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The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.

Mammalian septins are a family of guanosine triphosphate-binding proteins thought to play a role in a number of key cellular processes, such as cytokinesis, protein scaffolding and vesicle trafficking. Although their precise functions remain to be determined, electron microscopy has shown septin filament formation in vitro and a role as a cytoskeletal polymer has been proposed. Here, we present a 3D reconstruction of septin filaments determined using electron microscopy of negatively stained specimens and single-particle image processing. Septin was isolated from rat brain as an approximately 240-kDa complex, from which immunoblotting and N-terminal sequencing identified the major components as septins 3, 5 and 7. Electron microscopy and single-particle analysis indicated that the majority of the septin filaments were approximately 27 nm long. A comparison of 3D volumes obtained using two independent starting models (a row of spheres or a helix) and projection matching techniques revealed no major differences at the final resolution of 27 A, and this structure was highly reproducible when the entire procedure was repeated several times. The reconstruction revealed three apparent subunits, each separated by a cleft; these subunits were similar, but not identical, possibly indicating multiple isoforms within each filament. In some views a smaller cleft appeared to separate the subunits into two smaller regions, perhaps reflecting the presence of septin dimers. This is the first 3D reconstruction of the native septin assembly, and appears compatible with the hypothesis that the septin complex is a hexamer consisting of dimers or heterotrimers. Further investigations are necessary to confirm how the structure of the filaments determined in the present study correlates with the roles of septins in vivo.

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.

Galpha(h) (transglutaminase II) is a bifunctional enzyme possessing transglutaminase and GTPase activities. To better understand the factors affecting these two functions of Galpha(h), we have examined the characteristics of purified Galpha(h) from membrane and cytosol. GTP binding activity of mouse heart Galpha(h) was higher in membrane than that from cytosol. Furthermore, phospholipase C-delta1 (PLC-delta1) activity and coimmunoprecipitation of Galpha(h)-coupled PLC-delta1 in the alpha(1)-adrenoceptor-Galpha(h)-PLC-delta1 complex preparations were increased by phenylephrine in the presence of membranous Galpha(h). On the other hand, transglutaminase activity of cytosolic Galpha(h) was higher than that from membrane Galpha(h). These results demonstrate that bifunctions of Galpha(h) are regulated by its localization that can reflect the cellular functions of Galpha(h)., (Copyright 2001 Academic Press.)

A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. K(m) and k(cat) at 70 degrees C were 833 microM and 9.3 min(-1), respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.

The hydrogenase accessory protein HypB, or nickelin, has two functions in the N(2)-fixing, H(2)-oxidizing bacterium Bradyrhizobium japonicum. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in B. japonicum, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.

Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione S-transferase (GST) fusion forms of Streptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of S. pneumoniae during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [kD], Km, and Vmax). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similar k(D). In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from S. pneumoniae was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound-activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of beta-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated beta-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with beta-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, beta-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15 degrees C. These data suggest that the Rab2 protein plays a role in the low-temperature-sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.

Era, an essential GTPase, is present in many bacteria and Mycoplasma spp. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'-terminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The full-length era gene was able to complement the E. coli mutant deficient in Era production when carried on pACYC184, while the truncated era gene failed to do so when carried on pACYC184, pBR322 or pUC18. The cellular amounts of the truncated Era and the full-length Era proteins in E. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein alpha subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein beta subunit (Gbeta5L). RGS9 and Gbeta5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein gamma-subunit like domain that can mediate their association with Gbeta5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307-13312). We report an example of such a complex whose cellular localization and function are clearly defined.

ERA is an essential GTPase widely conserved in bacteria. Homologues of ERA are also present in higher eukaryotic cells. ERA is involved in bacterial cell cycle control at a point preceding cell division. In order to aid the functional investigation of ERA and to facilitate structure-function studies, we have undertaken the X-ray crystallographic analysis of this protein. Here, we report the purification and crystallization procedures and results. The purified ERA exhibits nucleotide-binding activity and GTP-hydrolytic activity. ERA is one of the very few multi-domain GTPases crystallized to date.

The alpha subunit (Galpha) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Galpha structure essentially comprises a GTPase "Ras-like" domain (RasD) and a unique alpha-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Galpha (Galphat) and the closely related gustducin (Galphag), but not Galphai1, Galphas, or Galphaq synergistically enhance guanosine 5'-gamma[-thio]triphosphate bound Galphat (GalphatGTPgammaS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GalphatGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Galphat with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Galphat within the PDE catalytic core in addition to the sites for the inhibitory Pgamma subunits. The HD moiety of GalphatGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GalphatGTPgammaS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Galphat activation enhances the PDE activation produced by subsaturating levels of Galphat, suggesting a HD-moiety synergism from a transient conformation of Galphat. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein betagamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha and the beta subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha than for beta subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alphao was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein betagamma subunit complexes as well as purified alpha subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao), and 200 nM (alphat). In functional experiments, the steady state GTPase activity of isolated alphao was inhibited by phosducin by approximately 60% with an IC50 value of approximately 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approximately 90% with an IC50 value of approximately 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alphao as measured by single-turnover assays, but it inhibited the release of GDP from alphao; the rate constant of GDP release was decreased approximately 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approximately 100 nM. These data suggest that phosducin binds with high affinity to G-protein betagamma subunits and with lower affinity to G-protein alpha subunits. We propose that the alpha subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the betagamma subunit complex alone.

Mechanical forces arising from strain, pressure, and fluid shear stress are sensed by cells through an unidentified mechanoreceptor(s) coupled to intracellular signaling pathways. In vascular endothelial cells, fluid shear stress is transduced via pathway(s) involving heterotrimeric guanine nucleotide-binding proteins (G proteins) by molecular mechanisms that are unknown. In the present study, we investigated the activation of purified G proteins reconstituted into phospholipid vesicles. Vesicles containing G proteins were loaded with [gamma-32P]GTP and subjected to physiological levels of fluid shear stress in a cone-and-plate viscometer. Steady-state GTP hydrolysis was measured as an index of G protein function. Shear stress (0-30 dynes/cm2) activated G proteins in dose-dependent manner (0.48-4.6 pmol/min per microg of protein). Liposomes containing lysophosphatidylcholine (30 mol %) or treated with benzyl alcohol (40 mM), conditions that increase bilayer fluidity, exhibited 3- to 5-fold enhancement of basal GTPase activity. Conversely, incorporation of cholesterol (24 mol %) into liposomes reduced the activation of G proteins by shear. These results demonstrate the ability of the phospholipid bilayer to mediate the shear stress-induced activation of membrane-bound G proteins in the absence of protein receptors and that bilayer physical properties modulate this response.

A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography. The molecular mass of RGA1 bearing a His tag was approx. 49 kDa. Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots. It exists at particularly high levels in the immature embryo after pellicle extrusion. In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants. When bound to guanosine 5'-(gamma-thio)triphosphate (GTP[S]), the RGA1 protein was partially protected from tryptic proteolysis. In the presence of GTP[S], trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size. When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion. Photoaffinity labelling with [alpha-32P]GTP and a GTP[S]-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide. Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM). The rate of guanine nucleotide binding of RGA1 (kon,GTP[S]=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2. The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP.

We have previously identified a human nucleolar phosphoprotein p130 whose alterations during mitosis are correlated well with the nucleolar disassembly and reassembly. Further studies found that p130 in the cell lysates or after being purified by immunoprecipitation was able to form large complexes triggered by F- and Mg2+. These sodium dodecyl sulfate-insoluble p130 molecules were readily dissociated by adding EDTA to the complexes. It is known that F- and Mg2+ act on many GTPases and ATPases through the induction of a conformational transition mimicking the nucleoside triphosphate-bound state. These initial observations led us to discover that p130 functions as a GTP/ATP binding protein with intrinsic GTPase/ATPase activities. The rate of GTP hydrolysis by purified p130 under our experimental conditions was 0.8 mol/min/mol of p130. These results imply that p130, a novel nucleolar GTPase/ATPase, may switch its conformation in a nucleotide-dependent manner.

Era is an essential gene in E. coli, encoding a GTP-binding protein of unknown function. In the present work, a mutant designated Era-dE, for deletion of effector region is described. This is the first and only known era allele that confers a dominant-negative phenotype. Phenotypic analysis of the mutant showed that overproduction of Era-dE caused a dominant inhibition of growth when TCA cycle intermediates such as succinate, pyruvate, malate, alpha-ketoglutarate, and fumarate were provided as the sole carbon source. Examination of the macromolecular composition of cells overexpressing the mutant showed protein, DNA, and ATP levels expected for cells growing at slow rates. The response of cells expressing Era-dE to different stress conditions was studied by examining the rates of synthesis of stress-inducible proteins. Interestingly, when subjected to succinate starvation, cells expressing Era-dE showed a defective carbon starvation response, whereas response to glucose starvation was similar to that seen in control cells. Taken together with previous results, these studies indicate that Era is perhaps involved in multiple cellular processes and Era-dE disrupts more than one of these functions. Furthermore, it appears that some possible functions of Era include regulation of the TCA cycle and response to carbon starvation.

The small guanosine 5'-triphosphate (GTP)ase Rab4 has been suggested to play a role in insulin-induced GLUT4 translocation. Under insulin stimulation, GLUT4 translocates to the plasma membranes, while Rab4 leaves the GLUT4-containing vesicles and becomes cytosolic. Rab proteins cycle between a GTP-bound active form and a guanosine 5'-diphosphate (GDP)-bound inactive form. The intrinsic GTPase activity of Rab proteins is low and the interconversion between the two forms is dependent on accessory factors. In the present work, we searched for a GTPase activating protein (GAP) for Rab4 in 3T3-L1 adipocytes. We used a glutathione-S-transferase (GST)-Rab4 protein which possesses the properties of a small GTPase (ability to bind GDP and GTP and to hydrolyse GTP) and can be isolated in a rapid and efficient way. This GAP activity was observed in 3T3-L1 adipocyte lysates, and was able to accelerate the hydrolysis of the [alpha-32P]GTP bound to GST-Rab4 into [alpha-32P]GDP. This activity, tentatively called Rab4-GAP, was also present in 3T3-L1 fibroblasts. The Rab4-GAP activity was present in total membrane fractions and nearly undetectable in cytosol. Following subcellular fractionation, Rab4-GAP was found to be enriched in plasma membranes when compared to internal microsomes. Insulin treatment of the cells had no effect on the total Rab4-GAP activity or on its subcellular localization. Taking our results together with the accepted model of Rab cycling in intracellular traffic, we propose that Rab4-GAP activity plays a role in the cycling between the GTP- and GDP-bound forms of Rab4, and thus possibly in the traffic of GLUT4-containing vesicles.

Geranylgeranylation of Rab GTPases is an essential post-translational modification that enables Rabs to associate with intracellular membranes where they regulate exocytic and endocytic pathways. Geranylgeranylation is initiated by formation of a stable complex between newly synthesized Rab proteins and Rab escort protein (REP). The complex is recognized by Rab geranylgeranyl (GG) transferase, which transfers two GG groups to Rabs. The geranylgeranylated Rabs regulate vesicular movement by oscillating between an inactive GDP-bound form and an active GTP-bound form. In this study, I show that the kinetics of geranylgeranylation is influenced by the nucleotide status of nascent Rab. GDP-bound Rab is geranylgeranylated with 10-50-fold higher affinity than GTP-bound Rab (or GTP analog-bound Rab), as indicated by the apparent Km of the reaction. In vitro REP.Rab binding assays demonstrate that REP forms a stable complex only with the GDP-bound form of Rab but not the GTP-bound form, suggesting that the apparent Km effect in the prenylation reaction is due to a discrimination between the two different nucleotide-bound forms of Rab by REP. Inasmuch as Rabs are likely GTP-bound after synthesis and REP does not possess GTPase-activating protein activity, these results raise the possibility that a Rab GTPase-activating protein enhances the REP*Rab interaction prior to prenylation.

We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1.rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.

1,3-beta-D-glucan synthase [also known as beta(1-->3) glucan synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-beta-linked glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1p. Glucan synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with beta(1-->3)glucan synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of beta(1-->3)glucan synthase.

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the digeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP.monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP.diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP.Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP.Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP.monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second geranylgeranylation reaction, ensuring efficient digeranylgeranylation of Rab substrates.

The sgp gene of Streptococcus mutans was recently detected immediately downstream from the dgk gene within the same operon. In this study, the sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein at a level of 40% of total cellular protein. One-step amylose affinity chromatography purification of this fusion protein yielded a product of approximately 95% purity. SGP was purified from this fusion protein following cleavage with protease factor Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, recombinant SGP showed specific binding for GTP and GDP, but not ATP, CTP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Kinetic studies revealed that the SGP Km value for GTP in this reaction was approximately 5.9 microM. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labelling demonstrated that SGP was associated with both membrane and cytoplasmic fractions. SGP not only had structural similarities with other G proteins but also proved to have high-level intrinsic GTPase activity. Therefore, SGP appears to be a new member of the G protein superfamily and may participate in transmembrane signaling in the responses of S. mutans cells to environmental stimuli.

Rab9 is a Ras-like GTPase required for the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Rab9 occurs in the cytosol as a complex with GDP dissociation inhibitor (GDI), which we have shown delivers prenyl Rab9 to late endosomes in a functional form. We report here basal rate constants for guanine nucleotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate constants were influenced in part by the hydrophobic environment of the prenyl group. Guanine nucleotide dissociation and GTP hydrolysis rates were lower in the presence of lipid; detergent stimulated intrinsic nucleotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a KD of 60 nM in 0.1% Lubrol and 23 nM in 0.02% Lubrol. In 0.1% Lubrol, GDI-alpha inhibited GDP dissociation half maximally at 72 +/- 18 nM, consistent with the KD determinations. These data suggest that GDI-alpha associates with prenyl Rab9 with a KD of < or = 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 1.9-fold and bound prenyl Rab9 with a KD of 67 nM in 0.1% Lubrol.

Using a rapid amplification of cDNA ends (RACE) cloning approach, we have isolated a cDNA clone encoding Rab19, a novel small GTPase of the Rab subfamily contained within partial sequences previously described [Chavrier et al., Gene 112 (1992) 261-264]. Northern blot analysis of the distribution of the rab19 mRNA in various adult mouse tissues and NIH 3T3 fibroblasts revealed that rab19 is expressed in a tissue-specific manner. The rab19 transcript was detected at high levels in intestine, lung and spleen, and at a lower level in kidney. In contrast, liver, brain, heart and NIH 3T3 fibroblasts contain only very little or no detectable rab19 mRNA. Therefore, Rab19 is likely to represent a novel tissue- or cell type-specific small GTPase.

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+. In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column. The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

A method is described for purification of G protein alpha and beta gamma subunits from Sf9 cells infected with recombinant baculoviruses. The subunit to be purified is coexpressed with an associated subunit bearing a hexahistidine tag. After adsorption of the oligomer to a Ni(2+)-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AIF4-. The alpha subunits of G12, Gq, Gz, and Gi1 and the beta 1 gamma 2 subunit complex were easily and efficiently purified by this method. Results was superior to established procedures in all cases. Purified alpha 12 was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (kon, GTP gamma S = 0.01 min-1) and a very slow kcat for hydrolysis of GTP (0.1-0.2 min-1). GTP gamma S (guanosine 5' -3-O- (thio)triphosphate) alpha 12 does not influence the activity of several adenylyl cyclases or phospholipases. Activated alpha z inhibits the activity of type I and type V adenylyl cyclases. It is a somewhat more potent inhibitor of type V adenylyl cyclase than is activated alpha i1.

Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches. Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21ras cause tumors in man. A conserved glutamic residue in the alpha subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP. The results of mutagenesis of this residue (Glu-207) in Gi alpha 1 refute this hypothesis. Based on the structure of the complex of Gi alpha 1 with GDP, Mg2+, and AlF-4, which appears to resemble the transition state for GTP hydrolysis, we believe that Gln-204 of Gi alpha 1, rather than Glu-207, supports catalysis of GTP hydrolysis by stabilization of the transition state.

Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI). We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro. Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity. Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein. Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting. Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9. These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.

On the basis of conserved sequences characteristic of the Ran/TC4 subfamily of the GTPase superfamily, a fragment of the gene encoding a Plasmodium falciparum Ran/TC4 homologue was amplified in the polymerase chain reaction. The fragment was used to screen a cDNA library to obtain clones which allowed determination of the complete gene sequence. The gene, designated pfran (Plasmodium falciparum ras-like nuclear protein), has around 70% amino acid identity with previously characterised Ran/TC4 proteins. Like other malarial mRNAs, the pfran mRNA contains a long (at least 679 bp) 5' untranslated region. Southern blotting experiments show that pfran is a single copy gene located on chromosome 11. RNA hybridisation experiments indicate that pfran mRNA is abundant in late trophozoite and schizont stages, but present at very low levels in gametocytes and early asexual stages.

hGBP1 is an interferon-induced 67-kDa protein of human cells that readily binds to agarose-immobilized GTP, GDP, and GMP but not to other nucleotides. We cloned hGBP1 cDNA into a histidine-tagging vector, produced recombinant hGBP1 with 6 extra histidine residues at its N terminus in Escherichia coli, and purified this protein to near homogeneity from bacterial lysates. Purified hGBP1 hydrolyzed radiolabeled GTP but failed to hydrolyze ATP, UTP, or CTP at significant rates. Unexpectedly, the principal product of the GTP hydrolysis reaction was GMP rather than GDP. Although significant amounts of GDP were produced when the reaction was performed at 15 degrees C, GDP could not serve as substrate or as inhibitor of hGBP1. hGBP1 lacked guanylate cyclase and guanylyltransferase activity. Degradation of GTP to GMP most likely occurred via two consecutive cleavages of single phosphate groups, because pyrophosphate was not a reaction product, and because hGBP1 failed to hydrolyze GTP gamma S. In vitro modification assays with radiolabeled mevalonic acid and farnesyl pyrophosphate showed that the CaaX motif at the C terminus of hGBP1 functions as an isoprenylation signal. Thus, hGBP1 is a GTPase with novel biochemical properties that may be membrane-associated in eukaryotic cells.

Interferons induce a number of different proteins which mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. Interferon-induced Mx proteins, which confer resistance to influenza, vesicular stomatitis, and measles viruses, contain consensus GTPase sequence elements. Insect cell-produced purified murine Mx1 and human MxA proteins were found to hydrolyze GTP with Km = 65 microM (Vmax, 7.1 min-1) and 62 microM (Vmax, 3.1 min-1), respectively. The GTPase activity of Mx1 and MxA proteins was strictly dependent on Mg2+ ions. Murine Mx1 protein was inactivated at 10 degrees C lower temperatures than MxA protein. As analyzed, by filter binding assay, Mx1 protein (at 1 microM) showed a relatively high affinity for GDP (Kd = 1.0 x 10(-7) M) and approximately 340-fold lower affinity for guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) (Kd = 3.4 x 10(-5) M). The Kd values for MxA protein were 2.0 x 10(-7) M for GDP and 5.9 x 10(-6) M for GTP gamma S, showing approximately a 30-fold affinity difference. ATP, UTP, or CTP did not inhibit the Mx protein-dependent GTPase activity, suggesting that Mx1 and MxA proteins are highly specific for guanosine nucleotides. In conclusion recombinant nuclear murine Mx1 and cytoplasmic human MxA proteins show clear differences in their enzymatic activities and nucleotide binding characteristics. How these differences influence their cellular functions and antiviral potential is presently not known.

The small GTP-binding protein Rab3A (identical to smg p25A) is expressed in neural/exocrine/endocrine cells, is distributed between the cytosol and secretory vesicle membranes, and may cycle between these locations to regulate exocytosis. It is proposed that the GTP/GDP state of Rab3A controls this distribution. In PC12 cells, cytosolic Rab3A is predominantly GDP-bound, whereas membrane-associated Rab3A is approximately 50% GTP-bound. Two cytosolic factors, GDP dissociation inhibitor (GDI) and guanine nucleotide releasing factor (GRF), act only on GDP.Rab3A, and preferentially with post-translationally modified Rab3A. Rab3A GTPase-activating protein (GAP) does not preferentially act on processed Rab3A, and interacts selectively with GTP.Rab3A. GDI antagonizes GRF but not GAP activity toward Rab3A. These data are consistent with the concept of an ordered Rab3A cycle controlled by factors that regulate the guanine-nucleotide binding state of Rab3A.

SEC12 encodes an integral membrane glycoprotein essential for vesicle formation from the endoplasmic reticulum (ER) in yeast. The SAR1 gene was discovered as a multicopy suppressor of a sec12ts strain and encodes a 21-kDa GTP-binding protein also required for protein transport from the ER to the Golgi apparatus (Nakano, A., and Muramatsu, M. (1989) J. Cell Biol. 109, 2677-2691). We have purified Sar1p to apparent homogeneity from cells harboring a galactose-regulated recombinant SAR1. Purified Sar1p binds guanine nucleotides specifically and exhibits GTPase activity (0.001 min-1). Nucleotide exchange and hydrolysis rates are greatly increased in the presence of Mg2+ and nonionic detergents or phospholipids. An assay that measures the formation of a vesicle intermediate in ER to Golgi transport was devised that is dependent on the addition of purified Sar1p. This assay employs membranes prepared from wild-type cells and cytosol fractions depleted of Sar1p due to overproduction of Sec12p or by gel filtration chromatography. The gel-filtered cytosol requires the addition of Sar1p and GTP to support vesicle budding. Sar1p prebound with GTP gamma S inhibits Sar1p function in the vesicle formation assay. The results indicate a role for Sar1p in vesicle budding from the ER and suggest that GTP hydrolysis by Sar1p is required for this event.

A substitution of Gly for Val at position 19, which corresponds to oncogenic Gly13-->Val mutation of ras p21, was introduced in a low Mr GTP-binding protein, ram p25. The protein was expressed in cytosolic fraction of Escherichia coli and purified by using specific antibody raised against ram p25. The mutated protein had no guanine nucleotide-binding activity although [Val13]ras p21 was reported to have. The analysis of guanine nucleotide composition of the purified [Val19]ram p25 revealed that the protein was free of nucleotide whereas the normal ram p25 bound about 1 mol of GDP per mol of protein. These results strongly suggested that some part(s) of variable regions as well as the consensus regions are important for the biochemical properties of ram p25.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.