The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines

p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.     

Morphology of cell injury: An approach to the EDIT programme by the use of tobacco pollen tubes. Evaluation-guided Development of New In Vitro Test Batteries

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.     

Applications of monolithic silica capillary columns in proteomics

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research.     

Solution structure of hpTX2, a toxin from Heteropoda venatoria spider that blocks Kv4.2 potassium channel

HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel. We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge. A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected. Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin. The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue. Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (L24). The high density in aromatic side chains of the putative functional surface as well as the lack of an asparagine is proposed to be the structural basis of the specificity of HpTX2 toward Kv4.2 channel.     

Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis

The small GTPases of the Rho family play a key role in actin cytoskeletal organization. In plants, a novel Rho subfamily, called ROP (Rho of plants), has been found. In Arabidopsis, 12 ROP GTPases have been identified which differ mainly at their C-termini. To test the localization of two members of this subfamily (AtROP4 and AtROP6), we have generated translational fusions with the green fluorescent protein (GFP). Microscopic analysis of transiently transfected BY2 cells revealed a predominant localization of AtROP4 in the perinuclear region, while AtROP6 was localized almost exclusively to the plasma membrane. Swapping of the AtROP4 and AtROP6 C-termini produced a change in localization. As RhoGDIs are known to bind to the C-terminus of GTPases of the Rho family, we searched for ArabidopsisRhoGDI genes. We identified the AtRhoGDI1gene and mapped it to chromosome 3. AtRhoGDI1 encodes a 22.5 kDa protein which contains highly conserved amino acids in the isoprene binding pocket and exhibits 29% to 37% similarity to known mammalian RhoGDI homologues. The AtRhoGDI1 gene was expressed in all tissues studied. Using the yeast two-hybrid system, we showed specific interaction of AtRhoGDI1 with both AtROP4 and AtROP6 as well as with their GTP-locked mutants, but not with a GTPase of the RAB family. Recombinant GST-AtRhoGDI1 could bind GFP-AtROP4 from transgenic tobacco BY2 cell extracts, confirming the interaction observed with the two-hybrid system.these authors contributed equally to the work     

Archael phosphoproteins. Identification of a hexosephosphate mutase and the alpha-subunit of succinyl-CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus.

When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [gamma-32P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35- and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the alpha-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-Thr-Asp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein.