The ubiquitin-activating enzyme is required for lysosomal degradation of cellular proteins under stress.
ts85, a cell-line that harbors a mutant thermolabile ubiquitin-activating enzyme, E1, fails to degrade short-lived proteins at the restrictive temperature (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). It is not known whether the ubiquitin system is also involved in the degradation of long-lived proteins. In the present study we show that upon shifting the mutant cells to the restrictive temperature, there is no change in the rate of degradation of long-lived proteins. In contrast, shifting the wild-type cells (FM3A) to the high temperature is accompanied by a 2-fold increase in the rate of proteolysis of this group of proteins. This heat-induced accelerated degradation can be completely inhibited by NH4Cl and chloroquine. Similarly, exposure of the cells to starvation, a stimulus that activates the autophagic-lysosomal pathway, has no effect on the degradation of long-lived proteins in the mutant cells following inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. A revertant of the ts85 cells behaved in a similar manner to the wild-type cells. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20) (Kulka, R. G. et al. (1988) J. Biol. Chem. 263, 15726-15731). Cycloheximide and 3-methyladenine, inhibitors of formation of autophagic vacuoles, suppress the heat-induced accelerated degradation in the wild-type cells. Taken together, the results suggest that: 1. heat stress induces enhanced degradation of intracellular proteins, 2. the process occurs most probably in autophagic vacuoles, 3. activation of ubiquitin is required for enhanced degradation to occur, and 4. the activation is involved most probably in formation of the autophagic vacuoles.
Ciechanover
A
A
Department of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Gropper
R
R
Schwartz
A L
AL
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013312:Stress, Physiological / Q000378:metabolism; D013696:Temperature; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D011506:Proteins; C025946:3-methyladenine; D003513:Cycloheximide; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes; D000225:Adenine