407 Penetration of protein toxins into cells P l Falnes and Kirsten Sandvig AB toxins deliver their enzymatically active A domain to the cytosol Some AB toxins are able to penetrate cellular membranes from endosomes where the low pH triggers their translocation One such toxin is diphtheria toxin and important features of its translocation mechanism have been unraveled during the last year Other toxins depend on retrograde transport through the secretory pathway to the ER before translocation and recent findings suggest that these toxins take advantage of the ER translocation machinery normally used for transport of cellular proteins In addition the intracellular targets of many of these toxins have been identified recently Addresses Department of Biochemistry Institute for Cancer Research The Norwegian Radium Hospital Montebello 0310 Oslo Norway e mail pfalnes labmed uio no e mail kirsten sandvig labmed uio no Current Opinion in Cell Biology 2000 12 407 413 0955 0674 00 see front matter 2000 Elsevier Science Ltd All rights reserved Abbreviations CNF1 cytotoxic necrotizing factor 1 EF edema factor GPI glycosylphosphatidylinositol LF lethal factor PA protective antigen SNAP 25 synaptosomal associated membrane protein of 25 kDa TGN trans Golgi network VAMP vesicle associated membrane protein Introduction A number of proteins from plants and bacteria are highly toxic to mammalian cells because of their ability to enter the cytosol and attack essential constituents for a review see 1 The majority of these toxins are referred to as AB toxins because of their structural organization summarized in Figure 1 1 The A moiety generally has enzymatic activity and modifies a cellular target upon entry into the cytosol Table 1 1 which leads to cell death or other effects on cellular physiology The B moiety consisting of one or more subunits binds the toxin to cell surface receptors and can also play a role in the translocation of the A moiety to the cytosol Examples of intracellular targets are ribosomes actin small GTP binding proteins like Rho and heterotrimeric G proteins 1 A recent discovery is the finding that the anthrax toxin lethal factor cleaves mitogen activated protein kinase kinase MAPKK 2 3 Commonly an AB toxin is synthesised in an inactive form that 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 region between two cysteine residues 4 In other cases e g diphtheria toxin Shiga toxin and Pseudomonas exotoxin A Figure 1 S S A Diphtheria toxin B S S B Pseudomonas exotoxin A A B B Anthrax toxin Clostridium botulinum C2 toxin SS Shiga like toxins A Cholera toxin E coli heat labile toxin A SS S S Pertussis toxin A Clostridial neurotoxins tetanus and botulinum toxins A Plant toxins e g ricin abrin B B B B BB BB B SS SS A B B B A Large clostridial cytotoxins e g Clostridium difficile toxins A and B Rho activating toxins e g CNF1 A A B A Current Opinion in Cell Biology Structural organization of AB toxins Proteolytic cleavage of the toxin is required in many cases for activity and such cleavage often occurs in the region between the A black and the B gray subunits Either the toxin has been cleared by a protease from the plant or the bacterium that produces the toxin indicated by open arrows or a protease usually furin from the target cell cleaves the toxin indicated by closed arrows Proteolytic cleavage often results in a toxin with an enzymatically active part linked to the rest of the molecule by a disulfide bond However the cleavage of the anthrax toxin B moiety by furin leads to the dissociation of part of the molecule and exposure of a site where the A moiety binds In the cases where no cleavage is indicated it is possible that cleavage takes place such processing is performed by furin which is expressed by the target cell 4 Other toxins e g anthrax toxins and Clostridium botulinum C2 toxin depend on proteolytic processing of the B moiety to expose a site that then binds the A moiety non covalently 4 In this article we describe strategies employed by AB toxins when entering cells emphasizing recent discoveries in the field 408 Membrane permeability Table 1 Mode of action of some AB toxins Toxin Enzymatic activity Cellular target s Diphtheria toxin Pseudomonas exotoxin A Anthrax edema toxin Anthrax lethal toxin Clostridium botulinum C2 toxin Shiga toxin Cholera toxin Pertussis toxin Clostridial neurotoxins Plant toxins ricin abrin etc Clostridium difficile toxins A and B Cytotoxic necrotizing factor 1 CNF1 ADP ribosyl transferase ADP ribosyl transferase Adenylate cyclase Zinc endoprotease ADP ribosyl transferase N glycosylase ADP ribosyl transferase ADP ribosyl transferase Zinc endoprotease N glycosylase Glucosyl transferase Deamidase EF 2 EF 2 cAMP modulated proteins MAPKK G actin 28S rRNA Heterotrimeric G protein Heterotrimeric G protein VAMP synaptobrevin SNAP 25 syntaxin 1 28S rRNA Rho proteins Rho proteins Toxin receptors at the cell surface and their role in penetration and intoxication The AB toxins in many cases bind to specific receptor molecules at the cell surface For instance the receptor for diphtheria toxin is the uncleaved precursor of the heparinbinding EGF like growth factor 5 the receptor for Pseudomonas exotoxin A is the a2 macroglobulin receptor 6 Shiga toxin binds to the glycolipid Gb3 7 and cholera toxin binds to GM1 8 In the case of plant toxins like ricin they usually bind to carbohydrates 9 regardless of whether they are attached to lipids or proteins The receptor in addition to providing binding sites at the cell surface has several other functions in the intoxication process Firstly if the receptor is internalized efficiently for instance from clathrin coated pits see below the toxin is also rapidly taken up Importantly receptor expression might be under regulation by growth factors cytokines This is the case for the Shiga toxin receptor Gb3 9 and it seems to play an important role in hemolytic uremic syndrome HUS caused by Escherichia coli that synthesize Shiga like toxins and cause food poisoning 10 Secondly the toxin receptor is important for targeting of the toxin to the organelle before it enters the cytosol An example is the Shiga toxin receptor where the lipid composition of the receptor is essential for retrograde toxin transport 9 11 A third point is that the receptor may play a direct role in toxin penetration through the membrane In the case of