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David Chakravorty Timothy E. Gookin Matthew J. Milner Yunqing Yu Sarah M. Assmann 《Plant physiology》2015,169(1):512-529
Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca2+ cofactor requirement instead of an Mg2+ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought. 相似文献
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Antilisterial Activity of Peptide AS-48 and Study of Changes Induced in the Cell Envelope Properties of an AS-48-Adapted Strain of Listeria monocytogenes 总被引:5,自引:0,他引:5
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Ftima Mendoza Mercedes Maqueda Antonio Glvez Manuel Martínez-Bueno Eva Valdivia 《Applied microbiology》1999,65(2):618-625
The peptide AS-48 is highly active on all Listeria species. It has a bactericidal and bacteriolytic mode of action on Listeria monocytogenes CECT 4032, causing depletion of the membrane electrical potential and pH gradient. The producer strain Enterococcus faecalis A-48-32, releases sufficient amounts of AS-48 into the growth medium to suppress L. monocytogenes in cocultures at enterococcus-to-listeria ratios above 1 at 37°C or above 10 at 15°C. As the temperature decreases, the bactericidal effects of AS-48 are less pronounced, but at 2.5 μg/ml it still can inhibit the growth of listeria at 6°C. AS-48 is highly active on liquid cultures, although concentrations above 0.2 μg/ml are required to avoid adaptation of listeria. AS-48-adapted cells can be selected at low (but still inhibitory) concentrations, and they can be inhibited completely by AS-48 at 0.5 μg/ml. The adaptation is lost gradually upon repeated subcultivation. AS48ad cells are cross-resistant to nisin and show an increased resistance to muramidases. Their fatty acid composition is modified: they show a much higher proportion of branched fatty acids as well as a higher C15:0 An-to-C17:0 An ratio. Resistance to AS-48 is also maintained by protoplasts from AS48ad cells. Electron microscopy observations show that the cell wall of AS48ad cells is thicker and less dense. The structure of wild-type cells is severely modified after AS-48 treatment: the cell wall and the cytoplasmic membrane are disorganized, and the cytoplasmic content is lost. Intracytoplasmic membrane vesicles are also observed when the wild-type strain is treated with high AS-48 concentrations. 相似文献
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Natsumi Maruta Yuri Trusov Eric Brenya Urvi Parekh José Ramón Botella 《Plant physiology》2015,167(3):1004-1016
In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.Heterotrimeric GTP-binding proteins (G proteins), classically consisting of Gα, Gβ, and Gγ subunits, are essential signal transduction elements in most eukaryotes. In animals and fungi, ligand perception by G protein-coupled receptors leads to replacement of GDP with GTP in Gα, triggering activation of the heterotrimer (Li et al., 2007; Oldham and Hamm, 2008). Upon activation, GTP-bound Gα and Gβγ are released and interact with downstream effectors, thereby transmitting signals to multiple intracellular signaling cascades. Signaling terminates when the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP and the inactive heterotrimer reforms at the receptor. The large diversity of mammalian Gα subunits confers specificity to the multiple signaling pathways mediated by G proteins (Wettschureck and Offermanns, 2005). Five distinct classes of Gα have been described in animals (Gαi, Gαq, Gαs, Gα12 and Gαv), with orthologs found in evolutionarily primitive organisms such as sponges (Oka et al., 2009). Humans possess four classes of Gα involving 23 functional isoforms encoded by 16 genes (McCudden et al., 2005), while only a single prototypical Gα is usually found per plant genome (Urano et al., 2013). Multiple copies of Gα are present in some species with recently duplicated genomes, such as soybean (Glycine max) with four Gα genes (Blanc and Wolfe, 2004; Bisht et al., 2011). In the model plant Arabidopsis (Arabidopsis thaliana), a prototypical Gα subunit (GPA1) is involved in a number of important processes, including cell proliferation (Ullah et al., 2001), inhibition of inward K+ channels and activation of anion channels in guard cells by mediating the abscisic acid pathway (Wang et al., 2001; Coursol et al., 2003), blue light responses (Warpeha et al., 2006, 2007), and germination and postgermination development (Chen et al., 2006; Pandey et al., 2006).It is well established that heterotrimeric G proteins play a fundamental role in plant innate immunity. In Arabidopsis, two different Gβγ dimers (Gβγ1 and Gβγ2) are generally considered to be the predominant elements in G protein defense signaling against a variety of fungal pathogens (Llorente et al., 2005; Trusov et al., 2006, 2007, 2009; Delgado-Cerezo et al., 2012; Torres et al., 2013). By contrast, these studies attributed a small or no role to Gα, because mutants deficient in Gα displayed only slightly increased resistance against the fungal pathogens (Llorente et al., 2005; Trusov et al., 2006; Torres et al., 2013). The Gβγ-mediated signaling also contributes to defense against a model bacterial pathogen Pseudomonas syringae, by participating in programmed cell death (PCD) and inducing reactive oxygen species (ROS) production in response to at least three pathogen-associated molecular patterns (PAMPs; Ishikawa, 2009; Liu et al., 2013; Torres et al., 2013). Gα is not involved in PCD or PAMP-triggered ROS production (Liu et al., 2013; Torres et al., 2013). Nonetheless, Arabidopsis Gα plays a positive role in defense against P. syringae, probably by mediating stomatal function and hence physically restricting bacterial entry to the leaf interior (Zhang et al., 2008; Zeng and He, 2010; Lee et al., 2013). Given the small contribution from Gα, the involvement of heterotrimeric G proteins in Arabidopsis resistance could be explained in two ways: either the Gβγ dimer acts independently from Gα, raising a question of how is it activated upon a pathogen attack, or Gα is replaced by another protein for heterotrimer formation.The Arabidopsis genome contains at least three genes encoding Gα-like proteins that have been classified as extra-large G proteins (XLGs; Lee and Assmann, 1999; Ding et al., 2008). XLGs comprise two structurally distinct regions. The C-terminal region is similar to the canonical Gα, containing the conserved helical and GTPase domains, while the N-terminal region is a stretch of approximately 400 amino acids including a putative nuclear localization signal (Ding et al., 2008). GTP binding and hydrolysis were confirmed for all three XLG proteins, although their enzymatic activities are very slow and require Ca2+ as a cofactor, whereas canonical Gα utilizes Mg2+ (Heo et al., 2012). Several other features differentiate XLGs from Gα subunits. Comparative analysis of XLG1 and Gα at the DNA level showed that the genes are organized in seven and 13 exons, respectively, without common splicing sites (Lee and Assmann, 1999). XLGs have been reported to localize to the nucleus (Ding et al., 2008). Analysis of knockout mutants revealed a nuclear function for XLG2, as it physically interacts with the Related To Vernalization1 (RTV1) protein, enhancing the DNA binding activity of RTV1 to floral integrator gene promoters and resulting in flowering initiation (Heo et al., 2012). Therefore, it appears that XLGs may act independently of G protein signaling. On the other hand, functional similarities between XLGs and the Arabidopsis Gβ subunit (AGB1) were also discovered. For instance, XLG3- and Gβ-deficient mutants were similarly impaired in root gravitropic responses (Pandey et al., 2008). Knockout of all three XLG genes caused increased root length, similarly to the Gβ-deficient mutant (Ding et al., 2008). Furthermore, as observed in Gβ-deficient mutants, xlg2 mutants displayed increased susceptibility to P. syringae, indicating a role in plant defense (Zhu et al., 2009). Nevertheless, a genetic analysis of the possible functional interaction between XLGs and Gβ has not been established.In this report, we performed in-depth genetic analyses to test the functional interaction between the three XLGs and Gβγ dimers during defense-related responses in Arabidopsis. We also examined physical interaction between XLG2 and the Gβγ dimers using yeast (Saccharomyces cerevisiae) three-hybrid (Y3H) and bimolecular fluorescent complementation (BiFC) assays. Our findings indicate that XLGs function as direct partners of Gβγ dimers in plant defense signaling. To estimate relatedness of XLGs and Gα proteins, we carried out a phylogenetic analysis. Based on our findings, we conclude that plant XLG proteins most probably originated from a canonical Gα subunit and retained prototypical interaction with Gβγ dimers. They function together with Gβγ in a number of processes including plant defense, although they most probably evolved activation/deactivation mechanisms very different from those of a prototypical Gα. 相似文献
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The NMR structure of 31mer RNA domain of Escherichia coli RNase P RNA using its non-uniformly deuterium labelled counterpart [the 'NMR-window' concept]. 总被引:1,自引:1,他引:0
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C Glemarec J Kufel A Fldesi T Maltseva A Sandstrm L A Kirsebom J Chattopadhyaya 《Nucleic acids research》1996,24(11):2022-2035
The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia coli is reported. Severe spectral overlaps of the proton resonances in the natural 31mer RNA (1) were successfully tackled by unique spectral simplifications found in the partially-deuterated 31 mer RNA analogue (2) incorporating deuterated cytidines [C5 (>95 atom % 2H), C2' (>97 atom % 2H), C3' (>97 atom % 2H), C4' (>65 atom % 2H) and C5' (>97 atom % 2H)] [for the 'NMR-window' concept see: Földesi,A. et al. (1992) Tetrahedron, 48, 9033; Foldesi,A. et al. (1993) J. Biochem. Biophys. Methods, 26, 1; Yamakage,S.-I. et al. (1993) Nucleic Acids Res., 21, 5005; Agback,P. et al. (1994) Nucleic Acids Res., 22, 1404; Földesi,A. et al. (1995) Tetrahedron, 51, 10065; Földesi,A. et al. (1996) Nucleic Acids Res., 24, 1187-1194]. 175 resonances have been assigned out of total of 235 non-exchangeable proton resonances in (1) in an unprecedented manner in the absence of 13C and 15N labelling. 41 out of 175 assigned resonances could be accomplished with the help of the deuterated analogue (2). The two stems in 31mer RNA adopt an A-type RNA conformation and the base-stacking continues from stem I into the beginning of the loop I. Long distance cross-strand NOEs showed a structured conformation at the junction between stem I and loop I. The loop I-stem II junction is less ordered and shows structural perturbation at and around the G11 -C22 base pair. 相似文献
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Sánchez-Barrena MJ Martínez-Ripoll M Gálvez A Valdivia E Maqueda M Cruz V Albert A 《Journal of molecular biology》2003,334(3):541-549
The bacteriocin AS-48 is a membrane-interacting peptide, which displays a broad anti-microbial spectrum against Gram-positive and Gram-negative bacteria. The NMR structure of AS-48 at pH 3 has been solved. The analysis of this structure suggests that the mechanism of AS-48 anti-bacterial activity involves the accumulation of positively charged molecules at the membrane surface leading to a disruption of the membrane potential. Here, we report the high-resolution crystal structure of AS-48 and sedimentation equilibrium experiments showing that this bacteriocin is able to adopt different oligomeric structures according to the physicochemical environment. The analysis of these structures suggests a mechanism for molecular function of AS-48 involving a transition from a water-soluble form to a membrane-bound state upon membrane binding. 相似文献
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Bacteriocin AS-48 is a membrane-interacting peptide that acts as a broad-spectrum antimicrobial against Gram-positive and Gram-negative bacteria. Prior Nuclear Magnetic Resonance experiments and the high resolution crystal structure of AS-48 have suggested a mechanism for the molecular activity of AS-48 whereby the peptide undergoes transition from a water-soluble to a membrane-bound state upon membrane binding. To help interpret experimental results, we here simulate the molecular dynamics of this binding mechanism at the coarse-grained level. By simulating the self-assembly of the peptide, we predict induction by the bacteriocin of different pore types consistent with a “leaky slit” model. 相似文献
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Dieter Hackenberg Pierre-Fran?ois Perroud Ralph Quatrano Sona Pandey 《Plant physiology》2016,172(2):1154-1166
In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.In all known eukaryotes, cellular signaling involves heterotrimeric GTP-binding proteins (G-proteins), which consist of Gα, Gβ, and Gγ subunits (Cabrera-Vera et al., 2003). According to the established paradigm, when Gα is GDP-bound, it forms a trimeric complex with the Gβγ dimer and remains associated with a G-protein coupled receptor. Signal perception by the receptor facilitates GDP to GTP exchange on Gα. GTP-Gα dissociates from the Gβγ dimer, and both these entities can transduce the signal by interacting with different effectors. The duration of the active state is determined by the intrinsic GTPase activity of Gα, which hydrolyzes bound GTP into GDP and inorganic phosphate (Pi), followed by the reassociation of the inactive, trimeric complex (Siderovski and Willard, 2005).In plants, G-protein signaling has been studied in only a few angiosperms to date at the functional level, although the proteins exist in the entire plant lineage (Hackenberg and Pandey, 2014; Urano and Jones, 2014; Hackenberg et al., 2016). Interestingly, while the overall biochemistry of the individual G-protein components and the interactions between them are conserved between plant and metazoan systems, deviations from the established norm are also obvious. For example, the repertoire of canonical G-proteins is significantly limited in plants; the human genome codes for 23 Gα, 5 Gβ, and 12 Gγ proteins, whereas most plant genomes, including those of basal plants, typically encode 1 canonical Gα, 1 Gβ, and three to five Gγ proteins (Urano and Jones, 2014). The only exceptions are some polyploid species, such as soybean, which have retained most of the duplicated G-protein genes (Bisht et al., 2011; Choudhury et al., 2011). Moreover, even in plants that possess only a single canonical Gα and Gβ protein, for example Arabidopsis (Arabidopsis thaliana) and rice, the phenotypes of plants lacking either one or both proteins are relatively subtle. The mutant plants exhibit multiple developmental and signaling defects but are able to complete the life cycle without any major consequences. These observations have questioned the significance of G-protein mediated signaling pathways in plants.Interestingly, plants also possess certain unique variants of the classical G-protein components such as the type III Cys-rich Gγ proteins and extra-large GTP-binding (XLG) proteins, which add to the diversity and expanse of the G-protein signaling networks (Roy Choudhury et al., 2011; Chakravorty et al., 2015; Maruta et al., 2015). The XLG proteins are almost twice the size of typical Gα proteins, with the C-terminal region that codes for Gα-like domain and an extended N-terminal region without any distinctive features. Plant XLGs are encoded by entirely independent genes and therefore are different from the mammalian extra-long versions of Gα proteins such as XLαs and XXLαs, which are expressed due to the use of alternate exons (Abramowitz et al., 2004). Three to five copies of XLG proteins are present in the genome of most angiosperms. At the functional level, the XLG proteins have been characterized only from Arabidopsis, to date, where recent studies suggest that the proteins compete with canonical Gα for binding with the Gβγ dimers and may form functional trimeric complexes (Chakravorty et al., 2015; Maruta et al., 2015). The XLG and Gβγ mutants of Arabidopsis seem to function in the same pathways during the regulation of a subset of plant responses, for example primary root length and its regulation by abscisic acid (ABA); the root waving and skewing responses; sensitivity to Glc, salt, and tunicamycin; and sensitivity to certain bacterial and fungal pathogens (Ding et al., 2008; Pandey et al., 2008; Chakravorty et al., 2015; Maruta et al., 2015). However, many of the phenotypes of Arabidopsis Gα and Gβγ mutants are also distinct from that of the xlg triple mutants. For example, compared to the wild-type plants, the canonical G-protein mutants exhibit altered response to gibberellic acid, brassinosteroids, and auxin and show changes in leaf shape, branching, flowering time, and stomatal densities (Ullah et al., 2003; Chen et al., 2004; Pandey et al., 2006; Zhang et al., 2008; Nilson and Assmann, 2010). The xlg triple mutants behave similarly to wild-type plants in all these aspects of development and signaling. Moreover, whether the XLG proteins are authentic GTP-binding and -hydrolyzing proteins and the extent to which they directly participate in G-protein-mediated signaling pathways remains confounding (Chakravorty et al., 2015; Maruta et al., 2015). Even in plants with a limited number of G-protein subunits such as Arabidopsis, one Gα and three XLGs potentially compete for a single Gβ protein, and the analysis of null mutants is not straightforward, that is, it is not possible to delineate whether the phenotypes seen in the Gα null mutants are truly due to the lack of Gα and/or because of an altered stoichiometry or availability of Gβ for the XLG proteins.As a bryophyte, Physcomitrella patens occupies a unique position in the evolutionary history of plants. It lacks vasculature but exhibits alteration between generations, which is dominated by a gametophytic (haploid) phase and a short sporophytic (diploid) phase (Cove et al., 2009). Many of the pathways related to hormone signaling, stress responses, and development are conserved between angiosperms and P. patens (Cove et al., 2009; Sun, 2011; Komatsu et al., 2013; Yasumura et al., 2015). It is also an intriguing example in the context of the G-protein signaling, because its fully sequenced genome does not encode a canonical Gα gene, although genes coding for the Gβ and Gγ proteins exist. A single gene for a potential XLG homolog also exists in the P. patens genome. This unique assortment of proteins predicts several alternative scenarios for G-protein signaling in P. patens. For example, the P. patens Gβγ proteins might be nonfunctional due to the loss of canonical Gα and are left in the genome as evolutionary artifacts. Alternatively, the Gβγ proteins of P. patens might maintain functionality regardless of the existence of a canonical Gα protein in pathways not regulated via classic G-protein signaling modes. Finally, a more likely scenario could be that the potential XLG protein can substitute for the Gα function in P. patens.To explore these possibilities and understand better the conserved and unique mechanisms of G-protein signaling pathways in plants and their significance, we examined the role of G-protein subunits in P. patens. We provide unambiguous evidence for the genetic coupling of XLG and Gβ proteins in controlling P. patens development. In contrast to all other plant species analyzed to date, where G-proteins are not essential for growth and survival, the XLG or one of the Gβ proteins is required for the sporophyte formation and life cycle completion in P. patens. Furthermore, one of the Arabidopsis XLG proteins, XLG2, and the canonical Gβ protein AGB1 can functionally complement the P. patens mutant phenotypes. These data provide new insights in the evolutionary breadth and the spectrum of signaling pathways regulated by G-proteins in plants. 相似文献
10.
M Angeles Jiménez Ana C Barrachi-Saccilotto Eva Valdivia Mercedes Maqueda Manuel Rico 《Journal of peptide science》2005,11(1):29-36
Bacteriocin AS-48 is a 70-residue cyclic polypeptide from Enterococcus faecalis that shows a broad antimicrobial spectrum against both Gram-positive and Gram-negative bacteria. The structure of bacteriocin AS-48 consists of a globular arrangement of five helices with a high positive electrostatic potential in the region comprising helix 4, the turn linking helix 4 and 5, and the N-terminus of helix 5. This region has been considered to participate in its biological activity and in particular in membrane permeation. To understand the mechanism of the antibacterial activity of AS-48 and to discriminate the several mechanisms proposed, a simplified bacteriocin was designed consisting of 21 residues and containing the high positively charged region. A disulfide bridge was introduced at an appropriate position to stabilize the peptide and to conserve the helix-turn-helix arrangement in the parent molecule. According to (1)H and (13)C NMR data, the designed simplified bacteriocin fragment adopts a significant population of a native-like helical hairpin conformation in aqueous solution, which is further stabilized in 30% TFE. The designed peptide does not show any antibacterial activity, though it is shown to compete with the intact native bacteriocin AS-48. These results suggest that the mechanism of membrane disruption by bacteriocin is not as simple as being driven by a deposition of positively charged molecules on the plane of the bacterial membrane. Some other regions of the protein must be present such as, for instance, hydrophobic regions so as to enhance the accumulation of the peptide and favour membrane permeation. 相似文献
11.
Takao Ojima Tomokatsu Ohta Kiyoyoshi Nishita 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,129(4)
The complete amino acid sequence of squid Todarodes pacificus troponin C (TnC), which was shown to bind only 1 mol Ca2+/mol, was determined by both the Edman and cDNA methods. The squid TnC is composed of 147 amino acids including an unblocked Pro at the N-terminus and the calculated molecular weight is 17 003.9. Among the four potential Ca2+-binding sites, namely sites I–IV from the N-terminus, only site IV completely satisfied the consensus amino acid sequence for the active Ca2+-binding loop. This indicates that squid TnC possesses a single Ca2+-binding site at the site IV as scallop TnCs [Nishita et al., J. Biol. Chem. 269 (1994) 3464–3468; Ojima et al., Arch. Biochem. Biophys. 311 (1994) 272–276). The sequence homology of squid TnC to TnCs of scallop, arthropods, and rabbit was 61%, 31–38%, and 31%, respectively. In the sequence of the central D/E-helix region of squid and scallop TnCs, a deletion of three amino acids was required to maximize the homology with the other TnCs. 相似文献
12.
This minireview in memory of Daniel I. Arnon, pioneer in photosynthesis research, concerns properties of the first and still only known alternative photophosphorylation system, with respect to the primary phosphorylated end product formed. The alternative to adenosine triphosphate (ATP), inorganic pyrophosphate (PPi), was produced in light, in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, when no adenosine diphosphate (ADP) had been added to the reaction mixture (Baltscheffsky H et al. (1966) Science 153: 1120–1122). This production of PPi and its capability to drive energy requiring reactions depend on the activity of a membrane bound inorganic pyrophosphatase (PPase) (Baltscheffsky M et al. (1966) Brookhaven Symposia in Biology, No. 19, pp 246–253); (Baltscheffsky M (1967) Nature 216: 241–243), which pumps protons (Moyle J et al. (1972) FEBS Lett 23: 233–236). Both enzyme and substrate in the PPase (PPi synthase) are much less complex than in the case of the corresponding adenosine triphosphatase (ATPase, ATP synthase). Whereas an artificially induced proton gradient alone can drive the synthesis of PPi, both a proton gradient and a membrane potential are required for obtaining ATP. The photobacterial, integrally membrane bound PPi synthase shows immunological cross reaction with membrane bound PPases from plant vacuoles (Nore BF et al. (1991) Biochem Biophys Res Commun 181: 962–967). With antibodies against the purified PPi synthase clones of its gene have been obtained and are currently being sequenced. Further structural information about the PPi synthase may serve to elucidate also fundamental mechanisms of electron transport coupled phosphorylation. The existence of the PPi synthase is in line with the assumption that PPi may have preceded ATP as energy carrier between energy yielding and energy requiring reactions. 相似文献
13.
14.
Saskia C. F. Milton R.C. de Lisle Milton Steven A. Kates Charles Glabe 《Letters in Peptide Science》1999,6(2-3):151-156
Fmoc-amino acid fluorides are highly efficient coupling agents for solution and solid-phase peptide synthesis and this property was used advantageously for the manual solid-phase assembly of the Alzheimer's A 1–42 peptide [Milton et al., In Marshak, D. (Ed.) Techniques in Protein Chemistry, Vol. VIII, Academic Press, Orlando, FL, 1997, pp. 865–873]. Further studies comparing this methodology in the preparation of A 1–42 peptide analogs employing a fully automated continuous-flow peptide synthesizer are reported. 相似文献
15.
Antonio Gálvez Mercedes Maqueda Manuel Martínez-Bueno Mariam Lebbadi Eva Valdivia 《Applied microbiology and biotechnology》1993,39(4-5):438-442
An antifungal substance named peptide A12-C has been purified to homogeneity from supernatants of sporulated cultures of Bacillus licheniformis A12. It consists of a 0.77-kDa hydrophilic peptide containing two residues of Glu and one of Arg, Ala, Pro, Tyr and Orn. No fatty acids, phosphorus or carbohydrates have been detected. Peptide A12-C is active on several fungi (Microsporum canis CECT 2797, Mucor mucedo CECT 2653, M. plumbeus (CCM F 443, Sporothrix schenckii CECT 2799 and Trichophyton mentagrophytes CECT 2793) and bacteria (Bacillus megaterium, Corynebacterium glutamicum, Sarcina and Mycobacterium), although the latter are less sensitive.
Correspondence to: A. Gálvez 相似文献
16.
Valérie Brachet Gra?a Raposo Sebastian Amigorena Ira Mellman 《The Journal of cell biology》1997,137(1):51-65
Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three αβ dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting αβ dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-Ab αβ dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-Ab class II molecules in lysosomes. In leupeptin, I-Ab dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-Ab dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of αβ dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.The initiation of most immune responses requires antigen recognition by helper T lymphocytes. The antigen receptors on T cells can only recognize antigens as small peptides bound to major histocompatibility complex (MHC)1 class II molecules at the surface of antigen presenting cells (Cresswell, 1994; Germain, 1994). The complexes between class II molecules and antigenic peptides are formed intracellularly somewhere along the endocytic pathway (Germain, 1994; Wolf and Ploegh, 1995). This process requires the internalization of protein antigen and its delivery to a site suitable for the generation of antigenic peptides. In addition, the peptides must be generated within, or transferred to, a site to which newly synthesized MHC class II molecules are delivered and rendered competent for peptide binding (Davidson et al., 1991).Invariant (Ii) chain plays a central role in controlling the intracellular transport of MHC class II (Cresswell, 1996). In the ER, Ii chain is synthesized as a trimer that complexes with three αβ dimers of MHC class II (Roche et al., 1991). Its NH2-terminal cytoplasmic domain contains a wellknown targeting signal that directs class II–Ii chain complexes to endosomes after exit from the TGN (Bakke and Dobberstein, 1990; Lotteau et al., 1990; Neefjes et al., 1990; Odorizzi et al., 1994; Pieters et al., 1993). Once in endosomes, Ii chain is subjected to proteolysis by acid hydrolases (Roche and Cresswell, 1991). Degradation occurs in a stepwise fashion, resulting in the appearance of class II– bound NH2-terminal intermediates containing the Ii chain cytoplasmic domain, membrane anchor, and parts of its luminal domain (Newcomb and Cresswell, 1993). The intermediates accumulate in the presence of protease inhibitors that interfere with Ii chain processing such as the serinecysteine protease inhibitor leupeptin, treatment with which can also block the transport of at least some class II haplotypes to the cell surface (Amigorena et al., 1995; Blum and Cresswell, 1988; Neefjes and Ploegh, 1992). How leupeptin inhibits surface appearance is unknown.In human cells, Ii chain degradation intermediates include a 21–22-kD fragment (designated LIP [leupeptininducible peptide]) and a 10–12-kD fragment (designated SLIP [small leupeptin-inducible peptide]) (Blum and Cresswell, 1988; Maric et al., 1994). In murine cells, only a 10– 12-kD fragment has been identified (Ii-p10) (Amigorena et al., 1995). Ii-p10 remains as a trimer associated with three αβ dimers and blocks the binding of antigenic peptides (Amigorena et al., 1995; Morton et al., 1995). It is thus likely that Ii-p10 includes a luminal region of Ii chain (designated CLIP) known to occupy the peptide binding groove of αβ dimers. Cleavage of Ii-p10 by a leupeptinsensitive protease causes its dissociation from αβ dimers, while leaving CLIP in the peptide binding groove. The removal of CLIP is favored at acidic pH but is additionally catalyzed by a second MHC gene product, HLA-DM (Sloan et al., 1995; Denzin and Cresswell, 1995; Karlsson et al., 1994; Roche, 1995). In mutant cells lacking HLA-DM, there is defective loading of antigenic peptides and the appearance of CLIP-αβ dimers on the plasma membrane (Mellins et al., 1994; Riberdy et al., 1992).The precise site(s) where these events occur remains unclear. In A20 B cells, a specialized population of endosome-like vesicles designated CIIV (for class II vesicles) represents a site through which a majority of newly synthesized class II molecules pass en route to the cell surface and a place where antigenic peptides bind αβ dimers of the I-Ad haplotype (Amigorena et al., 1994, 1995; Barnes and Mitchell, 1995). CIIV are physically distinct from the bulk of endosomes and lysosomes and contain at least some HLA-DM (Pierre et al., 1996). Despite the fact that most of the αβ dimers reaching CIIV are newly synthesized, CIIV contain little or no intact Ii chain (Amigorena et al., 1995). Thus, Ii chain–αβ complexes first may be delivered to endosomes where Ii chain is cleaved before being delivered to CIIV. That peptide loading can occur in CIIV has been demonstrated by experiments showing that leupeptin causes CIIV to transiently accumulate Ii-p10– containing complexes, which can then bind peptide (Amigorena et al., 1995).In human Epstein-Barr virus–transformed B lymphoblasts, most class II molecules have been localized to structures collectively designated MIIC (for MHC class II compartment) (Peters et al., 1991; Tulp et al., 1994; West et al., 1994). MIICs differ from CIIVs in that the latter contain endosomal but not lysosomal markers, while MIICs have most or all of the features of lysosomes (Peters et al., 1991, 1995; Pierre et al., 1996). Interestingly, the distribution of class II between endosomal (CIIV) and lysosomal (MIIC) compartments varies widely among cell types. Since lysosomes are classically defined as terminal degradative organelles (Kornfeld and Mellman, 1989), such variations may reflect differences in the rates at which class II is turned over in different cell types. On the other hand, MIICs also contain the bulk of HLA-DM and can host the loading of antigenic peptides onto class II molecules (Sanderson et al., 1994). The extent to which these complexes escape degradation and reach the cell surface is unclear. Nor is it at all clear how different cell types regulate the intracellular distribution of class II molecules between early and late endocytic compartments.We now show that murine A20 cells expressing endogenous I-Ad and transfected I-Ab normally localize little class II in lysosomes. Selective lysosomal accumulation of I-Ab αβ dimers can be induced after leupeptin treatment. Interestingly, I-Ab dimers, but not I-Ad dimers, are induced by leupeptin to form stable complexes with Ii-p10. Upon removal of the inhibitor, the Ii-p10 was removed and class II molecules were slowly transported from lysosomes to the cell surface. Thus, the rate of dissociation of Ii chain intermediates can regulate whether newly synthesized class II molecules are transported to the plasma membrane or to lysosomes. 相似文献
17.
Response of Salmonella choleraesuis LT2 Spheroplasts and Permeabilized Cells to the Bacteriocin AS-48 总被引:4,自引:0,他引:4
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The bacteriocin AS-48 was not active against intact cells of Salmonella choleraesuis LT2 at neutral pH, but it was very effective on spheroplasts, suggesting that the outer membrane (OM) acts as a protective barrier. Cells sublethally injured by heat or treated with OM-permeabilizing agents (i.e., EDTA and Tris) became sensitive to AS-48. The combination of two or more treatments decreased the amount of AS-48 required for cell killing. The activity of AS-48 against heat-injured cells did not change significantly in the pH range of 4.0 to 8.0. AS-48 showed bactericidal activity against intact cells of Salmonella at pH 4.0. The potency of AS-48 increased greatly when the bacteriocin was dissolved at pH 9.0. 相似文献
18.
The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1 总被引:7,自引:0,他引:7
Loren D. Walensky Philippe Gascard Michael E. Field Seth Blackshaw John G. Conboy Narla Mohandas Solomon H. Snyder 《The Journal of cell biology》1998,141(1):143-153
We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds. 相似文献
19.
Permeation of bacterial cells, permeation of cytoplasmic and artificial membrane vesicles, and channel formation on lipid bilayers by peptide antibiotic AS-48. 总被引:4,自引:0,他引:4
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Peptide AS-48 induces ion permeation, which is accompanied by the collapse of the cytoplasmic membrane potential, in sensitive bacteria. Active transport by cytoplasmic membrane vesicles is also impaired by AS-48. At low concentrations, this peptide also causes permeability of liposomes to low-molecular-weight compounds without a requirement for a membrane potential. Higher antibiotic concentrations induce severe disorganization, which is visualized under electron microscopy as aggregation and formation of multilamellar structures. Electrical measurements suggest that AS-48 can form channels in lipid bilayers. 相似文献
20.
An experimental phylogeny was constructed using bacteriophage T7 and a propagation protocol, in the presence of the mutagen N-methyl-N′-nitro-N′-nitrosoguanidine, based on Hillis et al. [Hillis, D.M., Bull, J.J., White, M.E., Badgett, M.R., Molineux, I.J., 1992. Experimental phylogenetics, generation of a known phylogeny. Science 255, 589–592]. The topology presented in this study has a considerable variation in branch lengths and is less symmetric than the one presented by Hillis et al. [Hillis, D.M., Bull, J.J., White, M.E., Badgett, M.R., Molineux, I.J., 1992. Experimental phylogenetics, generation of a known phylogeny. Science 255, 589–592]. These features are known to present additional difficulties to phylogenetic inference methods. The performance of several phylogenetic methods (conventional and less conventional) was tested using restriction site and nucleotide data. Only methods that encompassed a molecular clock or those based on sequence signatures recovered the true phylogeny. Nevertheless a likelihood ratio test rejected the hypothesis of the existence of a molecular clock when the whole sequence data set was considered. This fact or the particular substitution pattern (mainly G → A and C → T) may be related to the unexpected performance of distance methods based on sequence signatures. To test if the results could have been predicted by simulation studies we estimated the evolution parameters from the real phylogeny and used them to simulate evolution along the same tree (parametric bootstrap). We found that simulation could predict most but not all of the problems encountered by phylogenetic inference methods in the real phylogeny. Short interior branches may be more prone to error than predicted by theoretical studies. 相似文献