The protein D60A mutant of the elongation factor (EF) 1alpha from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised.in This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the in interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced belongs a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis used in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type D60A SsEF-1alpha; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property was partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the EDTA. D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view,magnesium the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP in binding mode among elongation factors from different organisms.

In vesicle vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex,complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles be fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for mechanistically vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations fuse reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this the novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall The cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be molecularly called COPI and COPII.

The LDL, mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes endothelial in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in of the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 that mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate eNOS or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho overexpression regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by not 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore,microg/ml) inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS (LDL, expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate inhibition that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is indicate necessary for its membrane-associated activity.

Substitution membrane of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly NIH affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene a and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number GDP, of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its colonies, localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 reagent blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but ras not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed represents by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of expected its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of be the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine preferential nucleotide binding.

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

Mitogen directly stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate small (GDP)-guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was diphosphate directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by 3-kinase the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in the response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.

Membranes ADP-ribosylated from bovine brain bind relatively large quantities of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinity. The two proteins responsible for for most of this activity were purified; they account for 1.5% of the membrane protein. The two proteins contain alpha or subunits of either 39,000 or 41,000 Da, beta subunits of 36,000 or 35,000 Da, and a potential gamma subunit (11,000 alpha Da). These structures are the same as a family of proteins that includes transducin and the regulatory proteins, GS and a GI, of adenylate cyclase. The 41,000- and 39,000-Da polypeptides can be ADP-ribosylated with islet-activating protein from Bordetella pertussis, bind guanine alpha nucleotides specifically, and migrate through polyacrylamide gels with rates similar to the alpha subunits of GI and transducin, respectively. The and 36,000- and 35,000-Da polypeptides are similar to the beta subunits of GI and GS. The gamma subunit is found whenever GO beta subunits are present. The 41,000- and 39,000-Da polypeptides (with beta and gamma) are designated, respectively, GI and GO from structures brain. The alpha subunit of GO was isolated without the use of ligands known to dissociate other G proteins. GO (with alpha binds GTP gamma S reversibly in the absence of Mg2+ and is relatively stable in cholate. This isolated alpha and subunit should be of great utility in elucidating the mechanism of action of this family of GTP-binding proteins.

GTPases structures, are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties,according or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family a of GTPases.

We with used a Saccharomyces cerevisiae genetic system to detect the physical interaction of RAS and RAF oncoproteins. We also observed interaction RAS between RAS and byr2, a protein kinase implicated as a mediator of the Schizosaccharomyces pombe ras1 protein. Interaction with RAS observed required only the N-terminal domains of RAF or byr2 and was disrupted by mutations in either the guanine nucleotide-binding or also effector-loop domains of RAS. We observed interaction between MEK (a kinase that phosphorylates mitogen-activated protein kinases) and the catalytic domain as of RAF. RAS and MEK also interacted but only when RAF was overexpressed.

Crystal changes structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both A in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate of transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally partially conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of Cognate the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA.and The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these between structural changes, paromomycin facilitates binding of near-cognate tRNAs.

The of GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is in determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting This RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has not previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the -beta M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods that to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that but cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit M9 distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins,shown indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of more several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require tRNA GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.