Specific inhibition by NH 4Cl of autophagy-associated proteolysis in cultured fibroblasts

Abstract

Rat embryo fibroblasts, prelabeled with [ 14C]leucine, showed an enhanced degradation of cell protein as well as increased peptide release when placed in a serum-deficient medium. NH 4Cl inhibited only the induced proteolysis, but had no effect on basal protein turnover. Electron microscopy studies showed that enhanced proteolysis was associated with an increase in autophagic vacuoles containing amorphous and membranous debris, and that NH 4Cl markedly increased the number of these intracellular vacuoles. Upon release from NH 4Cl inhibition, these cells showed a compensatory enhanced release of 14C into the medium and a decrease in the number of intracellular degradative vacuoles. We conclude that enhanced proteolysis reflects an activation of the autophagic-lysosomal system in these cells and that NH 4Cl inhibits the final hydrolysis and release steps in this mechanism.