ATP-DEPENDENT TRANSLOCATION OF RICIN ACROSS THE MEMBRANE OF PURIFIED ENDOSOMES

Ricin translocation was demonstrated (using both fluorescence- and rad iolabel-based assays) across the membrane of endosomes purified from m ouse lymphocytes. Selectivity of the process was shown by the absence of translocation 'activity of transferrin and horseradish peroxidase u sed as membrane-bound and fluid-phase endosome labels, respectively. E ndocytosed I-125-ricin translocation was found to be strictly ATP- (K( m) almost-equal-to 4 mM) and temperature-dependent, with up to 30% end osomal I-125-ricin appearing in the external medium after 2 h at 37-de grees-C. No treatments neutralizing the acidic endosome pH (ammonium c hloride, nigericin, chloroquine) significantly impaired ricin transloc ation, and the pH gradient across the endosome membrane is not require d for this process. Chase experiments showed that the ability of I-125 -ricin to translocate increases with its depth in the endocytic system (i.e. plasma membrane << early endosomes < late endosomes). Both A an d B ricin chains displayed translocation ability as demonstrated by th e results of our assay on ricin, ricin B, transferrin-ricin A, and tra nsferrin-ricin B conjugates. Biological activity of both ricin chains is preserved after translocation as shown by the inhibitory effect of the A chain on cell-free protein synthesis and the binding of the B ch ain to lactose-agarose.