RESUMO
Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular , Proteínas NLR/genética , Fosfolipídeos , Doenças das Plantas , Imunidade VegetalRESUMO
NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hipocótilo/efeitos da radiação , Proteínas 14-3-3/genética , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Hipocótilo/metabolismo , Ácidos Indolacéticos/metabolismo , Luz , Fosforilação , Fototropismo/efeitos da radiação , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which - epsilon - is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development.
Assuntos
Proteínas 14-3-3/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Proteínas 14-3-3/genética , Arabidopsis/genética , Inativação Gênica , Marcação de GenesRESUMO
The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Sequência Conservada , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Ribosilação do ADP/química , Sequência de Aminoácidos , Catálise , Dimerização , Imunofilinas/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.