Figure 1.
Phylogeny of G protein components in the plant kingdom.
(A) Conservation of G protein components in the plant kingdom. *, Genomes have not been sequenced; N/I, Not identified; +/−, 7TM-RGS gene is identified in a date palm, but not in the grass-family. (B–D) Phylogram of the consensus maximum likelihood (ML) tree as determined for Gα, Gβ and multi transmembrane RGS protein sequences. The trees were rooted with Homo sapiens Gαi1, Gαq, Gβ1, Gβ5 or S. moellendorffii 7TM-RGS genes. Bootstrap values above 40 are shown near each branch. White, gray or black circles indicate that the branch was supported more than 90%, 80% or 70%, respectively. Species and gene names are shown in blue, green, yellow or purple colors, indicating eudicots, monocots, gymnosperms or non-vascular plants, respectively. Scale bar represents 0.1 substitutions per sites. See Figure S1, S2, S3 for aligned sequences used for creating the trees.
Table 1.
G-protein components found in genome or EST databases of the land plant kingdom.
Figure 2.
Activation of plant Gα subunits.
(A, B) Time course of [35S]GTPγS binding to 1 µM Gα at 20°C. Data are presented as the mean ± SEM of more than triplicates. (C, D) Intrinsic tryptophan fluorescence of Gα was measured at room temperature. 5 µM GTPγS was added to 400 nM Gα in the cuvette at time 0. Data are mean ± SEM of duplicate samples.
Table 2.
Rates of nucleotide exchange and GTP hydrolysis.
Figure 3.
Inactivation of plant Gα subunits.
(A–C) The activation and inactivation rates of Gα were monitored at room temperature by measuring the intrinsic tryptophan fluorescence. 800 nM GTP or 5 µM GTPγS was added to 400 nM Gα in the cuvette at time 0. Data are mean ± SEM of duplicate samples. Note that the turnover rate of Gα depends on stoichimometry of the concentrations of active Gα protein and GTP. The specific activity of the Gα subunits estimated by [35S]GTPγS binding assay were as follows: AtGPA1, 0.46 mol/mol protein; RGA1, 0.55 mol/mol protein; MpGPA1, 0.69 mol/mol protein. (D) Time course of single turnover [γ32P]GTP hydrolysis by 800 nM Gα. The mean ± SEM of duplicate samples is shown.
Figure 4.
Evolution of monocot Gα and monocot specific loss of a 7TM-RGS gene.
(A) Evolutionary process to delete a 7TM-RGS gene in monocots. 1. The angiosperm ancestor possessed a 7TM-RGS gene. 2. After separation from the palm tree lineage, the Gα in the grass lineage lost a functional and physical coupling with its partner RGS protein. This uncoupling occurred by a single amino acid mutation, thus releasing the RGS gene from evolutionary constraint. 3. RGS genes were gradually deleted from grass-family genomes. 4. The extant S.italica RGS gene may still be functional, despite the loss of the 7TM region. (B) Switch I region of Gα. Conserved residues are highlighted with orange. Substituted Asn residue in monocots (Asn195 of OsRGA1) is shown with green. See Table S1 for species names. (C) Predicted structures of OsRGA1 (green) overlayed on Rattus norvegicus Gαi1 and RGS4 (cyan and yellow, PDB: 1AGR [21]). Residues in purple (Gαi1) or red (OsRGA1) are at the binding surface to RGS4. Asn195 of OsRGA1 and Thr182 of Gαi1, discussed in this paper, are illustrated by a stick model. (D) The binding surface between Gαi1 and RGS4. Thr182 of Gαi1 and the interacting residues of RGS4 are shown in a stick model.
Figure 5.
Intrinsic properties of Gα mutants.
(A, B) Time course of [35S]GTPγS binding to 1 µM Gα at 20°C. Data are presented as the mean ± SEM of triplicates. (C, D) Time course of single turnover [γ32P]GTP hydrolysis by 800 nM Gα. The mean ± SEM of duplicate samples is shown.
Figure 6.
Affinity of plant Gα to AtRGS1 immobilized on the SPR biosensor.
Recombinant AtRGS1 (284–459 aa) was immobilized on sensor chip CM5. AlF4-bound Gα subunits or the Gβγ dimer control (A) flowed over the chip at seven different concentrations (6.25, 12.5, 25, 50, 100, 200, 400 nM). Kinetics determined with 1∶1 (Langmular) binding model is shown in Table 3. Wild type Arabidopsis Ga subunit (B), T194N mutant Arabidopsis Gα subunit (C), wild type rice Gα subunit (D), and 195T mutant rice Gα subunit (E).
Figure 7.
GAP activity of RGS toward plant Gα subunits.
(A–C) Time course of steady-state [γ32P]GTP hydrolysis by 500 nM Gα in the presence or absence of 750 nM AtRGS1 were measured over time after incubation at 20°C. Rate of Pi production (mol/mol Gα protein) were shown. Data are mean ± SEM of duplicate samples. (D–H) Single turnover [γ32P]GTP hydrolysis by 500 nM Gα proteins with AtRGS1 (0 nM (black), 5 nM (purple), 12.5 nM (yellow), 50 nM (orange), 125 nM (blue), 500 nM (red) and 750 nM (green)). Data are mean +/− SEM for more than two individual experiments, except OsRGA1 with 50 nM RGS at 1 min and 5 min. Dose dependency of single time point (2 min) was shown in (D).
Table 3.
Affinity of plant Gα to AtRGS1 immobilized on the SPR biosensor.
Table 4.
Rates of steady state GTP hydrolysis.
Figure 8.
Model of G protein activation in the plant kingdom.
Slow rate of GDP release and GTP hydrolysis is indicated by a thin arrow. A rapid rate is indicated by a thick arrow. In animals, a rate of GDP release from Gα is much slower than that of GTP hydrolysis. Thus, acceleration of the GDP release by GPCR changes the G protein from inactive to active. In eudicots and monocots, GDP release is rapid, and GTP hydrolysis is much slower than the GDP release. Thus, G protein can self-activate without the aid of a GPCR or other GEF. Instead, the eudicot G protein is regulated by a 7TM-RGS protein, which constitutively promotes GTP hydrolysis step on plasma membrane. However, some monocot genomes lack the 7TM-RGS gene, thus some monocot G protein must use an unknown mechanism to regulate activation. In addition, a 7TM-RGS gene is not expressed in a liverwort. However, a liverwort G protein has a rapid rate of both GDP release and GTP hydrolysis, which is likely to compensate for the loss of the 7TM-RGS gene.