Probing Pores with Peptide Plugs

Extracellular proteins are made in the cytosol and transported out of the cell. Certain proteins travel the opposite way, from the outside to the cytosol. Most of these proteins are toxins made by bacteria and plants, but evidence is accumulating that certain growth factors can do the same. Toxins that enter the cytosol can, according to current knowledge, be roughly classified into two main groups, those that enter from endo-somes and those that enter from the endoplasmic retic-ulum (Alouf and Freer, 1999). In both cases, the travel to the cytosol is initiated by binding of the toxin to cell surface receptors, followed by endocytosis. Diphtheria toxin, the protein responsible for the tissue damage in diphtheria, is the most famous and best studied of these toxins. Like many other toxins acting inside cells, the active form consists of two polypeptides linked by a disulfide bond. Because the toxin is synthesized by the bacterium as a single chain protein that is subsequently cleaved, the two parts are referred to as the A and the B fragments. The B fragment consists of two functional parts that form separate domains in the crystallized protein, the receptor-binding R domain and the T domain (transmembrane) that is involved in translocation of the catalytic A fragment (or C domain) to the cytosol. The A fragment is an enzyme that ADP-ribosylates elongation factor 2 in the cytosol, leading to protein synthesis inhibition and, consequently, to cell death. Upon endocytosis of the toxin-receptor complex , the low pH in the endosome triggers unfolding of the toxin molecule, leading to insertion of the T domain into the endosomal membrane and concomitant translocation of the A fragment to the cytosol. The process can be induced already at the cell surface when cells with bound toxin are exposed to low pH. The T domain is different from the rest of the molecule in that it consists almost entirely of ␣-helices. In this respect, it resembles certain pore-forming toxins such as colicin A and Bacillus thuringiensis ␦-endotoxin (Parker and Pattus, 1993). In fact, as was first detected in black lipid films (Kagan et al., 1981) and later confirmed in living cells (Eriksen et al., 1994), diphtheria toxin also has the ability to form ion-conducting channels in biological membranes. Expression and periplas-mic secretion of diphtheria toxin in Escherichia coli caused the bacteria to die at acidic pH, due to channel formation in the inner …