The major pathway for cytosolic constituents to enter lysosomes is by autophagy. We used two cytosolic proteins, CuZn superoxide dismutase (SOD) and carbonic anhydrase III (CAIII), as autophagic markers in male rat hepatocytes. We took advantage of the differential presence of the two proteins in autophagic vacuoles because of the high resistance of SOD to lysosomal degradation as compared with CAIII. This allows us to determine the sequence of autophagic vacuole formation. We have double immunogold-labeled SOD and CAIII in cryosections of fasted rat liver and calculated the ratios of SOD over CAIII labeling densities (SOD/CAIII) in autophagic vacuoles (AV), as compared with the cytoplasm. Different classes of AV were defined according to their SOD/CAIII, their morphology, and their additional immunolabeling for the lysosomal markers lgp120 and cathepsin D. Of all AV, 15% exhibited a cytosol-like SOD/CAIII, indicating that degradation had not yet begun. Most of these initial AV (AVi) showed two enveloping membranes. The formation of AVi was prevented by 3-methyladenine, a potent inhibitor of autophagy. Of all AV, 85% showed a SOD/CAIII that exceeded the cytosolic ratio. These single membrane-bound vacuoles were called degradative AV (AVd). Labeling for lysosomal markers allowed the characterization of AV that shared features with both AVi and AVd. These AVi/d had a cytosol-like SOD/CAIII and a double membrane, but showed some labeling for lysosomal markers. Probably these AVi/d represent the recipient compartment for lysosomal components. AVd were positive for cathepsin D and lgp120. We discerned two AVd subclasses. Early AVd with cytosol-like SOD labeling density while CAIII labeling density was consistently lower than in the cytosol. Their size was similar to AVi and AVi/d. Late AVd contained higher SOD concentrations and were mostly larger. Our findings suggest that AV acquire lysosomal constituents by fusion with small nonautophagic structures and that after subsequent elimination of the inner membrane of AVi, degradation starts resulting in the formation of early AVd and late AVd.

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