Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins

Abstract

Accumulation of misfolded oxidant-damaged proteins is characteristic of many diseases and aging. To understand how cells handle postsynthetically damaged proteins, we studied in Saccharomyces cerevisiae the effects on overall protein degradation of shifting from 30 to 38 degrees C, exposure to reactive oxygen species generators (paraquat or cadmium), or lack of superoxide dismutases. Degradation rates of long-lived proteins (i.e., most cell proteins) were not affected by these insults, even when there was widespread oxidative damage to proteins. However, exposure to 38 degrees C, paraquat, cadmium, or deletion of SOD1 enhanced two- to threefold the degradation of newly synthesized proteins. By 1 h after synthesis, their degradation was not affected by these treatments. Degradation of these damaged cytosolic proteins requires the ubiquitin-proteasome pathway, including the E2s UBC4/UBC5, proteasomal subunit RPN10, and the CDC48-UfD1-NPL4 complex. In yeast lacking these components, the nondegraded polypeptides accumulate as aggregates. Thus, many cytosolic proteins proceed through a prolonged "fragile period" during which they are sensitive to degradation induced by superoxide radicals or increased temperatures.

Figure 1.

Shifting cells from 30 to 38°C stimulates the degradation of recently synthesized proteins, but not of long-lived proteins, and by 60 min after synthesis, proteins no longer show their response to temperature shift. (A) Proteins were labeled with ³⁵S-Met for 5 min at 30°C to label recently synthesized proteins or 90 min to label long-lived proteins. After washing twice with chase media containing cyclohexamide, the cells were shifted to the indicated temperatures and rates of degradation were measured. The data shown in this and the subsequent experiments represent the means ± SD obtained from three experiments. (B) Cell proteins were labeled with ³⁵S-Met for 5 min, washed, resuspended in chase media, and shifted to 38°C immediately or 30, 60, or 90 min later. The amounts of protein degradation after 1 h were measured. Degradation rates after 2 or 3 h showed similar differences as after 1 h (not depicted).

Figure 2.

Exposure to paraquat or cadmium stimulates the degradation of recently synthesized but not long-lived proteins, and 60 min after synthesis, proteins no longer show this response to paraquat and cadmium. (A) During exponential growth at 30°C, yeast were exposed to 80 μg/ml paraquat or 50 μg/ml cadmium for 90 min, and then ³⁵S-Met was added for 5 min at 30°C to label recently synthesized proteins or for 90 min to label long-lived proteins. After washing and resuspension in chase medium, rates of protein degradation were measured as in Fig. 1 A. (B) Proteins were labeled with ³⁵S-Met for 5 min, washed, and then paraquat and cadmium were added immediately or 60 min after synthesis. The amounts of degradation after 1 h were measured. Degradation rates after 2 and 3 h showed similar differences as after 1 h (not depicted).

Figure 3.

Although long-lived proteins are not degraded more rapidly after exposure to paraquat, they are oxidatively damaged, and a lack of SOD1 also enhances the degradation of recently synthesized proteins. (A) Yeast were grown exponentially, and a portion of cells were treated with cycloheximide for 2 h to allow the degradation of short-lived proteins (the times studied in Fig. 2 A). Both the control and treated cells were then exposed to paraquat for 90 or 150 min. The presence of carbonylated proteins in equal amounts of cell proteins was assayed after derivitization with DNP-hydrazine (DNPH) and then Western blotting with an anti–DNP-hydrazone antibody. A control lane without the treatments with DNPH and paraquat is included to show the specificity of the antibody. Equal loading of lanes was shown with an eIF5A antibody. (B) WT, Δsod1 mutant, and the Δsod1 mutant expressing SOD1 from a plasmid were labeled with ³⁵S-Met for 5 min at 30°C. Rates of protein degradation were measured at 30°C as in Fig. 1 A.

Figure 4.

Shift to 38°C stimulates the degradation of recently synthesized proteins in WT but not in Δrpn10 and Δubc4Δubc5 strains. Cells were labeled with ³⁵S-Met for 5 min at 30°C and shifted to 38°C or maintained at 30°C. Rates of degradation were measured as in Fig. 1 A.

Figure 5.

Paraquat and cadmium fail to stimulate the degradation of recently synthesized proteins in Δrpn10 and Δubc4Δubc5 strains and these strains contain higher amounts of oxidant-damaged proteins than WT during growth and after exposure to paraquat. (A) WT and mutants were grown exponentially at 30°C and treated with paraquat or cadmium for 90 min. Then cell proteins were labeled with ³⁵S-Met for 5 min, and rates of degradation measured as in Fig. 2 A. (B) WT and mutants were grown exponentially, and a portion was exposed to paraquat for 90 min. The presence of carbonylated proteins in equal amounts of cell extracts was assayed by OxyBlot as in Fig. 3 A. To visualize oxidant damage in the control cells, approximately three times more protein was loaded than in cells treated with paraquat.

Figure 6.

Shifting cells to 38°C and exposure to paraquat or cadmium fail to stimulate the degradation of recently synthesized proteins in the cdc48-3 mutant, and the cdc48-3 strain contains higher amounts of oxidant-damaged proteins than WT during growth and after exposure to paraquat. (A) WT and cdc48-3 strains were labeled with ³⁵S-Met for 5 min at 30°C, and rates of degradation at 30 or 38°C were measured as in Fig. 1 A. To measure the degradation of oxidant-damaged proteins, WT and cdc48-3 strains were exposed to paraquat or cadmium for 90 min, followed by labeling with ³⁵S-Met for 5 min, and rates of degradation were measured as in Fig. 2 A. (B) WT and the cdc48-3 mutant were grown exponentially, and a portion was exposed to paraquat for 90 min. The presence of carbonylated proteins in equal amounts of cell proteins was assayed by OxyBlot as in Fig. 3 A.

Figure 7.

Shifting cells to 38°C and exposure to paraquat or cadmium fail to stimulate the degradation of recently synthesized proteins in the ufd1-1 mutant. WT and ufd1-1 strains were labeled with ³⁵S-Met for 5 min at 30°C, and rates of degradation were measured at 30 or 38°C as in Fig. 1 A. To measure the degradation of oxidant-damaged proteins, WT and ufd1-1 strains were exposed to paraquat or cadmium for 90 min, followed by labeling with ³⁵S-Met for 5 min, and rates of degradation were measured as in Fig. 2 A.

Figure 8.

NPL4 is required for increased degradation at 38°C and after exposure to paraquat or cadmium. WT and npl4-1 strains were labeled with ³⁵S-Met for 5 min at 30°C, and rates of degradation at 30 or 38°C and upon exposure to paraquat or cadmium were measured as in Fig. 6 A.

Figure 9.

UBC7 and UFD4 are not required for increased degradation at 38°C and after exposure to paraquat or cadmium. (A) WT and Δubc7 strains were labeled with ³⁵S-Met for 5 min at 30°C, and rates of degradation at 38°C and upon exposure to paraquat or cadmium were measured as in Fig. 6 A. (B) WT and Δufd4 strains were labeled with ³⁵S-Met for 5 min at 30°C, and rates of degradation at 38°C and upon exposure to paraquat or cadmium were measured as in Fig. 6 A.

Figure 10.

After shift to 38°C or exposure to paraquat to cause oxidant damage, recently synthesized proteins not degraded in Δrpn10 and Δubc4Δubc5 mutants selectively accumulate in the particulate fraction, unlike recently synthesized proteins in WT or mutant strains at 30°C without oxidant present. (A) WT, Δrpn10, and Δubc4Δubc5 strains were labeled with ³⁵S-Met for 5 min, washed, and shifted to 38°C or treated with paraquat at 30°C. At the indicated times, lysates were prepared, and equal amounts of lysate proteins were subjected to differential centrifugation. The amounts of labeled protein present in pellets obtained by centrifuging for 20 min at 9,300 g and by ultracentrifugation at 275,000 g for 60 min were measured. To determine the amounts of labeled long-lived proteins that accumulate in these fractions, cells were shifted to 38°C or exposed to paraquat 3 h after labeling. (B) WT, Δrpn10, and Δubc4Δubc5 strains were labeled with ³⁵S-Met for 5 min at 30°C. The amounts of radiolabeled protein present in 275,000 g pellet were measured as described in Fig. 10 A. (C) Yeast were grown exponentially and treated with paraquat as in Fig. 10 A. Cell lysates were subjected to centrifugation and the presence of carbonylated proteins in the 275,000 g pellet assayed by OxyBlot as in Fig. 3 A.