the complexes described are directly phosphorylated, wild-type and ae– amyloplast lysates were incubated with 1 mM [γ32P]-ATP, and anti-SSIIa antibodies used to immunoprecipitate the protein complexes. Figure 5C shows that, in wild-type amyloplasts, 32P-labelled SBEIIb co-precipitated with SSIIa and, in ae– plastid lysates, SSIIa co-precipitated phosphorylated SBEI and SP. No 32P-labelling of SSI or SSIIa was detected in the immunoprecipitates from either wild-type or ae– amyloplast lysates. A number of non-specifically 32P-labelled proteins were also observed within the immunocomplexes (Fig. 5C). Previous work suggests that many of these proteins are components of the immunoglobulins and serum proteins, as well as unidentified plastidial phosphoproteins associating with the immunocomplexes or their target proteins. No further analysis or identification of these proteins was undertaken in the present study. Analysis of granule-associated proteins Proteins remaining attached to starch granules following extensive washing with buffer, SDS, and acetone are termed granule-associated proteins (Rahman et al., 1995; Mu-Forster et al., 1996). The granule-associated proteins of wild-type and ae– maize endosperms were analysed by SDS-PAGE and immunoblotting (Fig. 6). Many of the silver-stained granule-associated proteins were identified by mass spectrometry (MS, Fig. 6B), showing that GBSSI, SSI, and SSIIa were present in both wild-type and ae– starch granules, SSIIa at significantly higher levels in ae– granules. SBEIIb was found to be granule-associated in wild-type starch, but was not present in ae– granules (see also Fig. 6C). Silver-stained polyacrylamide gels of granule-associated proteins showed a number of additional proteins associated with ae– starch, that were either not present, or not detectable by MS analysis in wild-type granules (Fig. 6A). These included SBEI, SBEIIa, and SSIII. In addition, an abundant polypeptide of approximately 110 kDa was present exclusively in ae– starch granules. Starch granule-associated proteins were further analysed by immunoblotting using available antibodies (Fig. 6C). Immunoblot analysis of granule-associated proteins essentially confirmed the proteomic analysis described in Fig. 6A and B. GBSSI, SSI, SSIIa, and SBEIIb were present in wild-type starch granules, whereas ae– starch granules lacked SBEIIb but contained SBEI and SBEIIa. The 110 kDa polypeptide associated with ae– starch granules cross-reacted with anti-maize SP antibodies (Fig. 6C), although it was not possible to identify this polypeptide using MS. Immunoblot experiments using anti-maize Iso-1 antibodies indicated that Iso-1 was not present as a starch granule-associated protein in either wild-type or ae– endosperm. Discussion This paper presents a detailed biochemical characterization of the amylose extender (ae–) mutant of maize, and provides new insights which contribute to an understanding of the observed starch phenotype of the mutant, not only in terms of loss of a specific branching enzyme isoform, but also in terms of alterations in the interactions between components of the starch biosynthetic machinery within the amyloplast. In addition, it has been demonstrated that the assembly of heteromeric protein complexes in the wild type and the mutant involves protein phosphorylation, and is reversible. The observations suggest that the association of specific proteins within the growing starch granule is a reflection of the formation of stromal, functional heteroprotein complexes involved in amylopectin biosynthesis. More detailed aspects of the data and their significance are discussed below. Western blot analysis of native- and SDS-PAGE gels of soluble proteins from amyloplast lysates show that the ae– mutant used in this study lacks the SBEIIb protein, whilst the protein levels of SSI, SSIIa, Iso-1, Iso-2, plastidial SP, and the other forms of SBE were unaltered compared with the wild type. The combined maximum catalytic activity of the major (soluble) classes of enzymes involved in amylopectin biosynthesis were measured in amyloplast lysates and revealed marked changes in the activities of these enzymes in the ae– mutation. In particular, the ae– mutation caused a significant increase in the measurable soluble activity of SS, indicating that one or more isoforms of SS is activated in the mutant (despite protein levels of SSI and SSIIa being unchanged), and significant reductions in both SBE and SP activities. These observations contrast with previous studies with ae– mutants of maize and rice which report a decrease in soluble SS activity (in particular, SSI activity) in whole cell extracts of ae– endosperm compared with wild-type extracts (Boyer and Preiss, 1978; Nishi et al., 2001). In addition, Nishi et al. (2001) observed no change in SP activity in the ae– endosperm of rice compared with the wild type. Analysis of the SBE zymogram (Fig. 1B) indicates that loss of SBEIIb may not be the sole cause of the reduction in overall soluble branching enzyme activity, as SBEI showed a visible reduction in measurable catalytic activity (see zymogram analysis) compared with the wild type, despite there being no difference in SBEI protein levels (as judged by native and denaturing Western blots). It should be noted, however, that the methods employed for the estimation of SBE activity (zymogram analysis and the phosphorylase a-stimulation assay) are not quantitative, and only provide a guide to relative activities of these enzymes. Previous studies with ae– maize have also reported reduced SBEI activity compared with wild-type activities (Colleoni et al., 2003). Recent analysis of the ‘high-amylose’ (Amo1) barley mutant showed similar alterations to key enzyme activities during the major stages of starch deposition as seen with the ae– mutant described in this study; namely marked reductions in the catalytic activities of SBEII and SP isoforms (Borén et al., 2008). Co-immunoprecipitation experiments with wild-type maize amyloplast lysates showed that SSI, SSIIa, and SBEIIb (but not SBEIIa) form a protein complex as has been observed previously (Hennen-Bierwagen et al., 2008). A similar protein complex of 260–300 kDa was identified in wheat amyloplasts, comprising SSI, SSIIa, and SBEIIa (the major form of SBEII in wheat and barley), or SSI, SSIIa and SBEIIb (Tetlow et al., 2008). Co-immunoprecipitation analysis of the ae– mutant revealed novel protein interactions involving SSI, SSIIa, SBEI, SBEIIa, and SP, which were not observed in wild-type maize plastids, or those of wheat (Tetlow et al., 2008). For example, anti-SBEI antibodies specifically immunoprecipitated SSI, SSIIa, SBEIIa, and SP in ae– plastid lysates and recombinant SBEI demonstrated the same interactions. The affinity chromatography experiments, using recombinant SBEIIa, and the co-immunoprecipitation studies, indicate that, in the absence of SBEIIb, SBEIIa is also able to interact with SSI, SSIIa, SBEI, and SP. In the wild type, SBEIIb is expressed at approximately 50 times the level of SBEIIa (Gao et al., 1997) and mutational analysis suggests that either SBEIIa does not play a critical role in amylopectin biosynthesis in maize endosperm, or that other SBE isoforms can compensate for its loss (Blauth et al., 2001). However, in the ae– mutant, the data indicate that SBEIIa is now able to compete more effectively for interaction domains involved in protein complex formation. Co-immunoprecipitation and affinity chromatography with recombinant SBEIIa and SBEI also indicate that SBEIIa, SBEI, and SP all potentially interact with each other in the ae– mutant, but not in wild-type amyloplasts. It is feasible that SBEIIa/SBEI/SP may exist as a discrete protein complex analogous to the SBEIIb/SBEI/SP complex observed in wheat endosperm (Tetlow et al., 2004). However, a general point when considering protein–protein interactions is that the analyses do not allow the stoichiometric relationship between different components of the complexes to be determined. Based on the data presented here, the novel protein interactions found in the ae– mutant could describe a single unit composed of SSI, SSIIa, SBEI, SBEIIa, and SP, but the possibility cannot be excluded that SSI and SSIIa form separate, combinatorial, tri- or tetrameric assemblies with SBEI, SBEIIa, and SP. Gel permeation chromatography experiments showed that SBEI in the mutant eluted in fractions which indicated that it was larger than the predicted monomeric mass (80 kDa) observed in the wild type. Recent analysis of ae– endosperm extracts by Hennen-Bierwagen et al. (2009) did not find evidence for SBEI in a multi-protein assembly. However, those studies were limited to the analysis of elution profiles following gel permeation chromatography with respect to studies of SBEI in the ae– mutant, and it may be that under these conditions SBEI-containing complexes are not as stable. Elution profiles of SP from wild-type and ae– endosperm extracts were similar, despite SP being present in high molecular mass protein complexes with isoforms of SS and SBE in the ae– mutant (Fig. 2D). Phosphorylases from various sources can be found as homotetrameric or homodimeric assemblies (Nakano and Fukui, 1986; Albrecht et al., 1998; Buchbinder et al., 2001). The broad elution profiles of SP from gel permeation chromatography observed here for both maize genotypes are consistent with a homotetrameric form of SP in the wild type and, possibly, also the mutant. The likely explanation of this is that in the ae– mutant, SP is present in heteroprotein complexes as a lower complexity multimer or monomer, and/or that the heterocomplexes account for only a small proportion of the SP protein detected following fractionation. In wild-type amyloplasts, SBEI was shown to interact only with SBEIIb, which has been noted previously in wheat endosperm (Tetlow et al., 2004). The presence of an SBEI/SBEIIb heterodimer was not apparent in gel permeation chromatography (Fig. 2C) as only monomeric SBEI was detected in wild-type plastid lysates. This may suggest that this protein complex assembly is of relatively low abundance or is less stable. Analysis of protein complexes at different stages of endosperm development indicate that, once formed, the various protein complexes are present in the amyloplast until relatively late in development (up to 35 DAP). However, at the early stages of maize endosperm development (9–12 DAP), notably when rates of starch synthesis are very low (Tsai et al., 1970), no protein complexes were detected in either genotype. This finding is in agreement with recent data from developing wheat endosperm (Tetlow et al., 2008) in which protein complexes comprising SS and SBE isoforms could not be detected earlier than 10 DAP. Importantly, the results also show that the interactions between SSs and SBEs in wild-type maize amyloplasts are phosphorylation-dependent. Previously, it has been shown that assembly of a similar protein complex consisting of SSs and SBEs in wheat endosperm amyloplasts is driven by protein phosphorylation (Tetlow et al., 2004, 2008), indicating a more general role for this regulatory process in the formation of protein complexes in cereal amyloplasts. Recent work by Hennen-Bierwagen et al. (2009) has shown that a large protein complex (of approximately 670 kDa) in maize endosperm involving SSIII interacting with SSIIa, SBEIIa, and SBEIIb is assembled in a phosphorylation-dependent manner. Whilst the stability of the novel protein–protein interactions observed here in ae– endosperm amyloplasts was unaffected by pretreatment with either ATP or phosphatase (Fig. 5A), experiments with maize amyloplasts using [γ32P]-ATP showed that components of protein complexes in the wild type and ae– (precipitated by anti-maize SSIIa antibodies) were phosphorylated. Phosphorylated SBEIIb was detected in the wild type, and in the ae– protein complex SBEI and SP were also labelled with 32P (Fig. 5C). Labelling of SBEIIa was not detected but cannot be excluded given the relatively low abundance of this protein. Analysis of starch granule-associated proteins (i.e. those proteins strongly bound to, or locked within the granule following extensive detergent and solvent treatment) revealed marked alterations in the complement of granule-associated proteins in the ae– mutant, in addition to the loss of SBEIIb. A core group of granule-associated proteins was detected in wild-type granules using mass spectrometry and immunoblot analyses as tools for identification. These proteins (SSI, SSIIa, SBEIIb, and GBSSI) are routinely found as granule-associated proteins in the starch granules of other cereals such as wheat, barley, and rice which also contain SBEIIa as a granule-associated protein (Rahman et al., 1995; Morell et al., 2003; Borén et al., 2004; Regina et al., 2005; Umemoto and Aoki, 2005). Notably, all of the granule-associated proteins known to be involved in amylopectin synthesis are also components of identified soluble protein complexes in the endosperms of wheat and maize (Hennen-Bierwagen et al., 2008; Tetlow et al., 2008). However, in addition to this core group of proteins, ae– starch granules also contained SBEI, SP, SBEIIa, and SSIII. Similar alterations in the composition of starch granule-associated proteins in maize mutants, including ae–, have recently been described by Grimaud et al. (2008). Consistent with the data shown in Fig. 5C, Grimaud et al. (2008) also found that SP was phosphorylated in the starch granules of the ae– mutant. Together, these observations reinforce the point that the regulation of the formation of stromal protein complexes and their subsequent deposition within granules involves protein phosphorylation. The amylopectin-synthesizing enzymes found as granule-associated proteins in the starch granules of cereal endosperms are all involved in the synthesis of short- to intermediate-length glucan chains which are known to form clusters, resulting in semi-crystalline lamellae (Ball and Morell, 2003). The gradual, periodic synthesis of amylopectin clusters joined via amorphous lamellae as proposed by the French (1984) and Hizukuri (1986) models has been suggested to be the cause of entrapment of proteins (specifically, starch biosynthetic enzymes) within granules. However, detailed kinetic analysis of some SS isoforms suggests that granule-association of these proteins, at least, may be a product of their increased affinity for longer glucan chains during catalysis (Commuri and Keeling, 2001). Analysis of granule-associated proteins in higher plants reveals that only a specific group of amylopectin-synthesizing proteins are consistently observed within granules (SSI, SSIIa, and isoforms of SBEII), whereas other enzyme classes, which likely play an important role in amylopectin biosynthesis are either absent (SSIV, SBEI, SP, isoforms of Iso, pullulanase-type DBE, and D-enzyme) or are found in the granule at very low levels (SSIII). SSIII and SBEI, respectively, elongate and branch relatively long glucan chains (Gao et al., 1998; Blauth et al., 2002), and it has been proposed that these enzymes function in glucan chain formation between clusters in the amorphous region of the granule (Nakamura, 2002; James et al., 2003). The operation (and co-operation) of SSI, SSIIa, and SBEII isoforms (working as one or more protein complexes) in semi-crystalline cluster formation, and SSIII and SBEI (probably working with other SSs and SBEs) involved in the synthesis of the cluster-connecting glucan chains in the amorphous zones fits well with the ‘two-step branching and improper branch clearing’ model proposed by Nakamura (2002). In this model, glucan extension and branching activities occur in both the cluster and amorphous regions of the granule. During the whole process DBEs play critical roles in trimming the cluster shape at the periphery of the growing granule, since they act on sparsely localized branch points more rapidly than those in the densely packed semi-crystalline lamellae (Ball et al., 1996; Mouille et al., 1996; Nakamura et al., 1997). Consequently, those enzymes involved in the synthesis of the amorphous region, and the trimming process remain as soluble proteins in the amyloplast stroma, or are removed during the washing of starch granules. Analysis of the ae– mutant strengthens the notion that stromal protein complexes are involved in α-polyglucan cluster formation, since the ae– complex's novel enzyme complement also becomes granule localized. The protein–protein interactions observed in ae– maize amyloplasts also help explain the unique starch phenotype of this mutant. In ae– amyloplasts, SSI and SSIIa are associated with SBEI and SP (and SBEIIa). SBEI is known to have a high affinity for relatively long glucan chains (including amylose) (Guan and Preiss, 1993; Takeda et al., 1993). Thus the replacement of SBEIIb by SBEI in the ae– mutant protein complexes would lead to branch points with relatively longer glucan chains than within the clusters of the wild type. The role of SP in the mutant complexes is unclear, as is the general role for this enzyme in starch synthesis. If SP plays a catalytic role within the protein complex, it might be speculated that it is involved in trimming the longer glucan chains branched by SBEI activity in order to maintain a constant cluster size. A trimming role for SP in amylopectin biosynthesis was proposed following studies with an SP-deficient mutant of Chlamydomonas reinhardtii (Dauvillée et al., 2006). Such a role could explain the fact that peak chain lengths in wild-type and ae– starches are equivalent (Nishi et al., 2001; Klucinec and Thompson, 2002). In summary, this study has demonstrated that the complement of starch biosynthetic enzymes found in granules is a reflection of soluble, phosphorylation-dependent, protein complexes formed in the stroma of the amyloplast. We hypothesize that these complexes are the functional components involved in amylopectin cluster formation and become entrapped within the semi-crystalline lamellae during synthesis (see Fig. 7). Analysis of the maize ae– mutant shows that the loss of a key component of the amylopectin-synthesizing machinery (SBEIIb) results in some compensation by the inclusion of SBEI, SBEIIa, and SP within the glucan cluster-forming protein complexes, contributing to the altered phenotype of the amylopectin component and their entrapment as granule-associated proteins. Further biochemical analysis of other mutants will prove valuable in explaining the various starch phenotypes and the specific roles of heteroprotein complexes. Fig. 7. Open in new tabDownload slide Summary of novel protein–protein interactions formed between amylopectin-synthesizing enzymes in maize endosperm following loss of SBEIIb in the ae- mutation. In wild-type maize endosperm (A) the major form of SBEII (SBEIIb) forms a phosphorylation-dependent protein complex with SSI and SSIIa. Dephosphorylation with APase causes dissociation of this protein complex assembly. In the ae– mutant (B) the SSI/SSIIa core recruits SBEI, SBEIIa, and SP in place of SBEIIb via unknown mechanisms. The figure shows these proteins as a single protein complex, but the data are also consistent with the existence of smaller complexes made up of different combinations of these proteins (see text). None of the interactions observed in the ae– amyloplasts are noticeably affected by treatment with ATP or APase. In vitro [γ32P]-ATP-labelling experiments with amyloplasts showed phosphorylation of SBEIIb in the wild type, and phosphorylation of SBEI, and SP in the ae– mutant (denoted by ‘P’ labels in the diagram). The involvement of protein phosphorylation in the complex assembly in the ae– mutant is as yet unclear. For each of the genotypes, the components of the protein complexes found in the plastid stroma are also found in the respective starch granules. It is proposed that the protein–protein interactions summarized in Fig. 7 are involved in assembly of the clusters of short- to intermediate-sized glucan chains which form the semi-crystalline lamellae of starch granules within which the proteins become entrapped. Abbreviations Abbreviations ADP-Glc ADP-glucose ae– amylose extender AGPase adenosine 5′ diphosphate glucose pyrophosphorylase APase alkaline phosphatase DAP days after pollination DBE debranching enzyme D-enzyme disproportionating enzyme DTT dithiothreitol GBSSI granule-bound starch synthase Glc glucose Glc-1P α-d-glucose 1-phosphate Iso isoamylase MS mass spectrometry NP-40 nonyl phenoxylpolyethoxyl ethanol PMSF phenylmethyl sulphonyl fluoride SBE starch branching enzyme SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SP starch phosphorylase SS starch synthase Triton X-100 polyethylglycol p-t-octylphenol This work was supported by the Ontario Ministry of Agriculture and Food Bio-Products Research Grant (project no. 026262 to MJE and IJT), Natural Sciences and Engineering Research Council (NSERC) Discovery Grants (no. 262209 to MJE, and no. 400167 to IJT), and a NSERC Strategic Grant (no. 048237 to MJE and IJT). 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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/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) © 2009 The Author(s).
TI - The amylose extender mutant of maize conditions novel protein–protein interactions between starch biosynthetic enzymes in amyloplasts
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erp297
DA - 2009-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-amylose-extender-mutant-of-maize-conditions-novel-protein-protein-MRCz0emNXN
SP - 4423
EP - 4440
VL - 60
IS - 15
DP - DeepDyve
ER -