Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 17;49(11):e390.
doi: 10.1038/emm.2017.128.

Modulating cellular balance of Rps3 mono-ubiquitination by both Hel2 E3 ligase and Ubp3 deubiquitinase regulates protein quality control

Youjin Jung  1 , Hag Dong Kim  1   2 , Hee Woong Yang  1 , Hye Jin Kim  1 , Chang-Young Jang  3 , Joon Kim  1   2
Affiliations

Modulating cellular balance of Rps3 mono-ubiquitination by both Hel2 E3 ligase and Ubp3 deubiquitinase regulates protein quality control

Youjin Jung et al. Exp Mol Med. .

Abstract

When a ribosome complex is stalled during the translation elongation process in eukaryotes, the mono-ubiquitination of Rps3 has recently been shown to be critical to ribosome quality control. We have discovered that the regulatory role of Rps3 mono-ubiquitination is controlled by a deubiquitinase. We also showed that an autophagic signal appears to be coupled to the mono-ubiquitination of Rps3p through the entrance of Ubp3p into the autophagosome in yeasts. The mono-ubiquitination of the Rps3 protein is tightly modulated by reciprocal action between the Hel2p E3 ligase and the Ubp3p deubiquitinase in yeasts and the reciprocal action between the RNF123 E3 ligase and the USP10 deubiquitinase in mammalian cells. We also found that the Ubp3p/USP10 deubiquitinases critically modulate Hel2p/RNF123-mediated Rps3p mono-ubiquitination. In addition, we found that Hel2p/RNF123 and Ubp3p/USP10 appeared to be differently localized in the ribosome complex after ultraviolet irradiation. Together, our results support a model in which coordinated ubiquitination and deubiquitination activities can finely balance the level of regulatory Rps3p mono-ubiquitination in ribosome-associated quality control and autophagy processes.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Ubiquitination of K212 on Rps3p is increased by starvation and translation inhibition. Each sample was harvested and lysed for western blotting. The proteins were confirmed by immunoblotting using an anti-Rps3p antibody. The anti-pgk1p antibody was used as a loading control. (a) The BY4741 cells that were grown to the stationary phase in YPD media were reseeded and grown to the early log phase. After cells were treated with each translation inhibition drug such as Cycloheximide (100 μg ml−1), Emetine (10 μM), Blasticidin S (25 μg ml−1), Anisomycin (100 μM) or exposed to UV (200 J m−2), they were incubated for 1 h. (b) The JS143-7D and BY4741 strains were grown to stationary phase in SC media or YPD media. The cells were reseeded into each appropriate media and grown to the early log phase. The media was changed to SC media that lacked Trp and His (tryptophan, histidine depletion), SC media that contained a lower (1/10) or higher concentration of amino acids (10 ×), nitrogen starvation media, SC-His media containing 80 mM 3-AT, or YPD media that contained 100 ng ml−1 rapamycin (Tor kinase inhibition). The incubation time was 1 h, after which the cells were harvested by centrifugation. (c) The lysates from the Rps3 or Rps3K212R cells that were exposed to UV stress were analyzed by western blotting with indicated antibodies. The anti-eIF2α~p antibody was used as a positive control for UV stress. (d) The total extracts from the Rps3 and Rps3K212R mutant cells that were exposed to UV stress (+) or not (−) were subjected to centrifugation in a 5~45% sucrose gradient and analyzed by UV absorbance at 254 nm. The fractions were collected and analyzed by immunoblotting with anti-Rps3p, anti-ubiquitinated Rps3p, and anti-Pgk1p antibodies.
Figure 2
Figure 2
Hel2p is an E3 ligase of ribosomal protein s3. (a) The wild-type (wt) and hel2Δ strains were grown to early log phase in YPD media, exposed to UV stress (200 J m−2) and incubated for 1 h. The whole cell lysates were analyzed by immunoblot with anti-Rps3p, ubiquitinated Rps3p, eIF2α~p, and Pgk1 antibodies. (b) The BY4741 hel2Δ strain was transformed with plasmid encoding FLAG (control), FLAG-Hel2p (WT-Hel2), or FLAG-Hel2pC79A, H81A, C87A (mutation of the RING domain). The cells expressing FLAG, FLAG-Hel2p, or FLAG-Hel2pC79A, H81A, C87A were grown to the stationary phase in SD (U−) media. The cells were reseeded in SRG (U−) media and then grown to the early log phase. The whole cells were analyzed by immunoblotting with the indicated antibodies. (c) The BY4741 hel2Δ strains harboring the vectors expressing FLAG or Hel2p-FLAG were grown to the early log phase in SC (H−) media. The cells grown to early log phase were exposed to UV stress (+) or not (−) and incubated for 3 min. The cell lysates (left) or the ribosomes that were purified by ribosome pelleting (right) were confirmed by western blotting with the indicated antibodies. The incubation time after UV stress was 1 h, except in the case of c.
Figure 3
Figure 3
Rps3p is deubiquitinated by Ubp3p. (a) The wild-type (WT) and ubp3Δ strains were grown to the early log phase in YPD media, exposed to UV stress (200 J m−2) and incubated for 1 h. (b) The BY4741 ubp3Δ strain was transformed with a plasmid expressing His6 (control), His6-Ubp3p (WT-Ubp3), or His6-Ubp3pC469A, H861A (catalytically inactive allele). The transformed cells were grown to the early log phase in SRG (U−) media for induction. Each cell was exposed to UV irradiation (+) or not (−). The whole cells were analyzed by immunoblotting with the indicated antibodies. (c) The indicated strains expressing Ubp3p-HA from its endogenous locus were grown to the early log phase in YPD media. The cells grown to the early log phase were exposed to UV stress (+) or not (−) and incubated for 3 min. The cell lysates (left) or the ribosomes that were purified by ribosome pelleting (right) were confirmed by western blotting with the indicated antibodies. (d) The indicated strain containing the UBP3 deletion was grown to the early log phase in YPD media. The proteins in the total extracts were analyzed by immunoblotting with the indicated antibodies. The incubation time after UV stress was 1 h, except in the case of c.
Figure 4
Figure 4
Ubiquitinated Rps3p is regulated byUbc4p, Hel2p and Ubp3p in vitro. (a) An in vitro ubiquitination assay was performed on purified His6-tagged E1 (human), Hel2p, Rps3p, Ubp3p, and ubiquitin. The indicated E2 conjugation enzyme was added into the mixtures. The mixtures were then analyzed by immunoblotting with anti-Rps3p and anti-ubiquitinated Rps3p antibodies. (b) The wild-type (wt) and ubc4Δ strains were grown to the early log phase in YPD media. The cells were exposed to UV stress (+) or not (−) and incubated for 1 h. The whole cell lysates were analyzed by immunoblotting with anti-Rps3p, anti-ubiquitinated Rps3p, anti-eIF2α~p, and anti-Pgk1p antibodies. (c) An in vitro ubiquitination assay was performed on purified His6-tagged E1 (human), UBCH5b, Hel2p, Rps3p, Ubp3p, and ubiquitin. The Hel2p was added (+) or not (−) into a solution that contained E1, UBCH5b, ubiquitin, and Rps3p (lanes 1 and 2). Ubp3p was added (+) or not (−) into a solution that contained E1, UBCH5b, ubiquitin, Hel2p, and Rps3p. The solutions were analyzed by immunoblotting with anti-Rps3p and anti-ubiquitinated Rps3p antibodies. The Rps3p and ubiquitinated Rps3p are indicated on the right.
Figure 5
Figure 5
Ubp3p-dependent autophagy is indispensable for rapamycin-induced Rps3p mono-ubiquitination. The wild-type (WT), Δubp3 (a), Δbre5 (b), Δatg8 (c), and Δatg7 (d) deletion strains were grown to the early log phase in YPD media and then treated with rapamycin for the indicated time. The cells were analyzed by western blotting with anti-Rps3p, eIF2α~p, Pgk1p, and ubiquitinated Rps3p antibodies. (e) The strains with Ubp3p-HA that was reintroduced at its endogenous locus were grown to the early log phase in YPD media and exposed to UV stress or treated with rapamycin for the indicated time. The whole cell lysates were immunoprecipitated with the Rps3p antibody. The immunoprecipitates were analyzed by western blotting with the indicated antibodies. The arrow denotes mono-ubiquitinated Rps3p, while the asterisk denotes a non-specific band.
Figure 6
Figure 6
RNF123 and USP10 are implicated in the status of human RPS3 mono-ubiquitination. (a) The HT1080 cells were treated with various translational inhibitors, including 50 μg ml−1 cycloheximide, 20 μg ml−1 blasticidin S, 2 μg ml−1 DON, 2 μg ml−1 anisomycin, 150 J m−2 UV, 5 μM pactamycin, 50 μg ml−1 G418, 50 μg ml−1 puromycin, 50 μg ml−1 hygromycin B, and 20 μM emetine for 2 h. The cell lysates were subjected to immunoblot analysis using the indicated antibodies. (b) The HT1080 cells stably expressing the Flag-RPS3 wild-type or the K214R mutant were UV-irradiated (200 J m−2), harvested after 2 h and subjected to immunoblotting with anti-RPS3 or anti-Flag antibodies. (c and d) The HT1080 cells were transfected with siRNAs against RNF123 or USP10, treated with 150 J m−2 of UV irradiation and harvested after being incubated in fresh media for 2 h. The lysates were assayed by immunoblotting with the indicated antibodies. (e) The UV-irradiated HT1080 cells were incubated for the indicated times, followed by ultracentrifugation with a 20% sucrose cushion to isolate the ribosomal pellets. Each cell lysate and ribosomal pellet were subjected to immunoblot analysis using the indicated antibodies. (f) The HT1080 cells stably expressing the Flag-RPS3 wild-type or the K214R mutant were transfected with RPS3 5’-UTR siRNA. After 48 h, these cells were treated (or not) with 150 J m−2 UV and incubated for 15 min. Each cell lysate was ultracentrifuged with a 20% sucrose cushion to isolate the ribosomal fraction. Each cell lysate and ribosomal fraction were separated by SDS–PAGE and subjected to immunoblot analysis using the indicated antibodies. SDS–PAGE, SDS–polyacrylamide gel electrophoresis; UTR, untranslated region.
Figure 7
Figure 7
Mono-ubiquitination of Rps3p is responsible for cell viability and apoptosis. (a) Serial dilutions of the WT or Rps3pK212R mutant cells were spotted onto solid YPD media (lower). Serial dilutions of the WT or Rps3pK212Rmutant cells that were exposed to UV stress were spotted onto solid YPD media (upper). The plates were incubated for 2 days at 30 °C (b and c). The HT1080 cells stably expressing the Flag-RPS3 wild-type or the K214R mutant were transfected with RPS3 5’-UTR siRNA. After 48 h, these cells were irradiated (or not) with 150 J m−2 UV. (b) Subsequently, these cells were labeled with 35S-Met/35S-Cys (250 μCi per ml) for 15 min. Each cell lysate and ribosomal pellet were then subjected to immunoblot analysis using an anti-ubiquitin antibody (left). To detect the poly-ubiquitinated nascent polypeptides, immunoprecipitation with an anti-ubiquitin antibody was performed for ribosomal pellets, as described in ‘Materials and Methods,’ and subjected to autoradiography (right). (c) The UV-irradiated cells were incubated for 8 h, harvested, lysed, and analyzed by immunoblotting with the indicated antibodies. UTR, untranslated region.
See this image and copyright information in PMC

References

    1. Ruggero D, Pandolfi PPDoes the ribosome translate cancer?Nat Rev Cancer 2003; 3: 179–192. - PubMed
    1. Naora H. Involvement of ribosomal proteins in regulating cell growth and apoptosis: translational modulation or recruitment for extraribosomal activity? Immunol Cell Biol 1999; 77: 197–205. - PubMed
    1. Fumagalli S, Di Cara A, Neb-Gulati A, Natt F, Schwemberger S, Hall J et al. Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction. Nat Cell Biol 2009; 11: 501–508. - PMC - PubMed
    1. Naora H, Takai I, Adachi M, Naora H. Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis. J Cell Biol 1998; 141: 741–753. - PMC - PubMed
    1. Kim J, Chubatsu LS, Admon A, Stahl J, Fellous R, Linn S. Implication of mammalian ribosomal protein S3 in the processing of DNA damage. J Biol Chem 1995; 270: 13620–13629. - PubMed

Publication types

LinkOut - more resources