Structural basis of wedging the Golgi membrane by FAPP pleckstrin homology domainsOverduin and colleagues present the NMR structures of free, micelle and PtdIns(4)P-bound FAPP1-PH domain. The micelle-bound structure reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. A hydrophobic element inserts into and bends membranes, and is conserved in pleckstrin homology domains of CERT and OSBP proteins.

Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a poreforming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence crosscorrelation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca 2؉ entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II.Pore-forming proteins (PFPs) 4 have been developed by many organisms ranging from bacteria and parasites to animals to disrupt membrane integrity of the target cell. The purpose of membrane permeabilization in many cases is to compromise cell survival, as it happens with toxins like ␣-toxin, actinoporins, or the proteins of the Bcl-2 family and perforin in human cells. However, it can also be related with the escape of certain bacteria from the phagosome, like listeriolysin, or with protein delivery into the cytosol, as in the case of colicins (1-3).PFPs are secreted by the producer cell in soluble form and bind to target cells via specific receptors. Then they undergo a conformational change that exposes hydrophobic residues and allows their insertion into the plasma membrane and oligomerization into a pore. The nature, stoichiometry, and size of that pore depend on the PFP (1). In vitro studies suggest that the toxins of the actinoporin family form a tetramer (4 -6), although it could also have a structure similar to those recently reported for cytolysin A or fragaceatoxin C (7, 8), with a higher oligomeric state. The nature of the pore is believed to be toroidal, with lipids exposed to the pore lumen (9). In red blood cells and on artificial lipid vesicles, actinoporins formed pores ϳ2 nm in diameter (10, 11).Many PFPs use molecules associated with lipid rafts as receptors (12). Rafts are lipid/protein domains enriched in cholesterol and sphingolipids that act as signaling platforms in the plasma membrane of the cell. During the last years, there has been extensive debate over what rafts are and their functional role. Different domains of raft nature have been observed in several temporal and spatial scales. As a consequence, there are many different kinds of domains that have been associated with rafts (13).Some examples of raft-associated toxin...

The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network.membrane tubulation ͉ PH domain ͉ phosphatidylinositol 4-phosphate ͉ trans-Golgi network ͉ small-angle x-ray scattering (SAXS)

We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1 (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-ADeltaenv-GFP, ROD-ADeltaenv-RFP, or ROD-ADeltaenv-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E).