Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest ␣-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well. Exotoxin A (PE)1 is one of the major virulence factors secreted by Pseudomonas aeruginosa. This toxin is able to kill a large range of mammalian cell lines by inhibiting their protein synthesis (1), and it is one of the favorite toxins to prepare immunotoxins that are promising agents for the treatment of cancers (2). PE is a single chain 66-kDa protein organized in three structural (3) and functional (4) domains successively involved in the intoxication process. First, domain I binds to the ␣ 2 -macroglobulin/low density lipoprotein receptor-related protein (5), enabling internalization via receptor-mediated endocytosis. Domain II will then mediate translocation into the cytosol of the entire toxin (6) or of a carboxyl-terminal fragment generated by furin proteolysis and encompassing domain III and most of domain II (1). Finally, domain III will catalyze the

Exotoxin A is a major virulence factor of Pseudomonas aeruginosa. This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of the toxin proteins with different unfolding abilities so that they inhibited or did not inhibit endosomal translocation of the chimera. We then assayed the amount of toxin fragments delivered to the cytosol during cell intoxication. Finally we used furin inhibitors and examined the fate and intracellular localization of the toxin and its receptor. The results showed that exotoxin cytotoxicity results largely from endosomal translocation of the entire toxin. We found that the C-terminal fragment was unstable in the cytosol.Several bacterial toxins are cleaved by cell proteases upon uptake by mammalian cells. Site-specific processing by furinlike endoproteases is often associated with toxin activation and is therefore a key step of the intoxication process. Indeed, toxins such as anthrax protective antigen, aerolysin, Shiga toxin, and diphtheria toxin (DT) require furin-mediated activation in order to intoxicate cells (47). Exotoxin A (PE) that is secreted by Pseudomonas aeruginosa can also be cleaved by furin in vivo and in vitro (19). PE is produced as a single polypeptide chain composed of three structural and functional domains (domains I, II, and III) that are successively involved in the intoxication process (3). This process starts with the binding of domain I to the low-density receptor-related protein (LRP) (25), a huge protein synthesized in a 600-kDa precursor form that is cleaved by furin into 515-and 85-kDa subunits (16). The LRP is the only functional receptor for PE since LRP-deficient cells are resistant to PE (53). This receptor enables endocytosis of PE and various other ligands (17). Once PE is internalized, exposure to a low endosomal pH triggers major conformational changes within its structure and leads to domain II-mediated insertion into the endosome membrane (32). After this step, two pathways have been documented for continuation of the PE intoxication process. Either the entire toxin could cross the endosome membrane (2, 32), or the toxin could be processed by furin after Arg279 within an exposed loop between the...

Gelsolin is a calcium-, pH- and lipid-dependent actin filament severing/capping protein whose main function is to regulate the assembly state of the actin cytoskeleton. Gelsolin is associated with membranes in cells, and it is generally assumed that this interaction is mediated by PPIs (polyphosphoinositides), since an interaction with these lipids has been characterized in vitro. We demonstrate that non-PPI lipids also bind gelsolin, especially at low pH. The data suggest further that gelsolin becomes partially buried in the lipid bilayer under mildly acidic conditions, in a manner that is not dependent of the presence of PPIs. Our data also suggest that lipid binding involves a number of sites that are spread throughout the gelsolin molecule. Linker regions between gelsolin domains have been implicated by other work, notably the linker between G1 and G2 (gelsolin domains 1 and 2 respectively), and we postulate that the linker region between the N-terminal and C-terminal halves of gelsolin (between G3 and G4) is also involved in the interaction with lipids. This region is compatible with other studies in which additional binding sites have been located within G4-6. The lipid-gelsolin interactions reported in the present paper are not calcium-dependent, and are likely to involve significant conformational changes to the gelsolin molecule, as the chymotryptic digest pattern is altered by the presence of lipids under our conditions. We also report that vesicle-bound gelsolin is capable of binding to actin filaments, presumably through barbed end capping. Gelsolin bound to vesicles can nucleate actin assembly, but is less active in severing microfilaments.