407AB toxins deliver their enzymatically active A domain to thecytosol. Some AB-toxins are able to penetrate cellularmembranes from endosomes where the low pH triggers theirtranslocation. One such toxin is diphtheria toxin and importantfeatures of its translocation mechanism have been unraveledduring the last year. Other toxins depend on retrogradetransport through the secretory pathway to the ER beforetranslocation, and recent findings suggest that these toxins takeadvantage of the ER translocation machinery normally used fortransport of cellular proteins. In addition, the intracellular targetsof many of these toxins have been identified recently.AddressesDepartment of Biochemistry, Institute for Cancer Research,The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway*e-mail: [email protected]†e-mail: [email protected] Opinion in Cell Biology 2000, 12:407–4130955-0674/00/$ — see front matter© 2000 Elsevier Science Ltd. All rights reserved.AbbreviationsCNF1 cytotoxic necrotizing factor 1EF edema factorGPI glycosylphosphatidylinositolLF lethal factorPA protective antigenSNAP-25 synaptosomal-associated membrane protein of 25 kDaTGN trans Golgi networkVAMP vesicle-associated membrane proteinIntroductionA number of proteins from plants and bacteria are highlytoxic to mammalian cells because of their ability to enterthe cytosol and attack essential constituents (for a reviewsee [1]). The majority of these toxins are referred to asAB-toxins because of their structural organization (sum-marized in Figure 1) [1]. The A moiety generally hasenzymatic activity and modifies a cellular target uponentry into the cytosol (Table 1) [1], which leads to celldeath or other effects on cellular physiology. The B moi-ety, consisting of one or more subunits, binds the toxin tocell-surface receptors and can also play a role in thetranslocation of the A moiety to the cytosol. Examples ofintracellular targets are ribosomes, actin, small GTP-bind-ing proteins like Rho, and heterotrimeric G-proteins [1]. Arecent discovery is the finding that the anthrax toxinlethal factor cleaves mitogen-activated protein kinasekinase (MAPKK) [2,3]Commonly, an AB-toxin is synthesised in an inactive formthat is activated by proteolytic processing (Figure 1, [4]).Some toxins are cleaved by the producing organism(e.g. cholera toxin, ricin, clostridial neurotoxins) at a regionbetween two cysteine residues [4]. In other cases (e.g.diphtheria toxin, Shiga toxin and Pseudomonas exotoxin A),such processing is performed by furin, which is expressedby the target cell [4]. Other toxins (e.g. anthrax toxins andClostridium botulinum C2 toxin) depend on proteolytic pro-cessing of the B moiety to expose a site that then binds theA moiety non-covalently [4]. In this article, we describestrategies employed by AB-toxins when entering cells,emphasizing recent discoveries in the field.Penetration of protein toxins into cellsPål Ø Falnes* and Kirsten Sandvig†Figure 1Structural organization of AB-toxins. Proteolytic cleavage of the toxin isrequired, in many cases, for activity, and such cleavage often occurs inthe region between the A (black) and the B (gray) subunits. Either thetoxin has been cleared by a protease from the plant or the bacteriumthat produces the toxin (indicated by open arrows) or a protease(usually furin) from the target cell cleaves the toxin (indicated by closedarrows). Proteolytic cleavage often results in a toxin with anenzymatically active part linked to the rest of the molecule by adisulfide bond. However, the cleavage of the anthrax toxin B moiety byfurin leads to the dissociation of part of the molecule and exposure of asite where the A moiety binds. In the cases where no cleavage isindicated, it is possible that cleavage takes place.SSSSSSSSSSSSSSDiphtheria toxinPseudomonas exotoxin AAnthrax toxinClostridium botulinum C2 toxinPlant toxins(e.g. ricin, abrin)Shiga(like) toxinsCholera toxinE.coli heat-labile toxinLarge clostridial cytotoxins(e.g. Clostridium difficile toxins A and B)Rho-activating toxinse.g. (CNF1)Clostridial neurotoxins(tetanus and botulinum toxins)Pertussis toxinAAAAAAAAAAABBBBBBBBBBBBBBCurrent Opinion in Cell BiologyBBBToxin receptors at the cell surface and their rolein penetration and intoxicationThe AB-toxins, in many cases, bind to specific receptormolecules at the cell surface. For instance, the receptor fordiphtheria toxin is the uncleaved precursor of the heparin-binding EGF-like growth factor [5]; the receptor forPseudomonas exotoxin A is the a2-macroglobulin recep-tor [6]; Shiga toxin binds to the glycolipid Gb3 [7] andcholera toxin binds to GM1 [8]. In the case of plant toxins,like ricin, they usually bind to carbohydrates [9], regardlessof whether they are attached to lipids or proteins.The receptor, in addition to providing binding sites at thecell surface, has several other functions in the intoxicationprocess. Firstly, if the receptor is internalized efficiently,for instance from clathrin-coated pits (see below), thetoxin is also rapidly taken up. Importantly, receptorexpression might be under regulation by growthfactors/cytokines. This is the case for the Shiga toxinreceptor (Gb3) [9], and it seems to play an important rolein hemolytic uremic syndrome (HUS) caused byEscherichia coli that synthesize Shiga-like toxins and causefood poisoning [10]. Secondly, the toxin receptor is impor-tant for targeting of the toxin to the organelle before itenters the cytosol. An example is the Shiga-toxin receptor,where the lipid composition of the receptor is essential forretrograde toxin transport [9,11]. A third point is that thereceptor may play a direct role in toxin penetrationthrough the membrane. In the case of diphtheria toxin,the intact receptor seems to play an important role in effi-cient translocation across biological membranes. If thetransmembrane or cytoplasmic domain of the receptor isreplaced by a GPI (glycosylphosphatidylinositol) anchor,the efficiency of toxin translocation across the membraneis considerably reduced [12]. Point mutations in thereceptor also appear to inhibit translocation [13]. Endocytosis and intracellular transport of proteintoxinsAlthough exceptions exist where a toxin seems to pene-trate directly from the cell surface into the cytosol(e.g. Bordetella pertussis invasive adenylate cyclase) [14],most toxins are endocytosed, although by different mech-anisms [9,15], before translocation to the cytosol. Uptakefrom the cell