The two AGPase subunits evolve at different rates in angiosperms, yet they are equally sensitive to activity-altering amino acid changes when expressed in bacteria

Abstract

The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis.

Figure 1.

Amino Acid Conservation Patterns of BT2 and SH2. BT2 and SH2 were aligned with 30 and 42 homologs, respectively. Tolerated amino acid changes created by error-prone PCR in this experiment are shown above the amino acid sequence of BT2 and SH2. Black, nonpolar; green, uncharged polar; red, basic, blue, acidic; turquoise, proline; gray box, poor alignment with homologous sequences; black box, absolutely conserved residues among homologous sequences.

Figure 2.

Estimates of AGPase Subunit Gene Trees and ω Values. The overall estimate of ω for each subunit and groups within each subunit were calculated by CODEML. Gene trees, used to estimate ω, were constructed using GARLI. Bootstrap values ≥50% are shown, and the bars indicate the number of nucleotide substitutions per site. (A) Small and Large subunit gene tree. (B) Small subunit gene tree. (C) Large subunit gene tree.

Figure 3.

Glycogen Contents of sh2 and bt2 Mutants Compared with Wild-Type AGPase. Positive clones exhibit some glycogen production as judged by iodine staining. Negative clones do not exhibit detectable amounts of glycogen as judged by iodine staining. Negative clones produced 3 to 4% or less glycogen compared with the wild type. Partially stained clones, classified as positive in this experiment, produced 5 to 40% glycogen compared with the wild type.

Figure 4.

Purification of BT2 Using a Monoclonal Antibody Column. (A) Formaldehyde cross-linking of BT2. Total proteins were extracted from maize endosperm with or without prior formaldehyde treatment. Extracted proteins were probed with a monoclonal BT2 antibody following SDS-PAGE. F, formaldehyde treatment before protein extraction. (B) SDS-PAGE of proteins from bt2-7410 (a bt2 loss-of-protein mutant) and wild-type maize endosperm bound to a monoclonal BT2 antibody column. Formaldehyde cross-linking was performed on maize endosperm before protein extraction. Formaldehyde cross-linking was reversed by heating (95°C for 20 min) after column purification and before loading the purified proteins on the gel. M, marker. White arrows point to proteins purified from both the wild type and bt2-7410. Black arrows point to proteins purified only from the wild type.

Figure 5.

Comparison of Glycogen Produced by Bt2 Mutants Expressed with Wild-Type Sh2 and Wild-Type Agplemzm in E. coli. (A) Glycogen produced by E. coli cells expressing Bt2 mutants with wild-type Sh2 or wild-type Agplemzm. Values are the percentage of glycogen produced compared with glycogen produced by cells expressing wild-type Bt2 with wild-type Sh2 or Agplemzm. Three replicates were used for determining glycogen production for each mutant. Error bars indicate 2 × se. The asterisk indicates a significant difference between means at P = 0.05. (B) Iodine staining of E. coli cells expressing two Bt2 mutants or wild-type Bt2 with either wild-type Sh2 or wild-type Agplemzm.

Figure 6.

Phylogenetic Tree of AGPase Isoforms from Angiosperms and Unicellular Green Algae. The topology of the tree was determined by ML using aligned amino acid sequences analyzed by PhyML. The length of the branches reflects numbers of amino acid substitutions per site estimated using the Dayhoff+Γ model of sequence evolution. ML bootstrap values are indicated below branches, and the bar shows the number of amino acid substitutions per site.