RESUMO
The heterotrimeric G-protein complex is minimally composed of Gα, Gß, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Parede Celular , Proteínas de Ligação ao GTP/metabolismo , Glicômica , Proteômica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Bases de Dados Genéticas , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Teste de Complementação Genética , Genótipo , Imunoprecipitação , Morfogênese/genética , Fenótipo , Mapeamento de Interação de Proteínas , Receptores Acoplados a Proteínas G/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.
Assuntos
Farnesiltranstransferase/metabolismo , Picea/enzimologia , Extratos Vegetais/biossíntese , Proteínas de Plantas/metabolismo , Terpenos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Farnesiltranstransferase/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Picea/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Fosfatos de Poli-Isoprenil/biossíntese , RNA de Plantas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , SesquiterpenosRESUMO
Dietary restriction (DR) promotes healthy aging in diverse species. Essential amino acids play a key role, but the molecular mechanisms are unknown. The evolutionarily conserved Sestrin protein, an inhibitor of activity of the target of rapamycin complex 1 (TORC1), has recently been discovered as a sensor of amino acids in vitro. Here, we show that Sestrin null mutant flies have a blunted response of lifespan to DR. A mutant Sestrin fly line, with blocked amino acid binding and TORC1 activation, showed delayed development, reduced fecundity, extended lifespan and protection against lifespan-shortening, high-protein diets. Sestrin mediated reduced intestinal stem cell activity and gut cell turnover from DR, and stem cell proliferation in response to dietary amino acids, by regulating the TOR pathway and autophagy. Sestrin expression in intestinal stem cells was sufficient to maintain gut homeostasis and extend lifespan. Sestrin is thus a molecular link between dietary amino acids, stem cell function and longevity.