Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.     

Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex

In the elongation cycle of protein biosynthesis, the nucleotide exchange factor eEF1Balpha catalyzes the exchange of GDP bound to the G-protein, eEF1A, for GTP. To obtain more information about the recently solved eEF1A-eEF1Balpha structure, we determined the structures of the eEF1A-eEF1Balpha-GDP-Mg2+, eEF1A-eEF1Balpha-GDP and eEF1A-eEF1Balpha-GDPNP complexes at 3.0, 2.4 and 2.05 A resolution, respectively. Minor changes, specifically around the nucleotide binding site, in eEF1A and eEF1Balpha are consistent with in vivo data. The base, sugar and alpha-phosphate bind as in other known nucleotide G-protein complexes, whereas the beta- and gamma-phosphates are disordered. A mutation of Lys 205 in eEF1Balpha that inserts into the Mg2+ binding site of eEF1A is lethal. This together with the structures emphasizes the essential role of Mg2+ in nucleotide exchange in the eEF1A-eEF1Balpha complex.     

Guanine nucleotide exchange factor -H1 promotes inflammatory cytokine production and intracellular mycobacterial elimination in macrophages

Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen that can live within macrophages owning to its ability to arrest phagolysosome biogenesis. The guanine nucleotide exchange factor H1 (GEF-H1) may contribute to the phagocytosis of bacteria by macrophages through mediating the crosstalk between microtubules and the actin cytoskeleton. Its role in Shigella infection has been determined but little is known about the role of GEF-H1 in mycobacterial infection. In the present study, we demonstrated that GEF-H1 functioned as a key regulator of the macrophage-mediated anti-mycobacterial response. We found that both mRNA and protein expression levels of GEF-H1 were significantly upregulated in macrophage during mycobacterial infection. Moreover, silencing of GEF-H1 with specific siRNAs reduced the phosphorylation of p38 mitogen-activated protein kinase and TANK binding kinase 1 as well as the expression of interleukin-1β (IL-1β), IL-6, and interferon-β (IFN-β), without affecting nitric oxide production or autophagy. Importantly, GEF-H1 depletion attenuated macrophages-mediated mycobacterial phagocytosis and elimination. Taken together, our data supported that GEF-H1 was a novel regulator of inflammatory cytokine production and mycobacterial elimination, and may serve as a novel potential target for clinical treatment of tuberculosis.     

EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.     

Unique classes of mutations in the Saccharomyces cerevisiae G-protein translation elongation factor 1A suppress the requirement for guanine nucleotide exchange

G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1Balpha, is essential for viability. The requirement for the TEF5 gene encoding eEF1Balpha can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1Balpha structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.     

Role of magnesium in nucleotide exchange on the small G protein rac investigated using novel fluorescent Guanine nucleotide analogues

Novel guanine nucleotide analogues have been used to investigate the role of Mg(2+) in nucleotide release and binding with the small G protein rac. The fluorescent analogues have 7-(ethylamino)-8-bromocoumarin-3-carboxylic acid attached to the 3'-position of the ribose via an ethylenediamine linker. This modification has only small effects on the interaction with rac. There are large fluorescence changes on binding of the triphosphate to rac, on hydrolysis, and then on release of the diphosphate. Furthermore, the fluorescence is sensitive to the presence of Mg(2+) in the active site. Using this signal, it was shown that, for a variety of conditions, the nucleotides dissociate by a two-step mechanism. Mg(2+) is released first followed by the nucleotide. With the diphosphate, Mg(2+) is fast and nucleotide release slow. For the fluorescent GMPPNP analogue, the rate of dissociation is limited by Mg(2+) release. In the latter case, Mg(2+) binds tightly with a K(d) of 61 nM, whereas for the diphosphate the K(d) is 11 microM (30 degrees C, pH 7.6).     

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225- 237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta- COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.     

Guanine nucleotide binding properties of rap1 purified from human neutrophils.

The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.     

Identification of potential mechanisms for regulation of p115 RhoGEF through analysis of endogenous and mutant forms of the exchange factor

Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucidated by the identification of p115 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by G alpha(13). Endogenous p115 RhoGEF was found mainly in the cytosol of serum-starved cells but partially localized to membranes in cells stimulated with lysophosphatidic acid. Overexpressed p115 RhoGEF was equally distributed between membranes and cytosol; either the RGS or pleckstrin homology domain was sufficient for this partial targeting to membranes. Removal of the pleckstrin homology domain dramatically reduced the in vitro rate of p115 RhoGEF exchange activity. Deletion of amino acids 252--288 in the linker region between the RGS domain and the Dbl homology domain or of the last 150 C-terminal amino acids resulted in non-additive reduction of in vitro exchange activity. In contrast, p115 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than the full-length exchange factor in vivo. These results suggest that p115 RhoGEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p115 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or sphingosine 1-phosphate was more active than when the enzyme was immunoprecipitated from untreated cells. This indicates an additional and potentially novel long lived mechanism for regulation of p115 RhoGEF by G protein-coupled receptors.     

Tetrahymena eukaryotic translation elongation factor 1A (eEF1A) bundles filamentous actin through dimer formation

Eukaryotic translation elongation factor 1A (eEF1A) is known to be a multifunctional protein. In Tetrahymena, eEF1A is localized to the division furrow and has the character to bundle filamentous actin (F-actin). eEF1A binds F-actin and the ratio of eEF1A and actin is approximately 1:1 (Kurasawa et al., 1996). In this study, we revealed that eEF1A itself exists as monomer and dimer, using gel filtration column chromatography. Next, eEF1A monomer and eEF1A dimer were separated using gel filtration column, and their interaction with F-actin was examined with cosedimentation assay and electron microscopy. In the absence of Ca2+/calmodulin (CaM), eEF1A dimer bundled F-actin and coprecipitated with F-actin at low-speed centrifugation, but eEF1A monomer did not. In the presence of Ca2+/CaM, eEF1A monomer increased, while dimer decreased. To examine that Ca2+/CaM alters eEF1A dimer into monomer and inhibits bundle formation of F-actin, Ca2+/CaM was added to F-actin bundles formed by eEF1A dimer. Ca2+/CaM separated eEF1A dimer to monomer, loosened F-actin bundles and then dispersed actin filaments. Simultaneously, Ca2+/CaM/ eEF1A monomer complexes were dissociated from actin filaments. Therefore, Ca2+/CaM reversibly regulates the F-actin bundling activity of eEF1A.     

Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3

The translation elongation machinery in fungi differs from other eukaryotes in its dependence upon eukaryotic elongation factor 3 (eEF3). eEF3 is essential in vivo and required for each cycle of the translation elongation process in vitro. Models predict eEF3 affects the delivery of cognate aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated tRNA from the ribosomal Exit site. To dissect eEF3 function and its link to the A-site activities of eEF1A, we have identified a temperature-sensitive allele of the YEF3 gene. The F650S substitution, located between the two ATP binding cassettes, reduces both ribosome-dependent and intrinsic ATPase activities. In vivo this mutation increases sensitivity to aminoglycosidic drugs, causes a 50% reduction of total protein synthesis at permissive temperatures, slows run-off of polyribosomes, and reduces binding to eEF1A. Reciprocally, excess eEF3 confers synthetic slow growth, increased drug sensitivity, and reduced translation in an allele specific fashion with an E122K mutation in the GTP binding domain of eEF1A. In addition, this mutant form of eEF1A shows reduced binding of eEF3. Thus, optimal in vivo interactions between eEF3 and eEF1A are critical for protein synthesis.     

A novel small molecule regulator of guanine nucleotide exchange activity of the ADP-ribosylation factor and golgi membrane trafficking

An image-based phenotypic screen was developed to identify small molecule regulators of intracellular traffic. Using this screen we found that AG1478, a previously known inhibitor of epidermal growth factor receptor, had epidermal growth factor receptor-independent activity in inducing the disassembly of the Golgi in human cells. Similar to brefeldin A (BFA), a known disrupter of the Golgi, AG1478 inhibits the activity of small GTPase ADP-ribosylation factor. Unlike BFA, AG1478 exhibits low cytotoxicity and selectively targets the cis-Golgi without affecting endosomal compartment. We show that AG1478 inhibits GBF1, a large nucleotide exchange factor for the ADP-ribosylation factor, in a Sec7 domain-dependent manner and mimics the phenotype of a GBF1 mutant that has an inactive mutation. The treatment with AG1478 leads to the recruitment of GBF1 to the vesicular-tubular clusters adjacent to the endoplasmic reticulum exit sites, a step only transiently observed previously in the presence of BFA. We propose that the treatment with AG1478 delineates a membrane trafficking intermediate step that depends upon the Sec7 domain.     

GEFT, A Rho family guanine nucleotide exchange factor, regulates lens differentiation through a Rac1-mediated mechanism

The Rho-family of small GTPase specific guanine nucleotide exchange factor, GEFT, is expressed at high levels in adult human excitable tissues including the brain, heart, and skeletal muscle. Previously, we demonstrated that GEFT is specifically expressed in the adult mouse hippocampus and cerebellum, and that overexpression of this protein can result in neurite and dendrite remodeling. This finding prompted us to explore the expression of GEFT in other tissues, which share common developmental ancestry to the nervous system, specifically the ocular system. Using immunohistochemical analysis specific for GEFT protein expression, we observed the highest ocular expression of GEFT occurring in the neuroblastic layer and differentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lens epithelium, and throughout the retina. Exogenous expression of GEFT in N/N1003A rabbit lens epithelial cells induced lens fiber differentiation as reflected by cell elongation and lentoid formation, as well as a strong increase in β-crystallin and filensin expression. Moreover, transfection of lens epithelial cells with GEFT resulted in a Rac-1 mediated up-regulation of αA-, αB-, βB-, γC-, or γF-crystallin promoter activities that is in part dependent on the nuclear localization of Rac1. Furthermore, pharmacological inhibition of Rac1 blocked GEFT-induced N/N1003A lens fiber differentiation and βB-crystallin expression in ex vivo mouse lens explants. These results demonstrate for the first time a role for GEFT in lens cell differentiation and mouse eye development. Moreover, GEFT regulation of lens differentiation and eye development occurs through a Rac1-dependent mechanism.     

The interaction between the archaeal elongation factor 1alpha and its nucleotide exchange factor 1beta.

In Sulfolobus solfataricus the binding of the exchange factor 1beta (SsEF-1beta) to SsEF-1alpha-GDP displaces the nucleotide and the SsEF-1alpha-SsEF-1beta complex is formed. The complex itself is stable, but it dissociates upon the addition of GDP or Gpp(NH)p but not ATP. Since the rate of the formation of the SsEF-1alpha-SsEF-1beta complex is significatively slower than the rate of the nucleotide exchange catalyzed by SsEF-1beta it can be inferred that in vivo the GDP/GTP exchange reaction proceeds via an SsEF-1alpha-SsEF-1beta interaction without involving the formation of a stable binary complex as an intermediate.     

Ccpg1, a novel scaffold protein that regulates the activity of the Rho guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.