RESUMO
Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.
Assuntos
Arabidopsis , Calmodulina , Calmodulina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ligação ProteicaRESUMO
In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs) and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13 and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13 and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+ . Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13 and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Saccharomyces cerevisiae/metabolismo , Sinalização do Cálcio , Ligação Proteica , Cálcio/metabolismoRESUMO
Tail-anchored (TA) proteins are a unique class of integral membrane proteins that possess a single C-terminal transmembrane domain and target post-translationally to the specific organelles at which they function. While significant advances have been made in recent years in elucidating the mechanisms and molecular targeting signals involved in the proper sorting of TA proteins, particularly to the endoplasmic reticulum and mitochondria, relatively little is known about the targeting of TA proteins to the plastid outer envelope. Here we show that several known or predicted plastid TA outer envelope proteins (OEPs) in Arabidopsis possess a C-terminal RK/ST sequence motif that serves as a conserved element of their plastid targeting signal. Evidence for this conclusion comes primarily from experiments with OEP7.2, which is a member of the Arabidopsis 7 kDa OEP family. We confirmed that OEP7.2 is localized to the plastid outer envelope and possesses a TA topology, and its C-terminal sequence (CTS), which includes the RK/ST motif, is essential for proper targeting to plastids. The CTS of OEP7.2 is functionally interchangeable with the CTSs of other TA OEPs that possess similar RK/ST motifs, but not with those that lack the motif. Further, a bioinformatics search based on a consensus sequence led to the identification of several new OEP TA proteins. Collectively, this study provides new insight into the mechanisms of TA protein sorting in plant cells, defines a new targeting signal element for a subset of TA OEPs and expands the number and repertoire of TA proteins at the plastid outer envelope.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Plastídeos/genéticaRESUMO
KEY MESSAGE: We show that the calcium sensor, CML39, is important in various developmental processes from seeds to mature plants. This study bridges previous work on CML39 as a stress-induced gene and highlights the importance of calcium signalling in plant development. In addition to the evolutionarily-conserved Ca2+ sensor, calmodulin (CaM), plants possess a large family of CaM-related proteins (CMLs). Using a cml39 loss-of-function mutant, we investigated the roles of CML39 in Arabidopsis and discovered a range of phenotypes across developmental stages and in different tissues. In mature plants, loss of CML39 results in shorter siliques, reduced seed number per silique, and reduced number of ovules per pistil. We also observed changes in seed development, germination, and seed coat properties in cml39 mutants in comparison to wild-type plants. Using radicle emergence as a measure of germination, cml39 mutants showed more rapid germination than wild-type plants. In marked contrast to wild-type seeds, the germination of developing, immature cml39 seeds was not sensitive to cold-stratification. In addition, germination of cml39 seeds was less sensitive than wild-type to inhibition by ABA or by treatments that impaired gibberellic acid biosynthesis. Tetrazolium red staining indicated that the seed-coat permeability of cml39 seeds is greater than that of wild-type seeds. RNA sequencing analysis of cml39 seedlings suggests that changes in chromatin modification may underlie some of the phenotypes associated with cml39 mutants, consistent with previous reports that orthologs of CML39 participate in gene silencing. Aberrant ectopic expression of transcripts for seed storage proteins in 7-day old cml39 seedlings was observed, suggesting mis-regulation of early developmental programs. Collectively, our data support a model where CML39 serves as an important Ca2+ sensor during ovule and seed development, as well as during germination and seedling establishment.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Calmodulina/metabolismo , Frutas/embriologia , Germinação , Sementes/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Calmodulina/genética , Flores/embriologia , Flores/genética , Frutas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/genética , Giberelinas/metabolismo , Mutação/genética , Permeabilidade , Dormência de Plantas , Regiões Promotoras Genéticas/genética , Sementes/genética , Transcrição GênicaRESUMO
XBAT35 belongs to a subfamily of Arabidopsis (Arabidopsis thaliana) RING-type E3s that are similar in domain architecture to the rice (Oryza sativa) XA21 Binding Protein3, a defense protein. The XBAT35 transcript undergoes alternative splicing to produce two protein isoforms, XBAT35.1 and XBAT35.2. Here, we demonstrate that XBAT35.2 localizes predominantly to the Golgi and is involved in cell death induction and pathogen response. XBAT35.2, but not XBAT35.1, was found to trigger cell death when overexpressed in tobacco (Nicotiana benthamiana) leaves and does so in a manner that requires its RING domain. Loss of XBAT35 gene function disrupts the plant's ability to defend against pathogen attack, whereas overexpression of XBAT35.2 enhances resistance to pathogens. XBAT35.2 was found to be unstable and promotes its own degradation, suggesting self-regulation. Inoculation with virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000 results in a drastic reduction in the levels of ubiquitinated XBAT35.2 and an increase in the abundance of the E3. This implies that pathogen infection prohibits XBAT35.2 self-regulation and stabilizes the E3. In agreement with a role in defending against pathogens, XBAT35.2 interacts with defense-related Accelerated Cell Death11 (ACD11) in planta and promotes the proteasome-dependent turnover of ACD11 in cell-free degradation assays. In accordance with regulation by a stabilized XBAT35.2, the levels of ubiquitinated ACD11 increased considerably, and the abundance of ACD11 was reduced following pathogen infection. In addition, treatment of transgenic seedlings with a proteasome inhibitor results in the accumulation of ACD11, confirming proteasome-dependent degradation. Collectively, these results highlight a novel role for XBAT35.2 in cell death induction and defense against pathogens.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/microbiologia , Pseudomonas syringae/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Morte Celular , Resistência à Doença , Complexo de Golgi/metabolismo , Células Vegetais/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Pseudomonas syringae/patogenicidade , Domínios RING Finger , Frações Subcelulares/metabolismo , Nicotiana/citologia , Nicotiana/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , VirulênciaRESUMO
Many signalling pathways in plants are regulated by the second messenger calcium (Ca(2+)). In the standard model, Ca(2+)-sensor proteins, such as CaM (calmodulin), detect Ca(2+) signals and subsequently regulate downstream targets to advance the signal transduction cascade. In addition to CaM, plants possess many CMLs (CaM-like proteins) that are predicted to function as Ca(2+) sensors, but which remain largely uncharacterized. In the present study, we examined the biochemical properties, subcellular localization and tissue-specific distribution of Arabidopsis CML43. Our data indicate that CML43 displays characteristics typical of Ca(2+) sensors, including high-affinity Ca(2+) binding, conformational changes upon Ca(2+) binding that expose hydrophobic regions and stabilization of structure in the presence of Mg(2+) or Ca(2+). In vivo localization analysis demonstrates that CML43 resides in cytosolic and nuclear compartments. Transgenic plants expressing a CML43:GUS (ß-glucoronidase) promoter reporter gene revealed that CML43 promoter activity is restricted almost exclusively to root tips under normal growth conditions. GUS reporter activity in these transgenic plants was strongly increased when exposed to the defence compound SA (salicylic acid). Furthermore, immunoblot analysis revealed that the CML43 protein accumulates following treatment with SA. Collectively, our findings suggest that CML43 functions as a Ca(2+) sensor in root tips during both normal growth and plant immune response.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis , Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácido Salicílico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Proteínas de Ligação ao Cálcio/química , Calmodulina/genética , Células Cultivadas , Sistema Imunitário/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Homologia de Sequência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , NicotianaRESUMO
Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.
Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Coenzima A/metabolismo , Proteínas Mitocondriais/metabolismo , Zea mays/genética , Antiporters/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Pisum sativum/genética , Pisum sativum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Zea mays/metabolismoRESUMO
Post-translational import of nucleus-encoded chloroplast pre-proteins is critical for chloroplast biogenesis, and the Toc159 family of proteins serve as receptors for the process. Toc159 shares with other members of the family (e.g. Toc132), homologous GTPase (G-) and Membrane (M-) domains, but a highly dissimilar N-terminal acidic (A-) domain. Although there is good evidence that atToc159 and atToc132 from Arabidopsis mediate the initial sorting step, preferentially recognizing photosynthetic and non-photosynthetic preproteins, respectively, relatively few chloroplast preproteins have been assigned as substrates for particular members of the Toc159 family, which has limited the proof for the hypothesis. The current study expands the number of known preprotein substrates for members of the Arabidopsis Toc159 receptor family using a split-ubiquitin membrane-based yeast two-hybrid system using the atToc159 G-domain (Toc159G), atToc132 G-domain (Toc132G) and atToc132 A- plus G-domains (Toc132AG) as baits. cDNA library screening with all three baits followed by pairwise interaction assays involving the 81 chloroplast preproteins identified show that although G-domains of the Toc159 family are sufficient for preprotein recognition, they alone do not confer specificity for preprotein subclasses. The presence of the A-domain fused to atToc132G (Toc132AG) not only positively influences its specificity for non-photosynthetic preproteins, but also negatively regulates the ability of this receptor to interact with a subset of photosynthetic preproteins. Our study not only substantiates the fact that atToc132 can serve as a receptor by directly binding to chloroplast preproteins but also proposes the existence of subsets of preproteins with different but overlapping affinities for more than one member of the Toc159 receptor family.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte/metabolismo , Cloroplastos/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Biblioteca Gênica , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X({X≠E})) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b 5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins.