Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.

Abstract

Abstract. Seven cytosolic enzymes with varying halflives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h, glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organdies (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeprin. Inhibitors of lysosomal fusion processes (vinhlasrine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolyric inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.

Autophagy of cytoplasmic bulk cargo does not require LC3

“…6 We simplified the analysis of hepatocellular LC3 dynamics by blocking de novo influx of LC3 with the protein synthesis inhibitor cycloheximide. Under these conditions, total LC3 (LC3-I C LC3-II) declined rapidly (half-life »2 h); 6 this early LC3 disappearance could largely be accounted for by an autophagic-lysosomal degradation that was suppressed by inhibitors of autophagic sequestration (3MA, TG) or lysosomal function (NH 4 Cl, bafilomycin A 1 , leupeptin) 6 (Fig. 1A, inhibitors added at 0 min).…”

“…3 The bulk nature of macroautophagy has been strikingly demonstrated by the fact that cytosolic enzymes with widely different half-lives are all sequestered into autophagic vacuoles at the same rate, which also make them potential probes for macroautophagic cargo sequestration. 4 We routinely use LDH (lactate dehydrogenase) for this purpose, since it is a cytosolic enzyme present in all cells, and is degraded exclusively by the autophagic-lysosomal pathway. 4 Our macroautophagic cargo sequestration assay, which can be adapted to any cell type, 5 measures the net transfer of LDH into sedimentable autophagic vacuoles in the presence of leupeptin or bafilomycin A 1 to prevent LDH degradation in amphisomes and lysosomes.…”

“…4 We routinely use LDH (lactate dehydrogenase) for this purpose, since it is a cytosolic enzyme present in all cells, and is degraded exclusively by the autophagic-lysosomal pathway. 4 Our macroautophagic cargo sequestration assay, which can be adapted to any cell type, 5 measures the net transfer of LDH into sedimentable autophagic vacuoles in the presence of leupeptin or bafilomycin A 1 to prevent LDH degradation in amphisomes and lysosomes. Cells are usually incubated in an amino acid-and serum-free buffer to ensure that the maximally attainable autophagic activity (autophagic capacity) is measured.…”

“…In contrast, re-added 3MA, while still blocking cargo sequestration, allowed some degradative LC3 flux, suggesting that LC3-equipped phagophores could traverse the autophagic-lysosomal pathway without carrying bulk cytoplasmic cargo. 7 After »100 min of uninterrupted LC3 turnover during cycloheximide treatment, LC3 degradation was no longer sensitive to 3MA, TG or NH 4 Cl, indicating that its autophagic-lysosomal flux had stopped (Fig. 1A, inhibitors added at 100 min).…”

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“…It has been assumed for a long time that macroautophagy is a non-selective process, in which macromolecules are randomly degraded in the same ratio as they occur in the cytoplasm (Kominami et al, 1983;Kopitz et al, 1990). However, recent observations strongly suggest that this may not always be the case, and that macroautophagy can be selective under some conditions.…”