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Science 23 December 2005:
Vol. 310. no. 5756, pp. 1960 - 1963
DOI: 10.1126/science.1121925
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Reports

Protein Synthesis upon Acute Nutrient Restriction Relies on Proteasome Function

Ramunas M. Vabulas* and F. Ulrich Hartl*

Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.


Fig. 1. Proteasome activity is required to sustain protein synthesis upon amino acid restriction. (A) Amounts of Ub-EGFP reporter protein were analyzed in transiently transfected HeLa cells at 0, 5, and 10 min after addition of dimethyl sulfoxide (DMSO) alone, 5 mM CHX, 100 µM MG132 in DMSO, or CHX and MG132 combined by anti-GFP immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Translation was followed by measuring the incorporation of 35S-Met into TCA-insoluble material (left) or analyzing newly synthesized proteins by 10% SDS-PAGE and phosphorimaging (right). At time 0, 50 µCi/ml 35S-Met and MG132 were added. Circles, normal medium (80 µM Leu); squares, deficient medium (0.8 µMLeu). Solid symbols, MG132 addition; open symbols, DMSO controls. (C) Translation was analyzed as in (B). In addition to MG132, 40 µM clasto-lactacystin-ß-lactone (CL-ß-lactone) or 40 µM epoxomicin were used, and translation was observed for 15 min (fig. S1, A and B). Protein synthesis is expressed in percentage of controls lacking inhibitor. Means + SD of three independent experiments are shown. Gray bars, normal medium; black bars, Leu-deficient medium. (D) Same as (B), except that Phe-sufficient (40 µM) and Phe-deficient (0.4 µM) media were used. (E) Same as (B), except that the amount of Met was varied from 20 µM (normal medium) to 0.2 µM (deficient medium) and labeling was with 100 µCi/ml 3H-Leu. Representative results of at least three independent experiments are shown. [View Larger Version of this Image (57K GIF file)]
 

Fig. 2. Proteasome activity is most important during the early phase of amino acid starvation. (A and B) HeLa cells were incubated in normal medium (prestarvation) or in Leu- and Met-deficient medium for 3 or 6 hours (h). Protein translation was then analyzed in Leu-deficient medium by adding 50 µM Ci/ml 35S-Met with either DMSO (open symbols), MG132 (A), or epoxomicin (B) (solid symbols) (13). (C) Same as (A), except that 10 mM 3-MA was added at the beginning of prestarvation. DMSO (open symbols) or MG132 (solid symbols) was added during labeling. Representative results of at least three independent experiments are shown. cpm, counts per minute. [View Larger Version of this Image (26K GIF file)]
 

Fig. 3. Immediate cellular effects of amino acid restriction and proteasome inhibition. (A) Activation of GCN2 kinase in HeLa cells by reducing the medium concentration of Leu from 80 to 0.8 µM and simultaneous proteasome inhibition. Activated GCN2 kinase (pGCN2) was detected by immunoblotting with an antibody to pGCN2. Asterisk, nonspecific band. Equal loading was confirmed with antibodies detecting GCN2 independent of its phosphorylation (GCN2). At time 0, 100 µM MG132 or DMSO was added. (B) Translation was analyzed in Leu-deficient medium (–Leu) or Phe-deficient medium (–Phe). Incorporation of 35S-Met into TCA-precipitable material was measured. 50 µCi/ml 35S-Met was added either together with MG132 (solid symbols) or with DMSO (open symbols). After 10 min (dashed line), the respective lacking amino acid (triangles) or control amino acid (circles) (Phe in case of –Leu medium, Leu in case of –Phe medium) was added to normal concentration. aa, amino acid. Representative results of at least three independent experiments are shown. [View Larger Version of this Image (50K GIF file)]
 

Fig. 4. Effect of proteasome activity on radiolabeling of newly synthesized proteins and degradation of faulty proteins. (A and B) Newly synthesized proteins in HeLa cells were labeled with 50 µCi/ml 35S-Met in Met-deficient medium (A), or with 100 µCi/ml 3H-Leu in Leu-deficient medium (B). Radioactivity of TCA-insoluble material was measured. MG132 (solid symbols) or DMSO (open symbols) were added with radioactive tracers at time 0. (Right) Protein synthesis after 8 min (A) or 10 min (B) of labeling is expressed in percentage of controls lacking inhibitor. Means + SD of three independent experiments are shown. (C) Labeling with 50 µCi/ml 35S-Met in normal medium lacking nonessential amino acids. Circles, cells in normal medium; squares, cells preincubated for 15 min in 20 mM L-azetidine-2-carboxylic acid (Azc) before and during labeling. Open symbols, DMSO; solid symbols, MG132-containing cultures. cpm, counts per minute. (D) Proteins were labeled as in (C). Gray bars, control cells; black bars, Azc-treated cells. After labeling for 10 min, a chase was performed with 20 mM unlabeled Met. Half of the cultures received 100 µM MG132 and 10 µM clasto-lactacystin-ß-lactone during labeling and chase (+ proteasome inhibitor), and the other half received DMSO (– proteasome inhibitor). TCA-precipitable radioactivity at time 0 was set as 100%. Means + SD of three experiments are shown. [View Larger Version of this Image (45K GIF file)]
 

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