Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes

Autophagic vacuoles (AVs) arise when membranes of the ER sequester parts of the cytoplasm, forming a new, double-membraned vacuole, to which lysosomal enzymes are then delivered. To investigate the mechanism of lysosomal enzyme delivery to nascent AVs, amino acid (AA) starvation and glucagon treatment were used to induce autophagy in a cultured cell system using rat hepatocytes (Fu5C8 cells). The induction of autophagy was assayed using biochemical, morphometric and immunocytochemical techniques. In these cells, AA starvation resulted in a fivefold increase in total cellular proteolysis, and sixfold and 4.5-fold increases in the volume and surface densities of AVs, respectively. Using an antibody against the mannose 6-phosphate receptor (MPR) and two sizes of colloidal gold to label separately and track the endosomal and lysosomal compartments, the time course of endosomal and lysosomal fusion with AVs was analyzed in detail. On the basis of these experiments, we found that AVs rapidly fuse with pre-existing lysosomes, but seldom with a prelysosomal compartment (PLC). Using immunoperoxidase, staining for the MPR was infrequently observed in association with any AVs. However, at early times following the induction of autophagy (less than 2 h), many autophagic vacuoles stained positively for the lysosomal enzyme cathepsin D. Consistent with these results, treatment of cells with tunicamycin had no effect on autophagy-induced proteolysis. We conclude that lysosomal enzyme delivery to nascent AVs occurs primarily by the fusion of pre-existing mature lysosomes, with a much smaller contribution by MPRs or the PLC.