AUTOPHAGIC VACUOLES FUSE WITH THE PRELYSOSOMAL COMPARTMENT IN CULTURED RAT FIBROBLASTS

We previously demonstrated both mannose 6-phosphate receptor (MPR) and cathepsin L in early autophagic vacuoles of cultured rat fibroblasts. This suggested that the enzyme may originate either from the receptor -enriched prelysosomal compartment (PLC) or from the trans-Golgi netwo rk (TGN). In the present ultrastructural study, we elucidated the role s of the PLC and TGN in lysosomal enzyme delivery to autophagic vacuol es. Firstly, we studied whether endocytic markers, cationized ferritin (CF), bovine serum albumin-gold or horseradish peroxidase (HRP), can be detected in autophagic vacuoles. Autophagy was induced by serum rem oval from the medium with or without leupeptin, an inhibitor of cystei ne proteinases. Endocytic markers were not detected in autophagic vacu oles after short uptake which filled the early endosome, but only afte r longer labeling which filled the PLC. The markers were usually found in advanced autophagic vacuoles containing partially degraded cytopla sm and complex internal membranes which are the characteristic of the PLC. HRP-positive vesicles were also observed in continuity with early autophagic vacuoles containing intact cytoplasm. After uptake and tra nsport of CF and HRP to the PLC, these markers were delivered to autop hagic vacuoles even if microtubules were disrupted in vinblastine befo re the induction of autophagy. Secondly, we studied whether MPRs trans port cathepsin L to autophagic vacuoles directly from the TGN. Two inh ibitors of MPR-mediated enzyme transport, tunicamycin and chloroquine, were used. Quantitative immunocytochemistry showed that neither of th ese drugs prevented cathepsin L delivery to autophagic vacuoles. The r esults suggest that a large proportion of lysosomal enzymes is deliver ed to autophagic vacuoles from the PLC by a microtubule-independent ma nner. The first enzymes may be transported in small PLC-derived vesicl es or tubules which are reached by HRP but not by CF and gold. Later, the autophaged cytoplasm is delivered to larger vacuolar parts of the PLC. Mannose 6-phosphate receptors transport no or only trace amounts of lysosomal enzymes to autophagic vacuoles directly from the TGN.