Protein degradation in the lysosome.

Autophagosome-lysosome fision measured by means o f electroinjected [ ‘“C] lactose While [‘“C] sucrose is an inert sequestration probe which eventually accumulates in lysosomes, [‘“c] lactose is hydrolysed immediately upon entry into the lysosome, by the lysosomal enzyme 0-galactosidase. Autophagically sequestered [“C] lactose therefore rapidly equilibrates at a low steady-state level, probably corresponding to radioactivity in autophagosomes. At a constant sequestration rate, this steady-state level will be inversely proportional to the rate of autophagosome-lysosome fusion, and can therefore be used, for example, to test the effect of inhibitors on the latter process. Vinblastine, a strong inhibitor of autophagic-lysosomal degradation (Table l) , had little effect on sequestration (of [‘“C] sucrose), but dramatically enhanced the accumulation of [‘4C]lactose, consistent with its proposed mechanism of action as an inhibitor of autophagosome-lysosome fusion (Kovacs et al., 1982). Similar studies with individual amino acids suggested that several of them might act as fusion inhibitors, in particular asparagine. Using [‘“C] sucrose and [‘*C] lactose as probes of autophagic sequestration and fusion, respectively, it should now be possible to investigate these hitherto experimentally inaccessible processes in some detail. This project is generously supported by The Norwegian Cancer Society.