Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57's active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H2O2 inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI). © 2012 Brymora et al

The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex

Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp. © 2007 Elsevier B.V. All rights reserved

Suspended battery locomotive GAD-1 with an innovative drive as an alternative solution of a combustion engine

GAD-1 suspended locomotive with battery electric drive equipped with the batteries of New generation can be advantageous alternative to diesel transportation machines. It can significantly improve air quality in underground mine workings, where in the result of increasing number of diesel drives in suspended monorails and floor-mounted railways the workers are exposed to high concentration of exhaust gases and generated heat. The locomotive can also generate pulling force in rack-and-pinion and friction drive system. Design of GAD-1 battery locomotive together with electric system as well as the method of drive replacement are discussed

ERp57-RalA interaction involves the switch II region of RalA.

<p>(A) GST-RalA or the indicated mutants were loaded with GDP or GTP and then used as bait for pull-down experiments using rat testis lysate (RTL). Bound proteins were analysed Western blot using anti-ERp57, anti-RalBP1, or anti-Sec6 antibodies. The experiment shows three blots from the same pull-down experiment and results are representative of at least three independent experiments for each construct. (B) Crystal structure of GDP-bound RalA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050879#pone.0050879-Nicely1" target="_blank">[55]</a>, indicating the switch I (cyan) and switch II (red) regions and the residues mutated for pull-down assays. (Pdb code: 1U90).</p

Isolation and identification of ERp57 as a GDP-RalA binding protein.

<p>(A) GST-RalA loaded with GDP or GTP was used as bait for pull-down experiments from the indicated rat tissue extracts. Bound proteins were analysed by SDS-PAGE and Coomassie blue staining. A GDP-dependent RalA binding protein, p58, is indicated. (B) MALDI-TOF mass spectrum of tryptic peptides derived from brain p58, showing peak identities, matched against theoretical digests with a mass accuracy of ±0.1 Da. (C) Western blotting of GST-RalA pull-down experiments from synaptosomes, whole brain or testis extracts, probed with anti-ERp57 or anti-RalBP1 antibodies, as indicated. (D) A whole testis lysate and a GST-RalA pull-down from testis extract were analysed by Western blot, probed with antibodies recognising ERp57, RalBP1 and PDI. Note that ERp57 was not clearly detected in the lysate in this experiment, due to the weak ERp57 antibody. Detection in pull-down assays was attributed to enrichment of ERp57 in these samples. In our subsequent work, we generated and used a stronger ERp57 antibody, which detects ERp57 in tissue extract (e.g., Fig. 3A).</p

Redox reagents alter the subcellular distribution of RalA.

<p>A431 cells were treated with 5 mM H₂O₂ for 30 minutes, 20 mM NAC for 18 hours, or sequential treatment with 20 mM NAC for 18 hours and then 5 mM H₂O₂ for 30 minutes. Cell lysates were then fractionated by ultracentrifugation. The cytosolic fraction (S100) was analysed by Western blotting for the presence of endogenous RalA (upper panel), and quantitated by densitometry (central panel). Error bars represent the SEM (<i>n</i> = 3). The lower panel shows total cell protein stain of each of the samples represented in the upper panel.</p