RESUMO
Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gß (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gßγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gß are known in mammals, no Gß effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gß-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Divisão Celular/fisiologia , Dioxigenases/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Hipocótilo/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Dioxigenases/genética , Etilenos/biossíntese , Subunidades beta da Proteína de Ligação ao GTP/genética , Genes Dominantes , Hipocótilo/genética , Metionina/genética , Metionina/metabolismo , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas RecombinantesRESUMO
Signal transduction involving heterotrimeric G proteins is universal among fungi, animals, and plants. In plants and fungi, the best understood function for the G protein complex is its modulation of cell proliferation and one of several important signals that are known to modulate the rate at which these cells proliferate is D-glucose. Arabidopsis thaliana seedlings lacking the beta subunit (AGB1) of the G protein complex have altered cell division in the hypocotyl and are D-glucose hypersensitive. With the aim to discover new elements in G protein signaling, we screened for gain-of-function suppressors of altered cell proliferation during early development in the agb1-2 mutant background. One agb1-2-dependent suppressor, designated sgb1-1(D) for suppressor of G protein beta1 (agb1-2), restored to wild type the altered cell division in the hypocotyl and sugar hypersensitivity of the agb1-2 mutant. Consistent with AGB1 localization, SGB1 is found at the highest steady-state level in tissues with active cell division, and this level increases in hypocotyls when grown on D-glucose and sucrose. SGB1 is shown here to be a Golgi-localized hexose transporter and acts genetically with AGB1 in early seedling development.