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. 2015 Apr 24;11(4):e1005197.
doi: 10.1371/journal.pgen.1005197. eCollection 2015 Apr.

Inhibiting K63 polyubiquitination abolishes no-go type stalled translation surveillance in Saccharomyces cerevisiae

Kazuki Saito  1 , Wataru Horikawa  1 , Koichi Ito  1
Affiliations

Inhibiting K63 polyubiquitination abolishes no-go type stalled translation surveillance in Saccharomyces cerevisiae

Kazuki Saito et al. PLoS Genet. .

Abstract

Incidental ribosome stalling during translation elongation is an aberrant phenomenon during protein synthesis and is subjected to quality control by surveillance systems, in which mRNA and a nascent protein are rapidly degraded. Their detailed molecular mechanisms as well as responsible factors for these processes are beginning to be understood. However, the initial processes for detecting stalled translation that result in degradation remain to be determined. Among the factors identified to date, two E3 ubiquitin ligases have been reported to function in distinct manners. Because ubiquitination is one of the most versatile of cellular signals, these distinct functions of E3 ligases suggested diverse ubiquitination pathways during surveillance for stalled translation. In this study, we report experimental evidences for a unique role of non-proteasomal K63 polyubiquitination during quality control for stalled translation. Inhibiting K63 polyubiquitination by expressing a K63R ubiquitin mutation in Saccharomyces cerevisiae cells markedly abolished the quality control responses for stalled translation. More detailed analyses indicated that the effects of K63R mutants were independent of the proteasome and that K63 polyubiquitination is dependent on Hel2, one of the E3 ligases. Moreover, a K63R ubiquitin mutant barely inhibited the quality control pathway for nonstop translation, indicating distinct mechanisms for these highly related quality control pathways. Our results suggest that non-proteasomal K63 polyubiquitination is included in the initial surveillance process of stalled translation and presumably triggers protein degradation steps upon translational stall. These findings provide crucial information regarding the detailed molecular mechanisms for the initial steps involved in quality control systems and their classification.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Comparisons of the effects of deleting Hel2 and Ltn1.
(A) Western blot analysis for Rluc-HIS3 reporters from the wild-type (WT), hel2 ∆, and ltn1 ∆ strains (BY4727, SKY61, S18-E01). A schematic image of reporter fusion genes, Rluc-HIS3, either with or without 12 CGA codon repeats, is illustrated above. The expression of fusion protein was detected using a Rluc antibody. PGK1 was used as a loading control. Arrowhead 1 indicates the Rluc protein alone, arrowhead 2 indicates the Rluc-blank-HIS3 protein, and arrowhead 3 indicates the Rluc-CGA x12-HIS3 protein. (B) Dual luciferase assay for Rluc-CGA x12-luc2 reporter in the wild-type (WT), hel2 ∆, and ltn1 ∆ strains (HRKW-2, SKY113, HRKW-6). A schematic image of reporter fusion genes, Rluc-luc2, either with or without 12 CGA codon repeats, is illustrated above. Bars indicate luc2/Rluc ratios. Percentages were standardized using the results of a dual luciferase assay for Rluc-blank-luc2 in the wild-type strain (HRKW-1). The inset indicates the results with the ltn1 ∆ strain with an appropriately adjusted range. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.
Fig 2
Fig 2. Effect of K63 polyubiquitination on the quality control for stalled translation.
(A) Structure of Ubiquitin protein and location of the lysine residues for polyubiquitination [id: 1UBQ]. K63 (red) and other lysine residues (pink) are shown in sticks. The structural model was generated using MolFeat version 3.5 (Fiatlux, Tokyo, Japan). (B) Schematic illustration for the inhibition of polyubiquitination by arginine mutants. Arginine mutants were expressed from expression plasmids in yeast cells harboring endogenous ubiquitin genes on its genome. An incorporation of an arginine mutant into the (poly-) ubiquitin chain terminates further elongation. (C) Cellular ubiquitination levels of wild-type and ubiquitin mutants with a single arginine substitution (K6R, K11R, K27R, K29R, K33R, K48R, K63R). Ubiquitin genes were fused with His-tag and expressed from plasmids in the wild-type strain (BY4727). Cellular ubiquitination levels were detected by antibody for His-tag. PGK antibody was used as loading control. (D) Dual luciferase assay for the effects of arginine mutants of ubiquitin protein on the Rluc-CGA x12-luc2 reporter. Plasmids that included ubiquitin arginine mutants were introduced into a wild-type strain (HRKW-2). Ubi-WT indicates wild-type ubiquitin. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.
Fig 3
Fig 3. Comparison of the effects of Hel2 deletion and K63R expression on protein and mRNA levels in stalled translation.
(A) Western blot analysis for the effects of K63R ubiquitin on Rluc-HIS3 reporter. blank indicates the result of the Rluc-blank-HIS3 reporter, and CGAx12 indicates the results of the Rluc-CAAx12-HIS3 reporter. WT indicates the wild-type strain (BY4727) without K63R ubiquitin, hel2∆ indicates the hel2∆ strain (SKY61), and K63R indicates the wid-type strain expressing K63R ubiquitin. Arrowhead 1 indicates the Rluc protein alone, arrowhead 2 indicates the Rluc-blank-HIS3 protein, and arrowhead 3 indicates the Rluc-CGA x12-HIS3 protein. PGK antibody was used as loading control. (B) Northern blot analysis for the effects of K63R ubiquitin on Rluc-HIS3 reporter. blank and CGAx12 as well as WT, hel2∆, and K63R are as in (A). The reporter mRNA was probed by HIS3 complementary DNA fragment. Arrowhead 1 indicates the cleaved HIS3 mRNA and arrowhead 2 indicates the full-length mRNA. SCR1 mRNA was detected as a loading control.
Fig 4
Fig 4. Effects of K0 and K63only ubiquitin mutants on stalled translation.
(A) Cellular ubiquitination levels of K0 and K63only ubiquitin mutants. Ubiquitinated proteins were detected as above. Amount of the protein sample from a cell expressing His-tagged wild-type ubiquitin are decreased to 1/16 and 1/4 dilution. (B) Dual luciferase assay for the of K0 and K63only ubiquitin mutants on the Rluc-luc2 reporter. Plasmids bearing K0 or K63only ubiquitin mutants were introduced into a wild-type strain harboring Rluc-luc2 reporter strain (HRKW-2). (single) indicates the transformant with ubiquitin expression from single copy plasmids, and (multi) indicates that from multiple copy plasmids. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements. (C) Genetic colony growth test for the effect of K0 and K63only ubiquitin mutants on the Rluc-HIS3 reporter (SKY24). Types of ubiquitin vectors are indicated on the left. Transformant colonies were streaked on the SC-Leucine (Leu) (middle), and SC-Leucine, Histidine (Leu, His) (right) plates, and colony growth was monitored for 4 days at 30°C.
Fig 5
Fig 5. Effects of proteasome inhibitors, MG132 and PS341, on stalled translation and K63R expression.
(A) Western blot analysis for effect of MG132. Vectors with Rluc-HIS3 reporters were introduced into a wild-type strain (BY4727). Yeast transformants were incubated for 5 h with either DMSO (e.g., 0 μM MG132) or 75 μM MG132. Arrowhead 1 indicates the Rluc protein only and arrowhead 2 indicates the Rluc-blank-HIS3 protein. PGK1 was used as a loading control. (B) Dual luciferase assay for the effect of MG132 on wild-type strain with Rluc-CGA x12-luc2 reporter (HRKW-2). MG132 was used as in (A). (C) The empty or K63R ubiquitin mutation plasmids were introduced into the pdr5 ∆ yeast strain harboring Rluc-luc2 reporter (SKY142). The transformants were incubated in the presence of MG132 or PS341 at indicated concentrations for 2 h. Fold differences between the percentages of luc2/Rluc ratios between the transformants with the absence (-K63R) or presence (+K63R) of K63R expression are indicated over the top of the bar graphs.
Fig 6
Fig 6. Temporal regulation of K63 polyubiquitination and Hel2 with other factors for stalled translation.
(A) The luc2/Rluc ratios for Rluc-luc2 reporter in the wild-type (WT) and single knockout strains (as indicated) in the presence (+K63R) or absence (-K63R) of K63R expression. The inset shows the results for the rqc1 ∆ and ltn1 ∆ strains. The average luc2/Rluc ratios and their standard deviations were obtained for three independent measurements. (B) The luc2/Rluc ratios for Rluc-luc2 reporter in the strains with double knockout of Hel2 and other factors involved in stalled translation. All strains are hel2 ∆. Thus, as examples, WT in this figure indicates a hel2 ∆ single knockout strain, and asc1 ∆ indicates the hel2 ∆ asc1 ∆ strain. The average luc2/Rluc ratios and their standard deviations were obtained for three independent measurements. *p < 0.05, **p < 0.01.
Fig 7
Fig 7. Effects of K63 polyubiquitination and Hel2 on quality control for non-stop translation.
(A) Genetic colony growth test for the nonstop HIS3 reporter. A schematic image of a nonstop HIS3 allele is illustrated above. Empty vector (empty), wild-type ubiquitin (Ubi-WT), or K63R ubiquitin (K63R) plasmids were introduced into the wild-type, ski3 ∆, hel2 ∆, or ltn1 ∆ strains harboring nonstop HIS3 reporter (S17-A08, S17-A09, SKY123, SKY137). Types of knockout alleles and vectors are indicated on the left. Transformant colonies were streaked on the SC-Uracil (Ura) (middle), and SC-Uracil, Histidine (Ura, His) (right) plates, and colony growth was monitored for 3 days at 30°C. (B) Western blot analysis for the nonstop HIS3 reporter. Protein samples were extracted from the transformants of the nonstop HIS3 reporter strains as in (A). The expression of HIS3 protein was detected using a HIS3 polyclonal antibody. PGK1 was used as a loading control. (C) Northern blot analysis for the nonstop HIS3 reporter. mRNA samples were extracted from the transformants of the nonstop HIS3 reporter strains as in (A). The nonstop HIS3 reporter mRNA was probed by HIS3 complementary DNA fragment. SCR1 mRNA was detected as a loading control.
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