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. 2009 Apr 17;284(16):10343-52.
doi: 10.1074/jbc.M808840200. Epub 2009 Feb 9.

Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome

Lyudmila N Dimitrova  1 , Kazushige Kuroha, Tsuyako Tatematsu, Toshifumi Inada
Affiliations

Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome

Lyudmila N Dimitrova et al. J Biol Chem. .

Abstract

The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stem-loop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4Delta mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5 E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome.

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Figures

FIGURE 1.
FIGURE 1.
Protein degradation by the proteasome in response to translation arrest caused by consecutive basic amino acid residues. A, construction of the pGPDp-GFP-X-FLAG-HIS3 reporter plasmids, which contain various sequences encoding 12 consecutive amino acids inserted between GFP and HIS3. B, consecutive basic amino acid sequences induce translation arrest coupled with protein degradation by the proteasome. Cells harboring the indicated pGPDp-GFP-X-FLAG-HIS3 plasmid were grown on SC-Ura medium, and the samples were analyzed using Western blotting with anti-GFP (top panel) or anti-eEF2 (bottom panel) antibodies. When indicated (+), cell extracts were prepared 2 h after the addition of 0.2 mm MG132. When determining the relative expression levels, we did not include shorter (degraded) products. C, proteasome activity is inhibited in the presence of MG132. W303 cells were grown on SC medium, and cell extracts were prepared 2 h after the addition of 0.2 mm MG132 when indicated (+). The samples were analyzed using Western blotting with anti-FK2 antibodies, which recognize monoubiquitin and polyubiquitin but not free ubiquitin. D, truncated mRNA is not detected in the presence of MG132. W303 cells harboring the indicated pGPDp-GFP-X-FLAG-HIS3 plasmid were grown on SC-Ura medium, and RNA samples were analyzed using Northern blotting with DIG-labeled GFP or SCR probes. E, the stabilities of GFP-K12(AAG)-FLAG-His3, GFP-R12-FLAG-His3, and GFP-K12(AAA)-FLAG-His3 were analyzed using Western blotting. Samples of W303 cells harboring the indicated plasmid were prepared at the indicated time points after the addition of cycloheximide (0.1 mg/ml). The levels of the remaining proteins were determined by Western blotting with anti-GFP antibodies.
FIGURE 2.
FIGURE 2.
Consecutive basic amino acid residues rather than rare codons induce translation arrest coupled with protein degradation. A, sequence specificity of the translation arrest-induced protein degradation process mediated by the proteasome. W303 cells harboring the indicated pGPDp-GFP-X-FLAG-HIS3 plasmids were grown on SC-Ura medium, and cell extracts were prepared. When indicated (+), cell extracts were prepared 2 h after the addition of 0.2 mm MG132. The relative levels of the reporter proteins were determined using Western blotting with anti-GFP or anti-eEF2 antibodies. The levels of the products derived from GFP-S12-HIS3, GFP-D12-HIS3, GFP-E12-HIS3, GFP-G12-HIS3, GFP-I12-HIS3, and GFP-P12-HIS3 were higher than that of the product derived from GFP-HIS3. Additionally, the relative levels of those products were determined using a standard curve of samples from cell harboring pGPDp-GFP-S12-HIS3 or pGPDp-GFP-HIS3. B, W303 cells harboring the indicated pGPDp-GFP-X-FLAG-HIS3 plasmid were grown on SC-Ura medium, and the samples were analyzed using Western blotting. When indicated (+), cell extracts were prepared 2 h after the addition of 0.2 mm MG132.
FIGURE 3.
FIGURE 3.
Not4p is required for the degradation of translation arrest products by the proteasome. A, Not4p is required for the proteasome-mediated degradation of the truncated product. W303 or W303not4Δ cells were transformed with the pGPDp-GFP-R12-FLAG-HIS3 plasmid. The cells were grown in SC-Ura, and samples were analyzed using Western blotting with anti-GFP antibodies. When indicated (+), cell extracts were prepared 2 h (W303) or 5 h (W303not4Δ) after the addition of 0.2 mm MG132. B, RNA samples were prepared from cells shown in A and analyzed using Northern blotting with DIG-labeled GFP or SCR probes. C, the level of truncated product derived from GFP-K12-FLAG-HIS3 was higher in the not4Δ mutant. W303 or W303not4Δ cells were transformed with the indicated pGPDp-GFP-X-FLAG-HIS3 plasmid. The cells were grown in SC-Ura, and samples were analyzed using Western blotting. D, W303, W303not4Δ, or W303rpn6–2 (YNK7) cells harboring pGPDp-GFP-R12-FLAG-HIS3 were grown in SC medium at 30 °C. The samples were prepared and analyzed using Western blotting with anti-GFP antibodies (lanes 1–3) or anti-FK2 antibodies that recognize monoubiquitin and polyubiquitin but not free ubiquitin (lanes 4–6). E, the level of the truncated product derived from GFP-R12-FLAG-HIS3 was higher in the not4Δ mutant. W303, W303not3Δ, W303not4Δ, or W303not5Δ cells were transformed with the pGPDp-GFP-R12-FLAG-HIS3 plasmid. The cells were grown in SC-Ura, and the samples were analyzed using Western blotting. F, RNA samples were prepared from the cells shown in D and analyzed using Northern blotting with DIG-labeled GFP or SCR probes. G, the level of truncated product derived from GFP-R12-FLAG-HIS3 was higher in the not4Δ mutant expressing the Not4L35A mutant (mutation in the RING finger domain). W303not4Δ cells containing pGPDp-GFP-R12-FLAG-HIS3 were transformed with pADHp-NOT4 or pADHp-not4L35A. The cells were grown in SC-UraLeu medium, and the samples were analyzed using Western blotting. H, FLAG-Not4p protein was distributed in the polysome fractions. W303 cells were transformed with pADHp-FLAG-NOT4. Cell extracts were prepared, and polysome analysis was performed as described previously (3). Protein samples prepared from each fraction were analyzed using Western blotting with anti-FLAG antibodies. WT, wild type.
FIGURE 4.
FIGURE 4.
Not4p is not involved in the degradation of nonstop products. W303 or W303not4Δ cells were transformed with the indicated pGPDp-GFP-FLAG-HIS3 (pSA156) or pGPDp-GFP-FLAG-HIS3-NS (pSA157) plasmids (4). A, the cells were grown in SC-Ura, and the samples were analyzed using Western blotting with anti-GFP antibodies. B, the cells were grown in SC-Ura, and the samples were analyzed using Northern blotting with DIG-labeled GFP and SCR probes. WT, wild type.
FIGURE 5.
FIGURE 5.
Endogenous sequences induce translation arrest coupled with protein degradation. A, construction of the pGPDp-GFP-X-FLAG-HIS3 reporter plasmids. These plasmids contain various sequences inserted between GFP and HIS3. The inserted amino acid sequences and original gene names are shown. B, endogenous sequence elements induce translation arrest coupled with protein degradation by the proteasome. W303 cells were transformed with pGPDp-GFP-X-FLAG-HIS3 plasmids that contained sequences from the indicated genes. The cells were grown in SC-Ura, and the samples were analyzed using Western blotting with anti-GFP antibodies. When indicated (+), cell extracts were prepared 2 h after the addition of 0.2 mm MG132. C, translation arrest products in the not4Δ mutant. W303not4Δ cells were transformed with pGPDp-GFP-X-FLAG-HIS3 plasmids that contained sequences from the indicated genes. The cells were grown in SC-Ura, and the samples were analyzed using Western blotting with anti-GFP antibodies.
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