Diphtheria toxin forms pores of different sizes depending on its concentration in membranes: Probable relationship to oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposur e to low pH. These pores may play a critical role in the translocation of t he A chain of the toxin into the cytoplasm. The effect of protein concentra tion on diphtheria toxin pore formation in model membrane systems was assay ed by using a new fluorescence quenching method. In this method, the moveme nt of Cascade Blue labeled dextrans of various sizes across membranes is de tected by antibodies which quench Cascade Blue fluorescence. It was found t hat at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglyc erol vesicles with a size that depends on protein concentration. At the low est toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore propertie s were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations i n the presence of cholesterol. The dependence of pore size on toxin concent ration suggests that toxin oligomerization regulates pore size. This behavi or may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements o f the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligo merization could assist proper membrane insertion of the toxin and other pr oteins that convert from soluble to membrane-inserted states are discussed.