The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins

R. Gropper, R. A. Brandt, S. Elias, C. F. Bearer, A. Mayer, A. L. Schwartz, Aaron Ciechanover

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Abstract

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). However, the involvement of the ubiquitin system in the degradation of long lived proteins (most cellular proteins fall in this category) has not been addressed. 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 inhibited completely 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 after inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20 (Kulka, R.G., Raboy, B., Schuster, R., Parag, H.A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem. 263, 15726-15731)). Cycloheximide and 3-methyladenine, known inhibitors of formation of autophagic vacuoles, inhibit the heat-induced accelerated degradation of long lived proteins in 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; and 3) activation of ubiquitin is required for the formation of these vacuoles. As there is no change in the basal rate of degradation of intracellular proteins in the mutant cells at the restrictive temperature, it appears that the ubiquitin system is not involved in their breakdown.

Original languageEnglish
Pages (from-to)3602-3610
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number6
StatePublished - 17 Jul 1991

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Ubiquitin-Activating Enzymes
Proteolysis
Degradation
Proteins
Ubiquitin
Vacuoles
Temperature
Hot Temperature
Mutant Proteins
Ports and harbors
Cells
Cell Line
Chloroquine
Cycloheximide
Starvation
Chemical activation

Cite this

Gropper, R., Brandt, R. A., Elias, S., Bearer, C. F., Mayer, A., Schwartz, A. L., & Ciechanover, A. (1991). The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. Journal of Biological Chemistry, 266(6), 3602-3610.
Gropper, R. ; Brandt, R. A. ; Elias, S. ; Bearer, C. F. ; Mayer, A. ; Schwartz, A. L. ; Ciechanover, Aaron. / The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 6. pp. 3602-3610.
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abstract = "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). However, the involvement of the ubiquitin system in the degradation of long lived proteins (most cellular proteins fall in this category) has not been addressed. 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 inhibited completely 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 after inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20 (Kulka, R.G., Raboy, B., Schuster, R., Parag, H.A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem. 263, 15726-15731)). Cycloheximide and 3-methyladenine, known inhibitors of formation of autophagic vacuoles, inhibit the heat-induced accelerated degradation of long lived proteins in 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; and 3) activation of ubiquitin is required for the formation of these vacuoles. As there is no change in the basal rate of degradation of intracellular proteins in the mutant cells at the restrictive temperature, it appears that the ubiquitin system is not involved in their breakdown.",
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Gropper, R, Brandt, RA, Elias, S, Bearer, CF, Mayer, A, Schwartz, AL & Ciechanover, A 1991, 'The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins', Journal of Biological Chemistry, vol. 266, no. 6, pp. 3602-3610.

The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. / Gropper, R.; Brandt, R. A.; Elias, S.; Bearer, C. F.; Mayer, A.; Schwartz, A. L.; Ciechanover, Aaron.

In: Journal of Biological Chemistry, Vol. 266, No. 6, 17.07.1991, p. 3602-3610.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins

AU - Gropper, R.

AU - Brandt, R. A.

AU - Elias, S.

AU - Bearer, C. F.

AU - Mayer, A.

AU - Schwartz, A. L.

AU - Ciechanover, Aaron

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N2 - 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). However, the involvement of the ubiquitin system in the degradation of long lived proteins (most cellular proteins fall in this category) has not been addressed. 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 inhibited completely 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 after inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20 (Kulka, R.G., Raboy, B., Schuster, R., Parag, H.A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem. 263, 15726-15731)). Cycloheximide and 3-methyladenine, known inhibitors of formation of autophagic vacuoles, inhibit the heat-induced accelerated degradation of long lived proteins in 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; and 3) activation of ubiquitin is required for the formation of these vacuoles. As there is no change in the basal rate of degradation of intracellular proteins in the mutant cells at the restrictive temperature, it appears that the ubiquitin system is not involved in their breakdown.

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Gropper R, Brandt RA, Elias S, Bearer CF, Mayer A, Schwartz AL et al. The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. Journal of Biological Chemistry. 1991 Jul 17;266(6):3602-3610.