Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic...

[14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). During autophagy lactose is present only at a low, steady-state level in pre-lysosomal vacuoles (probably autophagosomes), serving as a useful marker for these organelles. If autophagosome-lysosome fusion is blocked with vinblastine (Kovács et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.