Inhibition of protein synthesis by didemnin B: How EF-1 alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein syn thesis in cells and in vitro. The stage at which DB inhibits protein synthe sis in cells is not known, although dehydrodidemnin B arrests translation a t the stage of polypeptide elongation. Inhibition of protein synthesis by D B in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models o f protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1 alpha; however, the dependence upon EF-1 alpha was pre viously unexplained. It is shown here that DB binds only weakly to EF-1 alp ha/GTP in solution, but binds to ribosome.EF-1 alpha complexes with a disso ciation constant K-d = 4 mu M.'Fhus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1 alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated A DP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 b inding to pre-translocative ribosome EF-1 alpha complexes, thus preventing ribosomal translocation, Based on this model for protein synthesis inhibiti on by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1 alpha function in which this elongation factor does not fully depart the ribosome during polypepti de elongation.