Membrane-Localized Extra-Large G Proteins and Gβγ of the Heterotrimeric G Proteins Form Functional Complexes Engaged in Plant Immunity in Arabidopsis

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.     

Dissociated GαGTP and Gβγ Protein Subunits Are the Major Activated Form of Heterotrimeric Gi/o Proteins

Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.     

Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites.Heterotrimeric G proteins undergo cycles of GTP-dependent conformational rearrangements and alterations of their oligomeric αβγ form to convey receptor signals to downstream effectors that control diverse cellular processes ranging from movement to division and differentiation. Interaction with activated receptor promotes the exchange of GDP for GTP on the G protein α subunit (Gα) and its separation from its βγ subunit partners (Gβγ). Both isolated Gα and Gβγ then interact with downstream effectors. GTP hydrolysis deactivates Gα, which reassociates with Gβγ, becoming ready to restart the cycle. Each of these stages has been subjected to extensive crystallographic studies with high-resolution structures of Gα in complex with GDP, GTP analog, Gβγ, and, most recently, the G-protein-coupled receptors now available. These studies have provided extensive mechanistic insight. However, a number of important questions remain, including:

• How do the distinct conformations evident in the accumulated structures interconvert?
• How do disease-associated mutations affect the fidelity of these transitions?
• And, critically, how do distal functional sites responsible for nucleotide and protein partner binding allosterically coordinate their activities?

Here we describe a comprehensive analysis of the accumulated Gα crystallographic structures supplemented with extensive conventional (cMD) and accelerated molecular dynamics (aMD) simulations (1) that together map the structural and dynamical features of Gα in different nucleotide states. These enhanced sampling simulations reveal the spontaneous interconversion between GDP and GTP conformations and also characterize large-scale opening motions of the α-helical domain (HD) that were not accessible to previous simulation studies (2–5). Furthermore, the current simulations results reveal a distinctive pattern of collective motions that provide evidence for a nucleotide-dependent network of dynamic communication between the active site and the receptor and effector binding sites.Principal component analysis of 53 Gα experimental structures homologous to transducin (Gαt) reveals that the major variation in accumulated structures is the concerted association/disassociation of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII). An additional small-scale (<10°) rotation of the HD relative to the main catalytic Ras-like domain (RasD) is also apparent (see Fig. S1 in the Supporting Material). The distinct conformation of SI–SIII regions gives rise to nucleotide-associated segregation of GDP- and GTP-analog-bound experimental structures along the PC1-PC2 plane. Interestingly, both GDP- and GTP-bound structures display a skewed distribution along the PC1-PC2 plane that arises from HD rotation. In comparison, the distribution of the GTP-bound structures becomes more restricted and the skew decreases when the mapping is based on a principle component analysis that excludes the HD region (see Fig. S1).Recently, the HD region of Gαs (the α-subunit of the stimulatory G protein for adenyl cyclase) was shown to adopt a dramatically more open conformation in a crystal structure complex with the β2 adrenergic receptor (β2AR) (6). This clam-shell-like 127° opening in the absence of nucleotide and presence of receptor is consistent with electron microscopy (7) and double electron-electron resonance analysis (8). These results, together with recent hydrogen-deuterium exchange mass spectrometry data (9), indicate that there may be additional functional motions and inherent flexibility in the ensemble of native states beyond those apparent in the accumulated crystal structures of Gαt (9). To address this question, we performed multiple 100-ns aMD simulations of nucleotide-free Gαt. These simulations reveal a spontaneous large-scale opening and closing motion of larger magnitude (>60°) than those evident in the distribution of crystallographic structures (Fig. 1 A and see Fig. S2). In addition, the trajectory reveals two dominant modes of HD opening: an out-of-plane shifting (PC1 in Fig. S3) and an in-plane rotation (PC2 in Fig. S3). It is also notable that nucleotide-free aMD simulations sample both active (GTP-like) and inactive (GDP-like) structures (see Fig. S2) in an analogous manner to the spontaneous GDP to GTP interconversion sampled for Ras and Rho small G proteins with similar methods (10–12).Open in a separate windowFigure 1Nucleotide-associated differences in flexibility and dynamic coupling. (A) Mapping aMD simulation trajectories (blue points) onto the principal components obtained from analysis of Gα crystallographic GDP-bound (green) and GTP-analog bound (red) experimental structures. (Orange) Open β2AR-Gαs complex structure. (B) Results of dynamic coupling analysis mapped onto the average structure for each nucleotide state. (Spheres) Nodes for the nucleotide; the protein cartoon is colored by community structure. (C) Community network graph. (Circles) Communities, colored as in panel B. Radius of the circle indicates the number of residues in the community. Thickness of linking lines is determined by the maximum betweenness of the respective intercommunity edges (see the Supporting Material). (Red, blue, and green edges) Major topological difference between states.The low sequence identity between Gαt and Gαs (44.5%), as well as the absence of the receptor and Gβγ in the simulations, may explain the difference between the predicted ∼60° Gαt-HD rotation and that displayed in the β2AR-Gαs crystallographic structure (see Fig. S3). It is notable that, although the amplitude is much smaller, aMD simulations with bound nucleotide display similar dominant HD motions to those observed in the nucleotide-free simulations (see Fig. S4). This suggests that the interdomain flexibility of RasD and HD is likely an intrinsic feature of Gαt regardless of nucleotide state.The transition between distinct conformations (structural clusters; see Fig. S5) was observed to correspond to significant dynamical changes in side-chain contacts (see Fig. S6). Specifically, we found sequential contacts breaking during the HD in-plane rotation motion starting from the region between HD helix αD and RasD helix αG toward that between HD helix αE and RasD SIII and the P-loop. In comparison, for the out-of-plane shift, we found simultaneous breaking and formation of contacts in the region containing the loop between helices αB and αC, the N-terminus of αA, αE, and αF of HD; α1, SI, and the loop between strand β6 and helix α5 of RasD. Interactions highlighted in these regions as potentially important for the conformational transitions include D137::K276, S140::K273, S140::D227, Q143::R238, N145::E39, and D146::K266, the effect of which can be further evaluated by mutagenesis experiments and simulations.Dynamic network analysis methods developed by Sethi et al. (13) were used to examine whether the motions of one residue were correlated to the motions of another (distant) residue. In this approach, a weighted graph is constructed where each residue represents a node and the weight of the connection between nodes represents their respective correlation value. A clustering of edges is then used to define local communities of highly correlated residues that represent substructures that are highly intraconnected, but loosely interconnected. Applying this approach to multiple 40-ns cMD simulations initiated from GTP-, GDP-, and aMD-derived APO conformations revealed a consistent community composition as well as a distinct pattern of intercommunity connection between nucleotide states (Fig. 1, B and C).The dynamics of the RasD region can be decomposed into two main communities that stem from the nucleotide base and phosphate regions in GDP and GTP states: The first community is composed of residues from the P-loop, helix α1, strands β1–β3, and the phosphates of the nucleotide (orange in Fig. 1, B and C). The second community comprises residues from helix αG, strands β4–β6, and the nucleotide base region (tan in Fig. 1, B and C). This dynamic partitioning of the central β-sheet and central role of the nucleotide is consistent with the bilobal structure and dynamics previously reported for Ras (14). In the presence of GTP, the first community includes or is dynamically coupled to SI, SII, and SIII regions (see the orange node and the red edge in Fig. 1 C). Removal of the γ-phosphate of GTP disrupts this region, leading to decoupling of the switch regions from the nucleotide. Also evident for GDP states is an apparent tighter coupling of RasD and HD regions (blue edges in Fig. 1 C). We note that these findings are robust to the choice of initial simulation conditions and are observed in both cMD and aMD simulations (see Fig. S7 and Fig. S8). Nucleotide-free Gαt simulations display an altered dynamical network with respect to those of nucleotide bound states. In particular, RasD and HD regions lose connecting edges consistent with the large-scale opening of these domains (e.g., SIII-HD green edges in Fig. 1 C).A number of residues highlighted here as potentially important for mediating the coupling between prominent communities (see Table S1 in the Supporting Material) have been shown by previous mutagenesis studies to affect GDP release. For example, the double mutation A322S/R174M was found to significantly enhance the rate of GDP release (15). The current results indicate that these positions are involved in coupling the nucleotide and RasD. Also, mutations R144A and L232Q caused a faster basal GDP release rate in Gαi1 (16). The current analysis indicates that the equivalent positions in Gαt (S140 and M228) couple the RasD and HD, and suggests that their mutation could promote domain-domain motions. We also note the apparent coupling of α5 with the nucleotide base and Ploop-β1 with the phosphate regions of GDP. These direct connections of the receptor connecting N- and C-terminus to GDP are suggestive of potential routes for receptor-mediated GDP release. We expect further study of these sites and of receptor-bound dynamics to be informative in this regard.In conclusion, simulations suggest a flexible HD in Gαt similar to that found for Gαs. In particular, in the absence of nucleotide we observed the spontaneous large-scale opening and closing of HD relative to RasD, which was unseen in previous computational studies. Moreover, we found that the functional states of Gαt are associated with the distinct dynamical couplings of functional regions including SI–SIII, P-loop, α5, and the HD region. Finally, our results indicate that nucleotide may not directly induce large-scale conformational changes but, instead, act as a modulator of intrinsically accessible conformations and as a central participant in their associated dynamical couplings.     

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

Heparan Sulfates Mediate the Interaction between Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) and the Gαq/11 Subunits of Heterotrimeric G Proteins

The endothelial cell-cell junction has emerged as a major cell signaling structure that responds to shear stress by eliciting the activation of signaling pathways. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and heterotrimeric G protein subunits Gα_q and 11 (Gα_q/11) are junctional proteins that have been independently proposed as mechanosensors. Our previous findings suggest that they form a mechanosensitive junctional complex that discriminates between different flow profiles. The nature of the PECAM-1·Gα_q/11 interaction is still unclear although it is likely an indirect association. Here, we investigated the role of heparan sulfates (HS) in mediating this interaction and in regulating downstream signaling in response to flow. Co-immunoprecipitation studies show that PECAM-1·Gα_q/11 binding is dramatically decreased by competitive inhibition with heparin, pharmacological inhibition with the HS antagonist surfen, and enzymatic removal of HS chains with heparinase III treatment as well as by site-directed mutagenesis of basic residues within the extracellular domain of PECAM-1. Using an in situ proximity ligation assay, we show that endogenous PECAM-1·Gα_q/11 interactions in endothelial cells are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan syndecan-1 in complexes with PECAM-1 that are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in endothelial cells in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1·Gα_q/11 mechanosensitive complex contains an endogenous heparan sulfate proteoglycan with HS chains that is critical for junctional complex assembly and regulating the flow response.     

The Intriguing Evolution of the “b” and “G” Subunits in F-type and V-type ATPases: Isolation of the vma-10 Gene from Neurospora crassa

We have characterized the vma-10 gene which encodes the G subunit of the vacuolar ATPase in Neurospora crassa. The gene is somewhat unusual in filamentous fungi because it contains five introns, comprising 71% of the region between the translation start and stop codons. The 5 untranslated region of the gene contains several elements that have been identified in other genes that encode subunits of the vacuolar ATPase in N. crassa. A comparison of G subunits from N. crassa, S. cerevisiae, and animal cells showed that the N-terminal half of the polypeptide shows the highest degree of sequence conservation. Most striking is the observation that this region could form an alpha helix in which all of the conserved residues are clustered on one face. Subunit G appears to be homologous to the b subunit found in F-type ATPases. The major difference between the b and G subunits is the lack of a membrane-spanning region in the G subunit. We have also identified homologous subunits in the operons which encode V-type ATPases in a eubacterium, Enterrococcus hirae, and an archaebacterium, Methanococcus jannaschii. As in eukaryotic vacuolar ATPases the G subunit homologs lack a membrane-spanning region. Although the b and G subunits appear to be derived from a common ancestor, significant changes have evolved. In F-type and V-type ATPases these subunits can have zero, one, or two membrane-spanning regions and can also differ significantly in the number of copies per enzyme.     

Ontogeny of the Spitzenkörper in germlings of Neurospora crassa

The Spitzenkörper (Spk) is a highly dynamic and pleomorphic complex located at the hyphal apex of filamentous fungi. Most studies revealing the structure and behavior of the Spk have been conducted on mature vegetative hyphae of filamentous fungi, including both main leading hyphae and branches. However, these reports do not address whether the observations can be extended to germ tubes. By enhanced phase-contrast video-microscopy and laser scanning confocal microscopy we have analyzed the intracellular changes prior to the appearance of a Spk in germlings of Neurospora crassa. Observations began at the early stages of spore germination and were carried out until a conspicuous Spk could be observed at the apex of germ tubes. Before a Spk could be observed, young germ tubes (<150 μm) displayed a uniform distribution of organelles such as nuclei, mitochondria, and cytoplasmic granules along the length of the cells. Once the germlings started reaching lengths of more than approximately 150 μm, visible organelles experienced a displacement towards the subapical region of the cell and a small exclusion zone free of organelles (0.6 ± 0.3 μm) formed at the apex. The position of this exclusion zone within the apex seemed to determine the germling growth direction, which was highly erratic. Few minutes after it first appeared, upon growth of the germling, the exclusion zone started to become occupied by an accumulation of material that gradually concentrated into a light gray body that we describe as an immature Spk. During this phase the presence of a Spk in the apical dome was not constant. Approximately 30 min later, the immature Spk became more robust and gradually acquired its typical phase-dark appearance, while the growth direction of the germ tube became less wavering. The formation of a mature phase-dark Spk coincided with the stabilization of the growth direction of the germling, therefore suggesting that it is at this stage when the transition from germling to vegetative hypha occurs.     

Are carotenoids the blue-light photoreceptor in the photoinduction of protoperithecia in Neurospora crassa?

A triple albino mutant of Neurospora crassa with a measured content of carotenoids absorbing at 470 nm less than 0.5% of that of the wild type (calculated value less than 8·10^-4%) had the same threshold for photoinduction of protoperithecia as the wild type when illuminated with monochromatic light at 471 nm. This is strong evidence against the hypothesis that the bulk of carotenoids are the blue-light photoreceptor for this phenomenon. However, it is impossible to exclude traces of carotenoids acting as the photoreceptor at less than 3·10^-12 M in a very efficient sensory transduction chain.Abbreviations A absorbance - al albino mutant - WT wild type     

Genetic interactions between clock mutations in Neurospora crassa: can they help us to understand complexity?

Recent work on circadian clocks in Neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. However, a number of other genes are known that affect either the period or temperature compensation of the rhythm. These include the period (no relationship to the period gene of Drosophila) genes and a number of genes that affect cellular metabolism. How these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered in single-oscillator models. Recent evidence has pointed to multiple oscillators in Neurospora, at least one of which is predicted to incorporate metabolic processes. Here, the Neurospora clock-affecting mutations will be reviewed and their genetic interactions discussed in the context of a more complex clock model involving two coupled oscillators: a FRQ/WC-based oscillator and a 'frq-less' oscillator that may involve metabolic components.     

Anticooperative Nearest-Neighbor Interactions between Residues in Unfolded Peptides and Proteins

Growing evidence suggests that the conformational distributions of amino acid residues in unfolded peptides and proteins depend on the nature of the nearest neighbors. To explore whether the underlying interactions would lead to a breakdown of the isolated pair hypothesis of the classical random coil model, we further analyzed the conformational propensities that were recently obtained for the two guest residues (x,y) of GxyG tetrapeptides. We constructed a statistical thermodynamics model that allows for cooperative as well as for anticooperative interactions between adjacent residues adopting either a polyproline II or a β-strand conformation. Our analysis reveals that the nearest-neighbor interactions between most of the central residues in the investigated GxyG peptides are anticooperative. Interaction Gibbs energies are rather large at high temperatures (350 K), at which point many proteins undergo thermal unfolding. At room temperature, these interaction energies are less pronounced. We used the obtained interaction parameter in a Zimm-Bragg/Ising-type approach to calculate the temperature dependence of the ultraviolet circular dichroism (CD) of the MAX3 peptide, which is predominantly built by KV repeats. The agreement between simulation and experimental data was found to be satisfactory. Finally, we analyzed the temperature dependence of the CD and ³J(H^NH^α) parameters of the amyloid β_1–9 fragment. The results of this analysis and a more qualitative consideration of the temperature dependence of denatured proteins probed by CD spectroscopy further corroborate the dominance of anticooperative nearest-neighbor interactions. Generally, our results show that unfolded peptides—and most likely also proteins—exhibit some similarity with antiferromagnetic systems.     

δ-Aminolaevulinate dehydratase, the regulatory enzyme of the haem-biosynthetic pathway in Neurospora crassa

The activity of delta-aminolaevulinate dehydratase is very low in the mould Neurospora crassa compared with the activities detected in bacterial and animal systems. The enzyme is inducible in iron-deficient cultures by addition of iron and is repressed by protoporphyrin. The properties of the purified enzyme indicate its allosteric nature and susceptibility to feedback inhibition by coproporphyrinogen III. Neurospora extracts also contain a protein inhibitor of the enzyme and a small-molecule activator, which appears to be associated with the enzyme. The regulatory function of this enzyme in vivo is correlated with the accumulation of delta-aminolaevulinic acid in normal cultures of N. crassa. The decay curve of the iron-induced enzyme in vivo shows a biphasic pattern, with one of the components showing a half-life of 4-5 min.     

Analysis of Mutations in Neurospora crassa ERMES Components Reveals Specific Functions Related to β-Barrel Protein Assembly and Maintenance of Mitochondrial Morphology

The endoplasmic reticulum mitochondria encounter structure (ERMES) tethers the ER to mitochondria and contains four structural components: Mmm1, Mdm12, Mdm10, and Mmm2 (Mdm34). The Gem1 protein may play a role in regulating ERMES function. Saccharomyces cerevisiae and Neurospora crassa strains lacking any of Mmm1, Mdm12, or Mdm10 are known to show a variety of phenotypic defects including altered mitochondrial morphology and defects in the assembly of β-barrel proteins into the mitochondrial outer membrane. Here we examine ERMES complex components in N. crassa and show that Mmm1 is an ER membrane protein containing a Cys residue near its N-terminus that is conserved in the class Sordariomycetes. The residue occurs in the ER-lumen domain of the protein and is involved in the formation of disulphide bonds that give rise to Mmm1 dimers. Dimer formation is required for efficient assembly of Tom40 into the TOM complex. However, no effects are seen on porin assembly or mitochondrial morphology. This demonstrates a specificity of function and suggests a direct role for Mmm1 in Tom40 assembly. Mutation of a highly conserved region in the cytosolic domain of Mmm1 results in moderate defects in Tom40 and porin assembly, as well as a slight morphological phenotype. Previous reports have not examined the role of Mmm2 with respect to mitochondrial protein import and assembly. Here we show that absence of Mmm2 affects assembly of β-barrel proteins and that lack of any ERMES structural component results in defects in Tom22 assembly. Loss of N. crassa Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.     

Synchronization of DNA synthesis in Neurospora crassa by 2′-deoxyadenosine and spore selection

2-Deoxyadenosine (2 mM), a DNA inhibitor, was used to synchronize DNA synthesis in cultures of Neurospora crassa lys 3. The cultures recovered spontaneously from the inhibitor which had little or no effect on the synthesis of RNA, protein or carbohydrate or on the specific growth rate of the mould. The degree of synchrony of DNA synthesis obtained with 2-deoxyadenosine varied directly with the organism's specific growth rate when the latter was altered by temperature changes. A direct relationship was observed between the rate of synthesis of DNA during the S period and the organism's specific growth rate.Conidia of Neurospora crassa lys 3 were separated into different density classes using urografin gradients; the separation treatment did not have an appreciable effect on the subsequent germination or growth of conidia. Populations of large, less dense conidia produced germ tubes more rapidly and more synchronously than populations of small, dense conidia. Cultures inoculated with the large conidia displayed continuous synthesis of RNA and protein but discontinuous synthesis of DNA.     

Separation of chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa

Cells from the slime variant of Neurospora crassa were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum and vacuolar contaminants as demonstrated by determination of appropriate enzymatic markers.Abbreviations ER Endoplasmic reticulum - UDP-GlcNAc uridine-5-diphosphate N-acetyl glucosamine - GlcNAc N-acetyl glucosamine - SDS sodium dodecyl sulfate - PMSF phenyl methyl sulfonyl fluoride - EDTA ethylene diamino tetraacetic acid Investigador Nacional de Mexico. On leave from the Centro de Investigacion y Estudios Avanzados (IPN), and the Universidad de Guanajuato, Gto., Mexico     

Double Suppression of the Gα Protein Activity by RGS Proteins

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Is the Fatty Acid Composition of Phospholipids Important for the Function of the Circadian Clock in Neurospora crassa?

Effects of pheriylmethanol (PM), 2-phenylethanol (PE) and 3-phenyl-1-propanol(PP) on the conidiation rhythm and on the fatty acid compositionof phospholipids were examined in Neurospora crassa. The periodfor the conidiation rhythm was shortened in proportion to theconcentration used of PE of more than 1 mM. PM at 10 mM andPP at 3 mM, however, did not cause a decrease in the lengthof the period. PE did not affect the temperature compensationof the length of the period or the amplitude of the phase shiftby light. The ratio of linolenic acid to Hnoleic acid decreased in proportionto the concentrations of PE, PM, and PP in all the phospholipidsexamined. The proportion of phosphatidic acid+diphosphatidylglycerolto the total phospholipids also was decreased by these compounds.Period shortening by PE can not be explained by the change inthe phospholipid fatty acid composition. (Received April 18, 1983; Accepted June 22, 1983)     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

Functional Characterization of the Conserved “GLK” Motif in Mitochondrial Porin from Neurospora crassa

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a glycine-leucine-lysine (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [-³²P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective -barrel that is not directly involved in ATP binding.