AN INTACT TRANSMEMBRANE HELIX-9 IS ESSENTIAL FOR THE EFFICIENT DELIVERY OF THE DIPHTHERIA-TOXIN CATALYTIC DOMAIN TO THE CYTOSOL OF TARGET-CELLS

To investigate structure/function relationships involved in the delive ry of the diphtheria toxin (DT) catalytic (C) domain to the cytosol of target cells, we have constructed and characterized internal in-frame deletion mutants in the transmembrane (T) domain of the fusion toxin DAB(389)IL-2. This fusion protein is composed of the C and T domains o f DT to which human interleukin-2 (IL-2) has been genetically fused, T he mutant fusion toxins were compared to DAB(389)IL-2 with respect to cytotoxic potency, receptor binding affinity, channel formation in pla nar lipid membranes, and sensitivity to proteolytic digestion. We demo nstrate that genetic fusion of human IL-2 sequences to a diphtheria to xin-related fragment that contains less than full-length transmembrane helix 9 results in a fusion protein that binds to the high affinity I L-2 receptor, but has lost greater than or equal to 3 logs of cytotoxi c potency and has decreased ability to insert into planar membranes an d form stable channels. These observations are consistent with the hyp othesis that an intact transmembrane helix 9 is essential for the form ation of stable channels that are required for the efficient delivery of the C domain to the cytosol of target cells.