Stress-induced alterations in autophagic pathway: relationship to ubiquitin system

Abstract

The autophagic response of the cell to nutrient deprivation or heat stress is characterized by an increase in the rate of cellular protein degradation. Using temperature-sensitive mutant cell lines that harbor a mutation in the ubiquitin pathway, we have recently shown that this response is dependent on a functional ubiquitin-activating enzyme E1. The ubiquitin pathway is involved in a multitude of cellular events including protein degradation, the best understood of these. Herein the activation of the ubiquitin molecule via E1 is followed by its covalent conjugation to acceptor proteins followed by proteolysis. It is therefore important to study the linkage between the autophagic response and E1. Using these same cell lines, CHO E36 and CHO ts20, we demonstrate that after heat stress or nutrient deprivation there is a rapid and reversible decrease in the buoyant density of subcellular vesicles containing lysosomal hydrolases, a characteristic found to accompany autophagy. This stress-induced change is found in all cell lines examined, independent of the activity of the E1. The light-density vesicles, which comigrate with endosomes on colloidal silica gradients, are not accessible to the endocytic marker transferrin-horseradish peroxidase (HRP) after cellular uptake and subsequent HRP-mediated density shift analysis. Furthermore, morphology of the isolated fractions from control and stress-induced cells was similar. These results thus demonstrate the changes in hydrolase-containing intracellular vesicles that accompany nutritional deprivation or heat stress and support the notion that the linkage of the autophagic response to the ubiquitin system is at a step in autophagy which does not affect the formation of autophagic vesicles.