Maize Endosperm ADP–Glucose Pyrophosphorylase SHRUNKEN2 and BRITTLE2 Subunit Interactions

Correspondence to: L. Curtis Hannah, hannah{at}gnv.ifas.ufl.edu (E-mail), 352-392-5653 (fax).

	ABSTRACT

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ADP–glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.

	INTRODUCTION

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Biochemical and molecular analyses of the many nonallelic starch mutants of maize have provided seminal insights into the path of starch biosynthesis (reviewed in Creech 1965 ; Tsai et al. 1970 ; Nelson and Pan 1995 ; Hannah 1997 ). Shrunken2 (Sh2) and Brittle2 (Bt2) are two loci originally identified by their starch-deficient kernel phenotype (Tsai and Nelson 1966 ; Dickinson and Preiss 1969 ). Biochemical and molecular characterization of Sh2 and Bt2 clearly identified them as encoding the large and small subunits of maize endosperm ADP–glucose pyrophosphorylase (AGP; Hannah and Nelson 1976 ; Hannah et al. 1980 ; Bae et al. 1990 ; Bhave et al. 1990 ). Subsequent analysis of AGP from a multitude of plant tissues has revealed that all plant AGPs consist of two similar but distinct subunits that interact and polymerize into the native 2ß2 heterotetrameric enzyme structure (Smith-White and Preiss 1992 ). This structural complexity characteristic of higher plant AGP differs significantly from that of AGP isolated from prokaryotic organisms. These latter AGPs are more simple in structure, being composed of four identical subunits (reviewed in Preiss and Romeo 1994 ; Preiss and Sivak 1996 ).

AGP catalyzes a key metabolic step in the synthesis of starch in higher plants and glycogen in bacteria (reviewed in Preiss 1991 ; Preiss and Sivak 1996 ). Enzymatic activity is activated by 3-phosphoglycerate and inhibited by Pi in the leaves and sink tissues of most but not all organs of higher plants.

That AGPs from bacteria and higher plants catalyze an analogous reaction allowed the development of a bacterial expression system to study higher plant AGP (Iglesias et al. 1993 ). Recently, this heterologous bacterial expression system was utilized to elucidate important regions for substrate and allosteric binding (Greene et al. 1996a , Greene et al. 1996b ; Laughlin et al. 1998a , Laughlin et al. 1998b ; Okita et al. 1998 ).

In contrast, little information is available concerning the mechanism of assembly of higher plant AGPs. Work by Giroux and Hannah 1994 clearly shows that the stability of the SH2 or BT2 protein is dependent on the presence of the other subunit. Endosperm extracts from sh2 null mutants showed an increased lability of the BT2 protein. Similarly, the SH2 protein is more labile in bt2 mutants. Thus, subunit interaction is critical for the overall stability of the maize endosperm AGP as well as the individual subunits. A similar situation has been reported for Arabidopsis mutants deficient in AGP. Early work by Lin et al. 1988a , Lin et al. 1988b identified two classes of starch-deficient mutants in Arabidopsis. The adg1 mutant lacked both the large and the small subunit (Lin et al. 1988a ). In contrast, the adg2 mutant lacked only the large subunit, although it exhibited reduced levels of the small subunit (Lin et al. 1988b ). Recent molecular characterization of the adg2 mutant revealed that a missense mutation in the large subunit was responsible for the lowered AGP activity (Wang et al. 1997 ). Wang et al. proposed that the absence of the large subunit results from its inability to interact with the small subunit, rendering it highly unstable. This is supported by evidence that the adg1 mutant represents a mutation in the small subunit of AGP (Wang et al. 1997 ).

Because of the importance of the subunit interactions in AGP activity, we have focused directly on systems to more precisely define motifs necessary for assembly. Expression of SH2 and BT2 in the yeast two-hybrid system and analysis of null sh2 and bt2 mutants point to the fact that motifs necessary for AGP assembly are located throughout each subunit.

	RESULTS

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Yeast Two-Hybrid Expression
Translational fusion of SH2 and/or BT2 to the activation domain (AD) and/or binding domain (BD) of the Gal4 transcription factors allows us to directly study SH2 and BT2 protein interactions. Such interactions are monitored via the formation of a functional Gal4 activator and subsequent transcriptional activation of the ß-galactosidase (ß-gal) reporter gene driven by the Gal4 promoter (Fields and Song 1989 ). Protein interactions can be visualized using the colorimetric X-gal assay. Furthermore, the strength of the protein interaction can be quantitated using ß-gal enzymatic assays.

Results from the cloning and expression of SH2 and BT2 proteins in the yeast two-hybrid system show that these proteins interact. Y190 yeast cells containing the various constructs were selected by growth on a synthetic medium lacking Leu and Trp (Figure 1A). Protein interactions are visualized easily using the X-gal filter assay (Figure 1B). This assay is highly sensitive, and the colorimeteric change can be detected within 2 hr of incubation at 30°C. This is a rapid response when compared with the 1.5 hr needed to see staining with the pVA3/pTD1 positive control. Additional controls were run to confirm that the positive X-gal staining phenotype was reflective of a SH2:BT2 interaction and not an artifact of the system. As seen in Figure 1B, we detected a positive-staining phenotype in all possible combinations of full-length SH2 and BT2. Thus, a strong interaction occurs regardless of which subunit was fused to the AD or the BD.

View larger version (73K): [in this window] [in a new window]   Figure 1. Yeast Two-Hybrid Analysis of SH2 and BT2 Interactions. (A) and (D) Yeast two-hybrid expression of the maize endosperm AGP SH2 and BT2 subunits was used to monitor SH2:BT2 as well as SH2:SH2 and BT2:BT2 interactions. Y190 yeast cells expressing the designated plasmids are selected for on a synthetic growth medium without Leu and Trp. (B) and (E) X-gal filter lift assays of plates in (A) and (D) allow the visual identification of SH2 and BT2 interactions. pVA3/pTD1 and pGAD424/pGBT9 are included as positive and negative controls. In (B), only yeast cells expressing pBT2-BD/pSH2-AD and pBT2-AD/pSH2-BD give a positive X-gal staining phenotype indicative of protein–protein interactions. pSH2-AD/pGBT9, pSH2-BD/pGAD424, pBT2-AD/pGBT9, and pBT2-BD/pGAD424 are included as controls and show the lack of X-gal staining with the expression of a single subunit. In (E), lack of any detectable X-gal staining with Y190 cells expressing pSH2-AD/pSH2-BD and pBT2-AD/pBT2-BD indicates that there is no SH2:SH2 or BT2:BT2 interaction in this system. Figure 1. (continued). (C) and (F) Growth of yeast cells expressing the designated plasmids in (A) and (C) on a synthetic medium without Leu, Trp, and His plus 30 mM 3-AT provides an additional selection for protein–protein interaction. In (C), only yeast cells expressing the positive control, pVA3/pTD1, or the SH2:BT2 combinations are able to grow, adding additional support for protein–protein interaction between SH2 and BT2 subunits. In (F), no growth of yeast cells expressing SH2:SH2 and BT2:BT2 combinations on this medium confirms the lack of homosubunit interaction in this system.

A His biosynthetic gene fused to a promoter that also is activated by a functional Gal4 transcriptional activator provides a second marker for protein–protein interactions in Y190 yeast cells. Y190 cells expressing the various combinations of SH2 and BT2 were grown on a synthetic medium lacking Trp, Leu, and His and containing 30 mM 3-amino-1,2,4-triazole (3-AT). Only yeast cells expressing the positive control or SH2 and BT2 together exhibit growth (Figure 1C). Our results show that SH2 and BT2 interactions can be monitored using the yeast two-hybrid system.

ß-Gal assays of crude extracts of yeast cells expressing SH2 and BT2 allowed quantification of the SH2 and BT2 interaction (Figure 2). SH2:BT2 interaction led to 6.78 µmol/min of ß-gal activity or 34% (19.98 µmol/min of ß-gal activity from plasmids pVA3/pTD1) of the positive control.

View larger version (16K): [in this window] [in a new window]   Figure 2. The Strength of SH2 and BT2 Interactions Measured by ß-Gal Enzyme Assays. ß-Gal assays of extracts from yeast cells expressing pBT2-BD/pSH2-AD were used to quantitate the SH2:BT2 interaction compared with the positive control, pVA3/pTD1. Values are the result of two independent assays. Standard errors are shown. Yeast cells expressing pBT2-BD/pSH2-AD exhibit a high level of ß-gal activity (34% of the positive control) and clearly show that there is a strong interaction between the SH2 and BT2 subunits of maize endosperm AGP.

Expression of the potato small subunit alone in Escherichia coli yielded a catalytically active AGP (Ballicora et al. 1995 ). Compared with the heterotetramer, the homopolymeric AGP required higher levels of 3-phosphoglycerate for activation and was more sensitive to inhibition by Pi. This observation provided initial support for the notion that the small subunit is the catalytic subunit. Accordingly, we expressed the BT2 protein on both plasmids (pBT2-AD and pBT2-BD) to directly test whether BT2 subunits could interact in the yeast two-hybrid system (Figure 1D). Expression of ß-gal (Figure 1E) and growth on a synthetic medium lacking Trp, Leu, and His and containing 30 mM 3-AT (Figure 1F) were monitored. No interaction was noted, as evidenced by the negative X-gal staining phenotype and lack of growth on the minus Trp, Leu, and His selection media containing 30 mM 3-AT. Likewise, SH2 was expressed on both plasmids (Figure 1D), and no evidence for interaction was noted (Figure 1E and Figure 1F). The two-hybrid data suggest that the individual subunits do not interact and that the early steps in the polymerization involve a heterodimer intermediate. However, it should be noted that the orientation of the AD and BD fusions to SH2 or BT2 may affect their ability to interact in the N-terminal region (reviewed in Fields and Sternglanz 1994 ).

Terminal truncations of Sh2 and Bt2 were generated to determine whether sequences specific to either the C or N terminus are important for interaction. No staining was obtained when the pSH2AT-BD plasmid containing the first 244 amino acids of SH2 or the pSH2CT-AD plasmid containing amino acids 238 to 516 of SH2 were expressed with the full-length BT2 subunit. This indicates that protein motifs located in both termini of SH2 are essential for interaction (Figure 3A and Figure 3B). Similarly, no interaction was detected when the pBT2AT-BD plasmid containing the first 232 amino acids of BT2 was expressed with a full-length SH2 subunit (Figure 3A and Figure 3B). However, a weak interaction was identified when the pBT2CT-AD plasmid containing amino acids 223 to 475 of BT2 was expressed with the full-length SH2 subunit. This weak interaction was only detectable after an extended incubation (>48 hr) in the X-gal solution (data not shown). This weak interaction was also detected by slight growth in the absence of His (Figure 3B). Overall, the data show that both N- and C-terminal regions of both subunits are essential for efficient interaction, although a weak interaction is possible in the absence of the N terminus of the Bt2 subunit.

View larger version (51K): [in this window] [in a new window]   Figure 3. The N- and C-Terminal Regions of SH2 and BT2 Are Important for Subunit Interaction. (A) X-gal filter lift assays of yeast cells expressing full-length SH2 or BT2 with the C- or N-terminal regions of the BT2 or SH2 subunit, respectively, show that sequences specific to both termini are important for interaction. pBT2-BD/pSH2-AD and pGAD424/pGBT9 are included as positive and negative controls. pSH2CT-AD codes for amino acids 238 to 516 of the SH2 subunit, and pBT2CT-AD codes for amino acids 223 to 475 of the BT2 subunit, representing the C-terminal halves of each subunit, respectively. pSH2AT-BD codes for amino acids 1 to 244, and pBT2AT-BD codes for amino acids 1 to 232, representing the N-terminal halves of SH2 and BT2, respectively. Negative X-gal staining shows that both termini are needed for SH2:BT2 interaction. (B) Growth of yeast cells expressing constructs described in (A) on a synthetic medium without Leu, Trp, or His plus 30 mM 3-AT. Lack of any significant growth indicates that both termini are essential for efficient SH2:BT2 interaction. A low level of growth was identified when pBT2CT-AD was expressed with the full-length SH2 subunit.

Density Gradient Analysis
The yeast two-hybrid results point to the fact that BT2:BT2 or SH2:SH2 homopolymers do not form. Homopolymer interactions were tested independently by examining the aggregation states of each subunit in endosperm null mutants lacking the other subunit. Crude extracts from the wild type, W64Ax182E, and the null mutants sh2-R and bt2-B were subjected to glycerol density gradient centrifugation. Sh2 transcript and protein are devoid in sh2-R, while bt2-B totally lacks Bt2 transcript and protein (Giroux and Hannah 1994 ). Enzyme assays of gradient fractions from W64Ax182E showed that AGP activity peaked in fraction 7 (Figure 4), with the expected molecular mass of ~210 kD. Calf intestinal alkaline phosphatase (CIAP; ~140 kD), BSA (~68 kD), and horseradish peroxidase (HRP; ~44 kD) are included as molecular mass standards.

View larger version (18K): [in this window] [in a new window]   Figure 4. Enzymatic Analysis of Glycerol Density Gradient Fractions. Enzymatic analysis of glycerol density gradient fractions (1 to 20) was used to monitor the sedimentation of AGP (~210 kD) and molecular mass standards CIAP (~140 kD), BSA (~68 kD), and HRP (~44 kD). Fraction numbers are plotted on the x-axis. AGP activity (squares) from W64Ax182E was measured using the pyrophosphorylysis assay and plotted on the left y-axis. No AGP activity was detected in sh2-R and bt2-B extracts. CIAP (diamonds) and HRP (ovals) were quantitated using colorimetric assays and absorbance at 405 and 450 nm, respectively. BSA (triangles) was measured by absorbance at 595 nm. CIAP, BSA, and HRP are plotted on the right y-axis in absorbance at their respective wavelengths.

Protein samples for the gradient fractions were immobilized onto a membrane by using a dot blot apparatus and probed with either anti-SH2 or anti-BT2 antibodies. Results from the protein blots identified similar profiles for SH2 and BT2 antigen in the W64Ax182E gradient (Figure 5A and Figure 5B). As expected, these peaked with AGP activity. SH2 and BT2 antigens also were identified in the 100-kD range (fractions 10 and 11; Figure 5A and Figure 5B). This is consistent with a dimer molecular mass and suggests the existence of SH2:BT2 heterodimers.

View larger version (63K): [in this window] [in a new window]   Figure 5. Protein Blot Analysis of Glycerol Density Gradient Fractions. (A) Gradient fractions from W64Ax182E, sh2-R, and bt2-B were dot blotted and probed with an anti-BT2 polyclonal antibody. Numbers across the top represent gradient fractions. Row A represents fractions 1 to 10 of W64Ax182E, and row B represents fractions 11 to 20 for W64Ax182E. Rows C and D represent fractions 1 to 20 for sh2-R, and rows E and F represent fractions 1 to 20 for bt2-B, as described for W64Ax182E. (B) Protein blot in (A) stripped and probed with anti-SH2 antibody.

Maize endosperm null mutants sh2-R and bt2-B were used to further study BT2:BT2 and SH2:SH2 subunit interactions, respectively. Availability of such mutants allows us to directly test subunit interactions from the endosperm of maize kernels. Overall, the glycerol gradient analysis of mutant extracts correlates well with the results identified in W64Ax182E. Gradient fractions of sh2-R and bt2-B were probed with anti-SH2 or anti-BT2 antibody, as described above. Analysis of the protein blot with the anti-BT2 antibody showed that the wild-type BT2 protein in the sh2-R null mutant peaked in fractions 14 and 15 (Figure 5A), corresponding to a size of ~50 kD range. Hence, BT2 exists as a monomer in the absence of SH2. Therefore, the density gradient result supports the two-hybrid data in showing that BT2 subunits do not interact to form higher molecular mass aggregates. Furthermore, in contrast with the W64Ax182E BT2 profile, no BT2 antigen was identified in the 100-kD range (fractions 10 and 11; Figure 5A) in the sh2-R null mutant. Lack of any BT2 antigen in the 100-kD range in the sh2-R mutant shows that the 100-kD BT2 protein found in the W64Ax182E is SH2 dependent and points to the existence of SH2:BT2 heterodimers.

The bt2-B null mutant allowed us to monitor the presence of BT2-independent SH2 interactions in the maize endosperm. Significant SH2 antigen levels were detected in fractions 6 to 14, with the major peak in fractions 10 to 12 thus corresponding to a size of ~100 kD (Figure 5B). Furthermore, in the absence of the BT2 subunit, the bt2-B mutant shifts the predominant peak of the SH2 antigen from the 200-kD range to the 100-kD range and indicates that SH2 and BT2 subunits are essential for tetramer formation. Whether this represents SH2:SH2 dimers or SH2 complexes with other proteins is not known. However, the yeast two-hybrid results presented above provided no evidence for SH2:SH2 homodimers.

Subunit Integrity
Proteolysis of higher plant AGP subunits has been well documented (Plaxton and Preiss 1987 ; Kleczkowski et al. 1993 ; Hannah et al. 1995 ). Time- and temperature-dependent removal of a 1- to 2-kD protein from the terminus of each subunit has been achieved, and the addition of protease inhibitors can inhibit subunit cleavage. Recent work by Laughlin et al. 1998b shows that segmental deletions of either the large or small subunit alters assembly of potato AGP. Hence, we asked whether the lack of protein interactions noted in the glycerol gradients of the mutant preparations discussed above is due to proteolysis.

Subunit integrity was monitored using SDS-PAGE, and subsequent analysis of protein blots was performed using antibody to either SH2 or BT2. Five micrograms of the wild-type crude extract and 15 µL of W64Ax182E gradient fractions 5 to 9 were resolved through a 10% SDS–polyacrylamide gel, blotted to a nitrocellulose membrane, and probed with anti-SH2 and anti-BT2 antibodies (Figure 6A and Figure 6B). Protein blots clearly show that both subunits are intact and that there is no detectable proteolysis. Likewise, 5 µg of a sh2-R mutant extract and 15 µL of sh2-R gradient fractions 12 to 16 were subjected to SDS-PAGE and protein gel blot analysis. No degradation was observed (Figure 6A and Figure 6B). This shows that lack of BT2:BT2 interactions in the sh2-R mutant cannot be attributed to proteolysis. With bt2-B, 5 µg of the crude extract and 15 µL of fractions 4 to 15 were electrophoresed and probed (Figure 6C and Figure 6D). There was no detectable degradation in these fractions, indicating that the SH2 subunit was intact. The lack of protein interactions in the endosperm null mutants of maize is not due to proteolysis.

View larger version (69K): [in this window] [in a new window]   Figure 6. Integrity of SH2 and BT2 Subunits. (A) and (C) SDS-PAGE of specific glycerol gradient fractions. Proteins are visualized using SYPRO Orange fluorescence protein stain and digital image analysis. Gradient samples and fractions are labeled above the gels. CE indicates crude extract, and the numbers are indicative of the gradient fraction loaded. Molecular masses of the markers (M) are indicated at left in kilodaltons. (B) and (D) Protein gel blot analysis of gels shown in (A) and (C) probed with anti-BT2 or anti-SH2 antibody, as indicated above the blot. No SH2 antigen was detected in the sh2-R null mutant, and that portion of the blot was not included here. Likewise, no BT2 antigen was detected in the bt2-B mutant when probed with anti-BT2 antibody. No proteolysis was detected with either subunit.

Isolation and Characterization of Subunit Interaction Mutants
To further elucidate specific residues important for SH2:BT2 interactions, we took a chemical mutagenesis approach. Mutagenized pSH2-AD plasmid DNA was transformed into Y190 cells expressing pBT2-BD. Six putative Sh2 subunit interaction mutants (Sh2-sim) lacking X-gal activity were identified. Two mutants, pSh2-sim36-AD and pSh2-sim49-AD, were further characterized and exhibited a null X-gal staining phenotype after incubation for 24 hr at 30°C (Figure 7A) as well as a lack of growth on medium lacking Leu, Trp, and His and containing 30 mM 3-AT (Figure 7B).

View larger version (49K): [in this window] [in a new window]   Figure 7. Subunit Interaction Mutants Eliminate SH2 and BT2 Interaction. (A) X-gal filter lift assay of yeast cells expressing subunit interaction mutants pSh2-sim36-AD and pSh2-sim49-AD with pBT2-BD. pBT2-BD/pSH2-AD and pGAD424/pGBT9 were included as positive and negative controls. Negative X-gal staining exhibited by pSh2-sim36-AD and pSh2-sim49-AD indicates that the mutational lesion in the SH2 subunit has altered its ability to interact with the BT2 subunit. (B) The lack of growth of yeast cells expressing the constructs described in (A) on a synthetic medium without Leu, Trp, and His plus 30 mM 3-AT confirms that the mutated SH2 subunit in pSh2-sim36-AD and pSh2-sim49-AD can no longer interact with BT2.

Sequence analysis of pSh2-sim36-AD revealed the insertion of a single base pair at amino acid 417. The frameshift mutation added 14 non-SH2 amino acids distal to position 417 before a premature stop codon was reached. This generated a loss of the last 99 amino acids from the C terminus of SH2 (Figure 8). Loss of interaction with this mutant is consistent with previous data pointing to the importance of both termini for efficient interaction. Mutant pSh2-sim49-AD contains two point mutations that generate an Ala-to-Thr substitution at position 245 and a Gly-to-Arg change at position 455. Both residues are completely conserved in all sequenced AGPs, and the surrounding residues are also highly conserved (Nakata et al. 1991 ; Smith-White and Preiss 1992 ; Ainsworth et al. 1995 ). Gly 455 is also in the region lost in the pSh2-sim36-AD mutant. Furthermore, Gly 455 is in close proximity (30 amino acids) to at least three additional mutations that we have isolated that alter subunit interactions (T.W. Greene and L.C. Hannah, unpublished data). Taken as a whole, the C terminus of SH2 is critically important for SH2:BT2 interactions.

View larger version (10K): [in this window] [in a new window]   Figure 8. Genetic Lesions of pSh2-sim36-AD and pSh2-sim49-AD. Mutation sites of pSh2-sim36-AD and pSh2-sim49-AD compared with wild-type Sh2. White bars represent the coding region for each of the proteins. Hatched areas represent the AD translationally fused to the coding region. A single base pair insertion was identified with pSh2-sim36-AD that resulted in the premature termination of the SH2 subunit. Deletion of the last 99 amino acids in pSh2-sim36-AD negates its interaction with the BT2 subunit. Two point mutations in pSh2-sim49-AD that generate the replacement of Ala with Thr at position 245 and Gly with Arg at position 455 also disrupt SH2:BT2 interactions. A positive (+) or negative (-) interaction with the BT2 subunit is indicated at right.

	DISCUSSION

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Heterologous expression systems have proven quite powerful in assessing the role or function of various higher plant proteins. Bacterial expression of AGP is a prime example (Iglesias et al. 1993 ). A significant amount of structure–function information concerning AGP has been obtained using this bacterial expression system combined with a random mutagenesis approach (Greene et al. 1996a , Greene et al. 1996b ; Laughlin et al. 1998a ; Okita et al. 1998 ). Although the bacterial expression system is powerful, it is dependent on the measurement of AGP activity. In contrast, the yeast two-hybrid expression system established by Fields and Song 1989 allowed us to focus directly on interactions of the large and small subunits of AGP.

The power of a genetics system combined with a selectable screen allowed us to mutate an individual subunit to begin our analysis of SH2 and BT2 interactions and potentially map these sites of interface between the two subunits. Two such mutants have been characterized and clearly showed that we can use this system for such an analysis. These two mutants, pSh2-sim36-AD and pSh2-sim49-AD, provided significant information with regard to SH2 and BT2 interaction. First, pSh2-sim36-AD combined with the C-terminal deletion constructs definitively showed that the C terminus is essential for subunit interaction. Second, pSh2-sim49-AD showed that we were able to generate specific point mutations that are capable of disrupting SH2 and BT2 interaction. This is significant and demonstrates that this heterologous system can be used to map sites of interaction between SH2 and BT2. We currently have four additional Sh2-sim-AD mutants to analyze; with the ability to screen large numbers of yeast colonies, this system will generate additional information concerning such interactions.

These data, combined with other previously published data, point to an interesting fundamental difference among plant AGPs. In the case of the maize endosperm, only polymers containing the large and the small subunit are enzymatically active. Although none of the single sh2 or bt2 mutants totally abolishes all AGP activity, the residual activity remaining in these single mutants is not reduced when the other wild-type gene is replaced with a nonfunctional allele (Hannah and Nelson 1976 ; Hannah et al. 1980 ). Giroux and Hannah 1994 reported further evidence that the residual activity in single sh2 and bt2 mutants is not due to homopolymers encoded by the remaining functional gene, and they identified AGP transcripts not encoded by Sh2 and Bt2 in the maize endosperm. Furthermore, bacterial expression of SH2 alone or BT2 alone does not complement the E. coli glg-C mutation, and no AGP enzymatic activity could be detected in the representative bacterial crude extracts (J. Shaw and L.C. Hannah, unpublished data). In contrast, E. coli expression of the potato AGP small subunit resulted in a homotetramer with partial AGP activity (Ballicora et al. 1995 ). The catalytic activity of the potato small subunit homotetramer was greatly enhanced by mutagenesis (Okita et al. 1998 ).

The yeast two-hybrid data and the mutant analysis reported here provide additional evidence for the fundamental difference in the properties of the potato small subunit and the maize endosperm small (BT2) subunit. Density gradient analysis clearly shows that the BT2 protein remains as a monomer in the absence of SH2, and no interaction of BT2 with BT2 was found in the yeast two-hybrid experiments. This fundamental difference in the behavior of the potato and maize small subunits is surprising because plant AGP small subunits are quite evolutionarily conserved (Smith-White and Preiss 1992 ).

In contrast to BT2, gradient analysis of a bt2-B mutant extract showed that most of the SH2 antigen is found in the 100-kD range. This size is twice that of the SH2 monomer. Yeast two-hybrid data suggest that this is not a SH2:SH2 dimer because placement of all or parts of SH2 in the yeast expression plasmids provided no evidence for interaction. The nature of the SH2-containing polymer in the endosperm and whether it is necessary for the assembly of an active AGP await further investigation.

AGP is an important enzyme in starch biosynthesis in higher plants (Okita 1992 ; Preiss 1993 ; Martin and Smith 1995 ). Its pivotal role in starch metabolism has been verified in many photosynthetic and nonphotosynthetic tissues (reviewed in Nakata and Okita 1994 ; Hannah 1997 ). There are still many unanswered questions concerning the structure–function relationship of this enzyme. For example, higher plant AGPs have evolved into a more complex enzyme consisting of two large and two small subunits. The reason for such an evolution is still uncertain, but one postulation argues that this evolution reflects the differences in regulation between AGPs. Specifically how these subunits interact or polymerize into the native heterotetrameric structure is still unanswered and awaits elucidation of a three-dimensional structure. The yeast two-hybrid expression system and mutagenesis should begin to identify the regions of SH2 and BT2 that are important for such interactions. Furthermore, this additional structure–function information will be important in our ability to engineer superior versions of AGP for transgene expression and manipulation of starch biosynthesis.

	METHODS

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Plasmid Construction
Shrunken2 (Sh2)- and Brittle2 (Bt2)-specific primers and polymerase chain reaction introduced 5' SalI sites and BamHI sites 3' to the coding regions. The Sh2 5' primer (5'-GCCGGGGATCCGTATGGC- CCAGT T TGCACT TGCAT TGGAC-3'), Sh2 3' primer (5'-GCGCGT-CGACCTCTATATGACAGACCCATCGT TGATGG-3'), Bt2 5' primer (5'-GCCGGGGATCCGTATGGACATGGCT T TGGCGTCTAAAGCC-3'), and Bt2 3' primer (5'-GCGCGTCGACCT TCATATAACTGT TCCACT-AGGGAG-3') were obtained from Gibco BRL. Introduction of the restriction sites allowed the cloning of the coding region into either pGAD424 or pGBT9 yeast expression vectors and generated the translational fusion of SH2 or BT2 to either the GAL4 activation domain (AD; pGAD424) or binding domain (BD; pGBT9). Constructs were confirmed by sequence analysis.

C- and N-terminal constructs used the 5' and 3' primers outlined above plus two additional internal primers for Sh2 and Bt2 that are given below. The Sh2 3' 820 primer (5'-GCGCGTCGACCTGCTCGGCTCTCATCAACAGGAGCAC-3') and Sh2 5' primer were used to construct the pSH2AT-BD expression plasmid containing the N-terminal half (amino acids 1 to 244) of SH2. The Sh2 5' 798 primer (5'-GCCGGGGATCCGTGCTCCTGT TGATGAGAGCCGAGCT TC-3') and Sh2 3' primer were used to construct pSH2CT-AD containing the C-terminal half (amino acids 238 to 516) of SH2. The Bt2 3' 722 (5'-GCGCGTCGACCTCCATCAT TGCT T TCAACTGCTCTCCT T-TCGG-3') and Bt2 5' primers were used to construct the pBT2AT-BD (amino acids 1 to 232) expression plasmid, and Bt2 5' 692 (5'-GCC-GGGGATCCGTCCGAAAGGAGAGCAGT TGAAAGCAATGATGG-3') and Bt2 3' primers were used to construct the pBT2CT-AD (amino acids 223 to 475) expression plasmid. Internal primers Sh2 5' 798 and Bt2 5' 692 introduced a SalI site 5' into the coding region, and Sh2 3' 820 and Bt2 3' 722 primers introduced a BamHI site 3' of the coding region, as outlined above. N- and C-terminal halves were cloned into either pGAD424 or pGBT9 to generate AD or BD translational fusions, as described above. Constructs were confirmed by sequence analysis.

Yeast Manipulations
Transformation of constructs into the yeast strain Y190 followed the protocol outlined by Clontech (Palo Alto, CA). Y190 yeast cells containing both plasmids were selected on a synthetic minimal medium containing 6.7 g/L yeast nitrogen base (Difco, Detroit, MI) without amino acids, 2% glucose, amino acid dropout supplement without Leu and Trp (Clontech), and 20 g/L agar (plates only).

Yeast cells expressing constructs were grown at 30°C for 2 days on SD medium without Leu and Trp or 4 days on SD medium without Leu, Trp, and His plus 30 mM 3-amino-1,2,4-triazole (3-AT). Yeast cells were lifted from the plate onto a 0.7-cm Whatman No. 1 filter paper circle. The filter paper containing the yeast cells was quick frozen in liquid N₂ followed by thawing at room temperature. This procedure was repeated twice more. Thawed filters were layered on 0.7-cm filters presoaked in 1.5 mL of an X-gal solution consisting of 10 mL of Z buffer (6.1 g/L Na₂HPO₄·7H₂O, 5.5 g/L NaH₂PO₄·H₂O, 0.75 g/L KCl, and 0.246 g/L MgSO₄), 0.027 mL of ß-mercaptoethanol, and 0.167 mL of an X-gal stock solution (20 mg/mL X-gal in N,N-dimethylformamide). Filters were incubated at 30°C, and ß-galactosidase (ß-gal) activity was monitored by the formation of blue color.

Yeast cells expressing constructs were grown to an OD₆₀₀ of 0.5 to 0.7. Yeast cells (1.5 mL) were concentrated in a 1.5-mL Eppendorf tube by centrifugation at 14,000 rpm for 30 sec. Cells were washed in Z buffer and resuspended in 300 µL of Z buffer. Aliquots (100 µL) of cells were subjected to rapid freezing in liquid N₂ and rapid thawing by incubating in a 37°C water bath. Cell debris was removed by centrifugation, and supernatant was added to 0.7 mL of Z buffer ß-mercaptoethanol solution. An O-nitrophenyl ß-D-galactopyranoside (4 mg/mL in Z buffer) solution (0.2 mL) was added, and samples were incubated at 30°C until a color change was noticed. Reactions were terminated by the addition of 0.4 mL of a 1 M Na₂CO₃ solution. ß-gal activity was quantitated by the absorbance at 420 nm.

Density Gradient Analysis
Glycerol gradients (5 to 15%) were prepared and run as previously described (Greene et al. 1996a ), except that 20% ammonium sulfate was added to help stabilize ADP–glucose pyrophosphorylase (AGP) activity. Endosperm extracts of W64Ax182E or the mutants sh2-R or bt2-B were generated from three kernels harvested at 20 to 22 days after pollination. Kernels were ground to a fine powder by using liquid nitrogen and a mortar and pestle. Ground kernels were resuspended in 1 mL of extraction buffer: 50 mM Hepes, pH 7.5, 10 mM KPi, pH 7.5, 1 mM DT T, 5 mM MgCl₂, 5 mM EDTA, 5% glycerol, 20% ammonium sulfate, 1 µg/mL pepstatin, 1 µg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL chymostatin, 1 mM benzamidine, 5 µg/mL aprotinin, and 1 µg/mL antipain. Protein concentrations of endosperm extracts were determined by Bradford assays using BSA (Sigma) as a standard (Bradford 1976 ). An equal amount of protein (500 µg) from each extract was layered on the top of a gradient and centrifuged at 50,000 rpm for 20 hr at 4°C. Gradients were fractionated into 0.25-mL aliquots by using a peristaltic pump at a flow rate of 1 mL/min. Sedimentation of AGP was monitored using the pyrophosphorylysis assay (Greene et al. 1996a ).

The control gradient contained 1 unit of calf intestinal alkaline phosphatase (CIAP; ~140 kD; Life Technologies, Grand Island, NY), 1 unit of horseradish peroxidase (HRP; ~44 kD; Sigma), and 200 µg of BSA (~68 kD). CIAP activity was monitored using Sigma FAST ready-to-use p-nitrophenyl phosphate tablets. p-Nitrophenol formation was measured by reading the absorbance at 405 nm. HRP activity was detected using Sigma FAST O-phenylenediamine dihydrochloride tablets. HRP activity was quantified using absorbance at 450 nm. BSA was detected using Bradford assays.

Protein Blot Analysis of Gradient Fractions
Fifteen microliters of each fraction was diluted in 15 µL of a denaturing solution (100 mM Tris-Cl, pH 6.8, 4% SDS, and 200 mM DT T) and boiled for 3 min. Samples were vacuum blotted onto a nitrocellulose (Schleicher & Schuell) membrane by using a Hybri-Dot blot apparatus (Life Technologies). Membranes were washed three times for 10 min in a TBS–Tween (10 mM Tris-Cl, pH 7.2, 150 mM NaCl, and 0.1% Tween 20) solution with shaking. Dot blots were blocked for 1 hr in a TBS–Tween and 5% BSA solution. Blocking buffer was removed, and membranes were further incubated with a 1:2000 dilution of polyclonal antibody to either SH2 or BT2 in TBS–Tween and 5% BSA solution for 1 hr (Giroux et al. 1996 ). Blots were washed in a TBS–Tween solution with shaking as before. Blots were incubated with a 1:4000 dilution of donkey anti–rabbit secondary antibody conjugated with HRP (Amersham, Arlington Heights, IL). Cross-reacting proteins were visualized using an enhanced chemiluminescence kit (Amersham). Blots were stripped by incubating in a solution containing 62.3 mM Tris-Cl, pH 6.7, 2% SDS, and 100 mM ß-mercaptoethanol at 60°C for 30 min. Blots were washed three times for 10 min in a TBS–Tween solution with shaking.

SDS-PAGE
Five micrograms of crude extracts and 15 µL of gradient fractions were loaded and resolved on a 10% SDS–polyacrylamide gel (Laemmli 1970 ). Protein was visualized using SYPRO Orange protein stain (Bio-Rad). SYPRO Orange molecular mass markers (Bio-Rad) were myosin (200,000 D), ß-gal (116,000 D), phosphorylase b (97,400 D), BSA (66,200 D), ovalbumin (45,000 D), carbonic anhydrase (31,000 D), and soybean trypsin inhibitor (21,500 D). Acrylamide gels were incubated in a 1:5000 dilution of SYPRO Orange protein stain for 3 hr. The extended incubation helped to leach out the SDS and lower the background fluorescence. Protein was visualized using UV light and a digital imaging system (IS-1000; Alpha Innotech Corporation, San Leandro, CA). Protein was blotted onto a nitrocelluose membrane by using a Hoefer SemiPhor transfer apparatus (Pharmacia Biotechnology, Piscataway, NJ). Analysis of protein blots followed the procedure described above.

Mutagenesis and Selection
Subunit interaction mutants were generated by chemical mutagenesis of the pSH2-AD construct. Purified plasmid DNA was subjected to hydroxylamine–HCl mutagenesis, as described by Greene et al. 1996a . Mutated pSH2-AD plasmid DNA was then transformed into Y190 yeast cells expressing pBT2-BD. More than 3000 colonies were screened by the X-gal filter lift assay. Six colonies were isolated by their negative X-gal staining phenotype. BamHI and SalI fragments containing the coding regions for pSh2-sim36-AD and pSh2-sim49-AD were subcloned into an unmutated pGAD424 vector. These were then transformed into Y190 cells expressing the pBT2-BD construct. pSh2-sim36-AD and pSh2-sim49-AD were again tested for X-gal staining and growth on an SD medium without Leu, Trp, and His containing 30 mM 3-AT, as described above. Mutations in pSh2-sim36-AD and pSh2-sim49-AD were identified by sequence analysis.

	ACKNOWLEDGMENTS

We thank Drs. Robert Ferl and Paul Sehnke for the use of laboratory facilities and advice with the yeast two-hybrid expression system. Research in this laboratory is supported by the Florida Agricultural Experiment Station (R-06307), National Science Foundation Grants No. IBN-9316887 and No. MCB-9420422, and United States Department of Agriculture Competitive Grants No. 94-37300-453, No. 97-36306-4461, No. 95-37301-2080, and No. 98-01006.

Received April 2, 1998; accepted May 28, 1998.

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