RESUMEN
Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.
Asunto(s)
Biotinilación , Compartimento Celular , Transporte de Proteínas , Proteoma/análisis , Proteoma/química , Células Cultivadas , Conjuntos de Datos como Asunto , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Espectrometría de Masas , Mitocondrias/química , Mitocondrias/metabolismo , Orgánulos/química , Orgánulos/metabolismo , Proteoma/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteoma/metabolismo , ARN Mensajero/metabolismo , Animales , HumanosRESUMEN
mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.
Asunto(s)
Citoplasma/ultraestructura , Gránulos Citoplasmáticos/metabolismo , ARN Mensajero/metabolismo , Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Espacio Intracelular , Proteínas/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Estrés FisiológicoRESUMEN
Stress granules (SGs) are cytosolic biomolecular condensates that form in response to cellular stress. Weak, multivalent interactions between their protein and RNA constituents drive their rapid, dynamic assembly through phase separation coupled to percolation. Though a consensus model of SG function has yet to be determined, their perceived implication in cytoprotective processes (e.g., antiviral responses and inhibition of apoptosis) and possible role in the pathogenesis of various neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia) have drawn great interest. Consequently, new studies using numerous cell biological, genetic, and proteomic methods have been performed to unravel the mechanisms underlying SG formation, organization, and function and, with them, a more clearly defined SG proteome. Here, we provide a consensus SG proteome through literature curation and an update of the user-friendly database RNAgranuleDB to version 2.0 (http://rnagranuledb.lunenfeld.ca/). With this updated SG proteome, we use next-generation phase separation prediction tools to assess the predisposition of SG proteins for phase separation and aggregation. Next, we analyze the primary sequence features of intrinsically disordered regions (IDRs) within SG-resident proteins. Finally, we review the protein- and RNA-level determinants, including post-translational modifications (PTMs), that regulate SG composition and assembly/disassembly dynamics.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteoma , Humanos , Proteómica , Gránulos de Estrés , Esclerosis Amiotrófica Lateral/patología , ARNRESUMEN
Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.
Asunto(s)
Biotina , Proteínas de Unión al ADN , Biotinilación , Estreptavidina/química , Sefarosa , Tripsina , Biotina/químicaRESUMEN
The LSM domain-containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P-body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E-binding protein 4E-T and the DEAD-box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C-terminal fragment of 4E-T. The 4E-T C-terminus forms a bi-partite motif that wraps around the N-terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C-terminal RecA-like domain of DDX6. LSM14 binds DDX6 via a unique non-contiguous motif with distinct directionality as compared to other DDX6-interacting proteins. Together with mutational and proteomic studies, the LSM14-DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P-body assembly.
Asunto(s)
ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans , Cristalografía por Rayos X , ARN Helicasas DEAD-box/genética , Drosophila melanogaster , Factor 4E Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/metabolismo , Proteómica , Proteínas Proto-Oncogénicas/genética , Rec A Recombinasas/química , Proteínas Recombinantes/química , Ribonucleoproteínas/genética , Alineación de SecuenciaRESUMEN
Systematic analysis of affinity-purified samples by liquid chromatography coupled to mass spectrometry (LC-MS) requires high coverage, reproducibility, and sensitivity. While data-independent acquisition (DIA) approaches improve the reproducibility of protein-protein interaction detection as compared to standard data-dependent acquisition approaches, the need for library generation reduces their throughput, and analysis pipelines are still being optimized. In this study, we report the development of a simple and robust approach, termed turboDDA, to improve interactome analysis using spectral counting and data-dependent acquisition (DDA) by eliminating the dynamic exclusion (DE) step and optimizing the acquisition parameters. Using representative interaction and proximity proteomics samples, we detected increases in identified interactors of 18-71% compared to all samples analyzed by standard DDA with dynamic exclusion and for most samples analyzed by DIA with the MSPLIT-DIA spectral counting approach. In summary, turboDDA provides better sensitivity and identifies more high-confident interactors than the optimized DDA with DE and comparable or better sensitivity than DIA spectral counting approaches.
Asunto(s)
Proteómica , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Reproducibilidad de los ResultadosRESUMEN
Meiosis arrest female 1 (MARF1) is a cytoplasmic RNA binding protein that is essential for meiotic progression of mouse oocytes, in part by limiting retrotransposon expression. MARF1 is also expressed in somatic cells and tissues; however, its mechanism of action has yet to be investigated. Human MARF1 contains a NYN-like domain, two RRMs and eight LOTUS domains. Here we provide evidence that MARF1 post-transcriptionally silences targeted mRNAs. MARF1 physically interacts with the DCP1:DCP2 mRNA decapping complex but not with deadenylation machineries. Importantly, we provide a 1.7 Å resolution crystal structure of the human MARF1 NYN domain, which we demonstrate is a bona fide endoribonuclease, the activity of which is essential for the repression of MARF1-targeted mRNAs. Thus, MARF1 post-transcriptionally represses gene expression by serving as both an endoribonuclease and as a platform that recruits the DCP1:DCP2 decapping complex to targeted mRNAs.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Endorribonucleasas/metabolismo , Interferencia de ARN , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Endorribonucleasas/química , Endorribonucleasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , División del ARN , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/química , Transactivadores/genéticaRESUMEN
BACKGROUND: In addition to DNA, gametes contribute epigenetic information in the form of histones and non-coding RNA. Epigenetic programs often respond to stressful environmental conditions and provide a heritable history of ancestral stress that allows for adaptation and propagation of the species. In the nematode C. elegans, defective epigenetic transmission often manifests as progressive germline mortality. We previously isolated sup-46 in a screen for suppressors of the hexosamine pathway gene mutant, gna-2(qa705). In this study, we examine the role of SUP-46 in stress resistance and progressive germline mortality. RESULTS: We identified SUP-46 as an HNRNPM family RNA-binding protein, and uncovered a highly novel role for SUP-46 in preventing paternally-mediated progressive germline mortality following mating. Proximity biotinylation profiling of human homologs (HNRNPM, MYEF2) identified proteins of ribonucleoprotein complexes previously shown to contain non-coding RNA. Like HNRNPM and MYEF2, SUP-46 was associated with multiple RNA granules, including stress granules, and also formed granules on active chromatin. SUP-46 depletion disrupted germ RNA granules and caused ectopic sperm, increased sperm transcripts, and chronic heat stress sensitivity. SUP-46 was also required for resistance to acute heat stress, and a conserved "MYEF2" motif was identified that was needed for stress resistance. CONCLUSIONS: In mammals, non-coding RNA from the sperm of stressed males has been shown to recapitulate paternal stress phenotypes in the offspring. Our results suggest that HNRNPM family proteins enable stress resistance and paternally-mediated epigenetic transmission that may be conserved across species.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Epigénesis Genética , Células Germinativas/metabolismo , Canales de Potasio/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canales de Potasio/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1-resistance complex, resulting in ETI activation.
Asunto(s)
Acetiltransferasas/inmunología , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas Portadoras/inmunología , Fosfotransferasas/metabolismo , Pseudomonas syringae/inmunología , Acetiltransferasas/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Western Blotting , Proteínas Portadoras/metabolismo , Cromatografía Liquida , Clonación Molecular , Análisis por Conglomerados , Inmunoprecipitación , Fosfotransferasas/genética , Filogenia , Pseudomonas syringae/enzimología , Resonancia por Plasmón de Superficie , Espectrometría de Masas en Tándem , Técnicas del Sistema de Dos HíbridosRESUMEN
A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of synthetic dosage lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs, and literature-curated kinase-substrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale GI networks.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Fosfotransferasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sitios de Unión/genética , Western Blotting , Genoma Fúngico/genética , Genómica/métodos , Inmunoprecipitación , Modelos Genéticos , Mutación , Motivos de Nucleótidos/genética , Fosfotransferasas/metabolismo , Unión Proteica , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Intercellular communication in the nervous system occurs through the release of neurotransmitters into the synaptic cleft between neurons. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single-particle electron cryomicroscopy allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzyme's rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. ATP hydrolysis during vesicle loading results in a loss of the V1 region of V-ATPase from the SV membrane, suggesting that loading is sufficient to induce dissociation of the enzyme.
Asunto(s)
Vesículas Sinápticas , ATPasas de Translocación de Protón Vacuolares , Animales , Ratas , Proteínas Bacterianas/química , Encéfalo/ultraestructura , Encéfalo/enzimología , Colesterol/química , Microscopía por Crioelectrón , Hidrólisis , Vesículas Sinápticas/enzimología , Vesículas Sinápticas/ultraestructura , Sinaptofisina/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/aislamiento & purificación , ATPasas de Translocación de Protón Vacuolares/ultraestructura , Conformación ProteicaRESUMEN
The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.
Asunto(s)
Tamaño de la Célula , Proteína Quinasa Deficiente en Lisina WNK 1 , Humanos , Animales , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Evolución Molecular , Células HEK293 , Unión Proteica , Familia de Multigenes , Presión OsmóticaRESUMEN
Plant cell signaling triggers the abscission of entire organs, such as fruit, leaves and flowers. Previously, we characterized an ADP-ribosylation factor GTPase-activating protein, NEVERSHED (NEV), that regulates membrane trafficking and is essential for floral organ shedding in Arabidopsis. Through a screen for mutations that restore organ separation in nev flowers, we have identified a leucine-rich repeat receptor-like kinase, EVERSHED (EVR), that functions as an inhibitor of abscission. Defects in the Golgi structure and location of the trans-Golgi network in nev abscission zone cells are rescued by a mutation in EVR, suggesting that EVR might regulate membrane trafficking during abscission. In addition to shedding their floral organs prematurely, nev evr flowers show enlarged abscission zones. A similar phenotype was reported for plants ectopically expressing INFLORESCENCE DEFICIENT IN ABSCISSION, a predicted signaling ligand for the HAESA/HAESA-LIKE2 receptor-like kinases, indicating that this signaling pathway may be constitutively active in nev evr flowers. We present a model in which EVR modulates the timing and region of abscission by promoting the internalization of other receptor-like kinases from the plasma membrane.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Flores/crecimiento & desarrollo , Proteínas Quinasas/fisiología , Arabidopsis/fisiología , Proteínas Activadoras de GTPasa , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/metabolismoRESUMEN
Global quantitative analysis of genetic interactions is a powerful approach for deciphering the roles of genes and mapping functional relationships among pathways. Using colony size as a proxy for fitness, we developed a method for measuring fitness-based genetic interactions from high-density arrays of yeast double mutants generated by synthetic genetic array (SGA) analysis. We identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate single- and double-mutant fitness measurements, which rival the accuracy of other high-resolution studies. We applied the SGA score to examine the relationship between physical and genetic interaction networks, and we found that positive genetic interactions connect across functionally distinct protein complexes revealing a network of genetic suppression among loss-of-function alleles.
Asunto(s)
Aptitud Genética , Genoma Fúngico , Levaduras/genética , Algoritmos , Regulación Fúngica de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Mutagénesis , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Rayos Ultravioleta , Levaduras/efectos de la radiaciónRESUMEN
ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Sitios de Unión/genética , Retículo Endoplásmico/metabolismo , Células Eucariotas/metabolismo , Prueba de Complementación Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Microscopía Confocal , Mutación , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Through a sensitized screen for novel components of pathways regulating organ separation in Arabidopsis flowers, we have found that the leucine-rich repeat receptor-like kinase SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) acts as a negative regulator of abscission. Mutations in SERK1 dominantly rescue abscission in flowers without functional NEVERSHED (NEV), an ADP-ribosylation factor GTPase-activating protein required for floral organ shedding. We previously reported that the organization of the Golgi apparatus and location of the trans-Golgi network (TGN) are altered in nev mutant flowers. Disruption of SERK1 restores Golgi structure and the close association of the TGN in nev flowers, suggesting that defects in these organelles may be responsible for the block in abscission. We have also found that the abscission zones of nev serk1 flowers are enlarged compared to wild-type. A similar phenotype was previously observed in plants constitutively expressing a putative ligand required for organ separation, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), suggesting that signalling through IDA and its proposed receptors, HAESA and HAESA-LIKE2, may be deregulated in nev serk1 abscission zone cells. Our studies indicate that in addition to its previously characterized roles in stamen development and brassinosteroid perception, SERK1 plays a unique role in modulating the loss of cell adhesion that occurs during organ abscission.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Flores/crecimiento & desarrollo , Proteínas Quinasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mapeo Cromosómico , Flores/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Aparato de Golgi/metabolismo , Microscopía Electrónica de Transmisión , Mutación , Fenotipo , Proteínas Quinasas/genéticaRESUMEN
Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of P. syringae T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar (i.e., congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions.
Asunto(s)
Pseudomonas syringae/genética , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/genética , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cinesinas/metabolismo , Unión Proteica , Pseudomonas syringae/patogenicidad , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Proximity dependent biotin identification (BioID) has emerged as a powerful tool for studies of proteome architecture, including insoluble or membrane-associated proteins. The technique has been well established in mammalian cells but has yet to be applied to whole plant systems. Here we demonstrate the application of BioID on leaf tissues of the model plant Arabidopsis thaliana, thereby expanding the versatility of this important technique and providing a powerful proteomics tool for plant biologists.