Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment

A number of proteins are able to enter cells from the extracellular environ ment, including protein toxins, growth factors, viral proteins, homeoprotei ns, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence fro m the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragme nt (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment. The fusio n of the TAT-peptide to dtA converted the protein to a heparin-binding prot ein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficie ntly deliver enzymatically active dtA to the cytosol. Interestingly, the fu sed TAT-peptide potentiated the binding and cytotoxic effect of the corresp onding holotoxin. We made a fusion protein between VP22, another membrane-p ermeant protein, and dtA, and also in this case we detected association wit h cells in the absence of a cytotoxic effect. The data indicate that transp ort of dtA into the cell by the TAT-peptide and VP22 is inefficient.