The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Severe impairment of growth and differentiation in a Neurospora crassa mutant lacking all heterotrimeric G alpha proteins

Heterotrimeric G alpha proteins play a critical role in regulating growth and differentiation in filamentous fungi. No systematic analysis of functional relationships between subunits has been investigated. This study explores the relative contributions of Neurospora crassa G alpha subunits, gna-1, gna-2, and gna-3, in directing development by analyzing strains deleted for various combinations of these genes. Although viable, mutants lacking all G alpha subunits or gna-1 and gna-3 are severely restricted in apical growth, forming small colonies. These strains form little aerial hyphae during asexual development on solid medium and exhibit inappropriate sporulation in submerged cultures. Similar to all strains carrying the Delta gna-1 mutation, these mutants are female sterile. Defects attributed to gna-2 are observed only in conjunction with the loss of gna-1 or gna-3, suggesting a minor role for this G alpha in N. crassa biology. Results from analysis of adenylyl cyclase and epistatic studies with the cAMP-dependent protein kinase regulatory subunit (mcb) indicate separate functions for GNA-1 and GNA-3 in cAMP metabolism and additional cAMP-independent roles for GNA-1. These studies indicate that although G alpha subunits are not essential for viability in filamentous fungi, their loss results in an organism that cannot effectively forage for nutrients or undergo asexual or sexual reproduction.     

Overlapping Functions for Two G Protein α Subunits in Neurospora Crassa

Heterotrimeric G proteins, consisting of α, β and γ subunits, mediate a variety of signaling pathways in eukaryotes. We have previously identified two genes, gna-1 and gna-2, that encode G protein α subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gna-2. Results from Western analysis and measurements of gna-2 promoter-lacZ fusion activity indicate that gna-2 is expressed during the vegetative and sexual cycle of N. crassa in both A and a mating types. Activating mutations predicted to abolish the GTPase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of Δgna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gna-2 in a Δgna-1 strain accentuates the Δgna-1 phenotypes. Δgna-1 Δgna-2 strains have a slower rate of hyphal apical extension than Δgna-1 strains on hyperosmotic media. Moreover, Δgna-1 Δgna-2 mutants have more pronounced defects in female fertility than Δgna-1 strains. We propose that gna-1 and gna-2 have overlapping functions and may constitute a gene family. This is the first report of G protein α subunits with overlapping functions in eukaryotic microbes.     

GPR-4 is a predicted G-protein-coupled receptor required for carbon source-dependent asexual growth and development in Neurospora crassa

The filamentous fungus Neurospora crassa is able to utilize a wide variety of carbon sources. Here, we examine the involvement of a predicted G-protein-coupled receptor (GPCR), GPR-4, during growth and development in the presence of different carbon sources in N. crassa. Deltagpr-4 mutants have reduced mass accumulation compared to the wild type when cultured on high levels of glycerol, mannitol, or arabinose. The defect is most severe on glycerol and is cell density dependent. The genetic and physical relationship between GPR-4 and the three N. crassa Galpha subunits (GNA-1, GNA-2, and GNA-3) was explored. All three Galpha mutants are defective in mass accumulation when cultured on glycerol. However, the phenotypes of Deltagna-1 and Deltagpr-4 Deltagna-1 mutants are identical, introduction of a constitutively activated gna-1 allele suppresses the defects of the Deltagpr-4 mutation, and the carboxy terminus of GPR-4 interacts most strongly with GNA-1 in the yeast two-hybrid assay. Although steady-state cyclic AMP (cAMP) levels are normal in Deltagpr-4 strains, exogenous cAMP partially remediates the dry mass defects of Deltagpr-4 mutants on glycerol medium and Deltagpr-4 strains lack the transient increase in cAMP levels observed in the wild type after addition of glucose to glycerol-grown liquid cultures. Our results support the hypothesis that GPR-4 is coupled to GNA-1 in a cAMP signaling pathway that regulates the response to carbon source in N. crassa. GPR-4-related GPCRs are present in the genomes of several filamentous ascomycete fungal pathogens, raising the possibility that a similar pathway regulates carbon sensing in these organisms.     

Positive regulation of adenylyl cyclase activity by a galphai homolog in Neurospora crassa

GNA-1 and GNA-2 are two G protein alpha subunits from the filamentous fungus Neurospora crassa. Loss of gna-1 leads to multiple phenotypes, while Deltagna-2 strains do not exhibit visible defects. However, Deltagna-1Deltagna-2 mutants are more affected in Deltagna-1 phenotypes. Here we report a biochemical investigation of the roles of GNA-1 and GNA-2 in cAMP metabolism. Assays of Mg2+ ATP-dependent adenylyl cyclase activity (+/-GppNHp) in extracts from submerged cultures indicated that Deltagna-2 strains were normal, whereas Deltagna-1 and Deltagna-1Deltagna-2 strains had only 10-15% the activity of the wild-type control. Levels of the Gbeta protein, GNB-1, were normal in Deltagna-1 strains, excluding altered GNB-1 production as a factor in loss of adenylyl cyclase activity. Steady-state cAMP levels in Deltagna-1 and Deltagna-1Deltagna-2 mutants were reduced relative to wild-type under conditions that result in morphological abnormalities (solid medium), while levels in submerged culture were normal. cAMP phosphodiesterase activities in submerged cultures of Deltagna-1 and/or Deltagna-2 strains were lower than in wild-type; the individual deletions were additive in decreasing activity. These results suggest that in submerged culture, N. crassa, like mammalian systems, possesses compensatory mechanisms that maintain cAMP at relatively constant levels. Furthermore, the finding that Mg2+ATP-dependent adenylyl cyclase activity in wild-type cell extracts could be inhibited using anti-GNA-1 IgG suggests that GNA-1 directly interacts with adenylyl cyclase in N. crassa.     

Use of 1H nuclear magnetic resonance to measure intracellular metabolite levels during growth and asexual sporulation in Neurospora crassa

Conidiation is an asexual sporulation pathway that is a response to adverse conditions and is the main mode of dispersal utilized by filamentous fungal pathogens for reestablishment in a more favorable environment. Heterotrimeric G proteins (consisting of α, β, and γ subunits) have been shown to regulate conidiation in diverse fungi. Previous work has demonstrated that all three of the Gα subunits in the filamentous fungus Neurospora crassa affect the accumulation of mass on poor carbon sources and that loss of gna-3 leads to the most dramatic effects on conidiation. In this study, we used (1)H nuclear magnetic resonance (NMR) to profile the metabolome of N. crassa in extracts isolated from vegetative hyphae and conidia from cultures grown under conditions of high or low sucrose. We compared wild-type and Δgna-3 strains to determine whether lack of gna-3 causes a significant difference in the global metabolite profile. The results demonstrate that the global metabolome of wild-type hyphae is influenced by carbon availability. The metabolome of the Δgna-3 strain cultured on both high and low sucrose is similar to that of the wild type grown on high sucrose, suggesting an overall defect in nutrient sensing in the mutant. However, analysis of individual metabolites revealed differences in wild-type and Δgna-3 strains cultured under conditions of low and high sucrose.     

Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the three N. crassa Gα subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3^Q208L allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.     

Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa

Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.     

The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa.

Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility.     

The heterotrimeric G-protein subunits GNG-1 and GNB-1 form a Gbetagamma dimer required for normal female fertility, asexual development, and galpha protein levels in Neurospora crassa

We have identified a gene encoding a heterotrimeric G protein gamma subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian Ggamma genes, consisting of three exons and two introns, with introns present in both the open reading frame and 5'-untranslated region. The GNG-1 amino acid sequence displays high identity to predicted Ggamma subunits from other filamentous fungi, including Giberella zeae, Cryphonectria parasitica, Trichoderma harzianum, and Magnaporthe grisea. Deletion of gng-1 leads to developmental defects similar to those previously characterized for Deltagnb-1 (Gbeta) mutants. Deltagng-1, Deltagnb-1, and Deltagng-1 Deltagnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Deltagnb-1 mutants, loss of gng-1 negatively influences levels of Galpha proteins (GNA-1, GNA-2, and GNA-3) in plasma membrane fractions isolated from various tissues of N. crassa and leads to a significant reduction in the amount of intracellular cyclic AMP. In addition, we show that GNB-1 is essential for maintenance of normal steady-state levels of GNG-1, suggesting a functional interaction between GNB-1 and GNG-1. Direct evidence for a physical association between GNB-1 and GNG-1 in vivo was provided by coimmunoprecipitation.     

Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa

We have identified a new gene encoding the G protein alpha subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the Galpha proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the Deltagna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to Deltagna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submerged-culture conidiation is refractory to cAMP but is suppressed by peptone. In addition, Deltagna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. Deltagna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of Deltagna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the Deltagna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.     

A pheromone receptor gene, pre-1, is essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa

Neurospora crassa is a heterothallic filamentous fungus with two mating types, mat a and mat A. Its mating involves differentiation of female reproductive structures (protoperithecia) and chemotropic growth of female-specific hyphae (trichogynes) towards a cell of the opposite mating type in a pheromone-mediated process. In this study, we characterize the pre-1 gene, encoding a predicted G-protein-coupled receptor with sequence similarity to fungal pheromone receptors. pre-1 is most highly expressed in mat A strains under mating conditions, but low levels can also be detected in mat a strains. Analysis of pre-1 deletion mutants showed that loss of pre-1 does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. Protoperithecia from Deltapre-1 mat A mutants do not undergo fertilization; this defect largely stems from an inability of their trichogynes to recognize and fuse with mat a cells. Previous work has demonstrated that the Galpha subunit, GNA-1, and the Gbeta protein, GNB-1, are essential for female fertility in N. crassa. Trichogynes of Deltagna-1 and Deltagnb-1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Deltapre-1 mat A strains. However, the female sterility defect of the Deltapre-1 mat A mutant could not be complemented by constitutive activation of gna-1, suggesting additional layers of regulation. We propose that PRE-1 is a pheromone receptor coupled to GNA-1 that is essential for the mating of mat A strains as females, consistent with a role in launching the pheromone response pathway in N. crassa.     

Ric-8 regulation of heterotrimeric G proteins

Abstract

Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.     

Ablation of RIC8A Function in Mouse Neurons Leads to a Severe Neuromuscular Phenotype and Postnatal Death

Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed Ric8a conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (SynCre) into the floxed Ric8a (Ric8a ^F/F) background ablated RIC8A function in most differentiated neuron populations. Mutant SynCre ^+/- Ric8 ^lacZ/F mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of SynCre ^+/- Ric8a ^lacZ/F mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype.     

A Highlights from MBoC Selection: Drosophila Ric-8 interacts with the Gα12/13 subunit,Concertina, during activation of the Folded gastrulation pathway

Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein–coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα_12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure–function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.     

A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1^Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1^Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2^Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans.     

The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα?GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1?GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1?GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1?GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1?GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.     

Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf)

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short).