G Protein Activation without a GEF in the Plant Kingdom

Abstract

Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent Gα regulatory mechanisms in the plant kingdom. In the grasses, purifying selection on the regulatory gene was lost after the physical decoupling of the RGS protein and its cognate Gα partner. More broadly these findings show extreme divergence in Gα activation and regulation that played a critical role in the evolution of G protein signaling pathways.

Characterization of the Heterotrimeric G-Protein Complex and Its Regulator from the Green Alga Chara braunii Expands the Evolutionary Breadth of Plant G-Protein Signaling

“…It should be noted, however, that most plant species, including moss species, the genera Selaginella, Adiantum, and Pinus, Arabidopsis, and rice, possess only a few, if not only one, homologs of G-protein genes, and multiplicity is typically present with respect to the Gg proteins (Urano et al, 2012). Moreover, one type of Gg protein seems to be specific to higher plants.…”

“…Furthermore, many monocots do not possess an RGS protein homolog, and it is predicted to have been lost multiple times during evolution (Urano et al, 2012). Remarkably, a sequence homolog of AtRGS1 is present in the C. braunii transcriptome.…”

“…four Ga, four Gb, and 10 Gg proteins in soybean; Bisht et al, 2011;Choudhury et al, 2011;Pandey, 2011). Furthermore, at the biochemical level, the higher plant Ga proteins have significantly slower GTPase activity compared with the mammalian Ga proteins (Jones et al, 2011;Choudhury et al, 2012;Urano et al, 2012).…”

“…The liverwort Marchantia polymorpha contains the sequence homologs of Ga, Gb, and Gg proteins but not the regulatory protein, RGS. Interestingly, the Ga protein from M. polymorpha displays rapid GTP binding (similar to plants) but also rapid GDP release (similar to metazoans; Urano et al, 2012). Remarkably, the moss Physcomitrella patens encodes for the Gb and Gg proteins but no canonical Ga or RGS proteins.…”

“…The fully sequenced genomes of chlorophyte green algae, Volvox carteri, Chlamydomonas reinhardtii, Cocomyxa subellipsoidea C-169, Micromonas pusilla CCMP1545, M. pusilla RCC299, and Ostreococcus lucimarinus, do not contain G-proteins homologs (our analysis; www.phytozome.net). These observations have led to hypotheses that G-protein signaling in the green plant lineage evolved with the evolution of land plants or, alternatively, with the evolution of a sporophytic lifestyle (Urano et al, 2012(Urano et al, , 2013Bradford et al, 2013). It has also been reasoned that G-protein signaling in plants is independent of a receptordependent regulation because sequence homologs of G-protein-coupled receptor-like proteins from flowering plants are present in green algae, even though they do not encode for G-protein components themselves (Bradford et al, 2013;Urano et al, 2013).…”