ED: Investigation, Writing - review & editing
MT: Investigation, Writing - review & editing
YH: Investigation, Writing - review & editing
PS: Supervision, Writing - review & editing, Project administration
JT: Supervision, Writing - review & editing, Project administration
AK: Supervision, Project administration, Writing - review & editing
ER: Supervision, Project administration, Writing - original draft, Writing - review & editing, Funding acquisition
Multiple ubiquitin ligases with nuclear substrates promote regulated protein degradation and turnover of protein quality control (PQC) substrates. We hypothesized that two ubiquitin ligases with nuclear substrates – the anaphase-promoting complex/cyclosome with the Cdh1p substrate recognition factor (APC/C Cdh1p ) and the Slx5p/Slx8p SUMO-targeted ubiquitin ligase – contribute to PQC. We predicted yeast lacking subunits of these enzymes would exhibit compromised growth in the presence of hygromycin B, which reduces translational fidelity. We observed that loss of Cdh1p, Slx5p, or Slx8p sensitizes yeast to hygromycin B to a similar extent as loss of two ubiquitin ligases with characterized roles in nuclear PQC and hygromycin B resistance. In addition to their well-characterized function in regulated protein degradation, our results are consistent with prominent roles for both APC/C Cdh1p and Slx5p/Slx8p in PQC.
The ubiquitin-proteasome system (UPS) mediates both regulatory and quality control protein degradation (Finley et al., 2012; Kleiger and Mayor, 2014). In the UPS, ubiquitin ligase enzymes covalently attach multiple copies of the small protein ubiquitin to substrate proteins. Polyubiquitylated proteins are degraded by the 26S proteasome.
Several ubiquitin ligases contribute to the degradation of nuclear proteins in
We previously demonstrated that yeast lacking
The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit soluble ubiquitin ligase that mediates turnover of cell cycle-regulated proteins (e.g. cyclins) (Irniger et al., 1995; Sudakin et al., 1995). APC/C target specificity is determined by the identity of a coactivator subunit, which can be either Cdh1p or Cdc20p (Visintin et al., 1997). APC/C Cdh1p targets both cytoplasmic and nuclear proteins for degradation and regulates the mitosis-to-G1 transition (Schwab et al., 1997; Visintin et al., 1997). APC/C Cdh1p was recently shown to promote the turnover of an integral inner nuclear membrane protein in yeast, expanding the panel of ubiquitin ligases that participate in INMAD (Koch et al., 2019). Whether APC mediates PQC in addition to regulatory protein degradation is unknown.
The Slx5p/Slx8p heterodimer was initially characterized as a SUMO (Small Ubiquitin-like MOdifier)-targeted ubiquitin ligase (STUbL), marking for degradation proteins that have first been SUMOylated (Uzunova et al., 2007; Xie et al., 2007). Subsequent studies demonstrated a subset of Slx5p/Slx8p substrates are targeted in a SUMO-independent manner (Xie et al., 2010). Like Doa10p and the Asi complex, Slx5p/Slx8p promotes regulatory protein turnover (e.g. degradation of yeast transcription factor MATα2p (Xie et al., 2010) and SUMO ligase Siz1 (Westerbeck et al., 2014)) and degradation of aberrant proteins (e.g. destruction of a mutated variant of the Mot1p transcription factor (Wang and Prelich, 2009)). The extent to which Slx5p/Slx8p contributes to PQC relative to other nuclear ubiquitin ligases is uncharacterized.
To investigate potential contributions of APC/C
Cdh1p
and Slx5p/Slx8p to PQC, we cultured wild type yeast and yeast lacking
To validate the observation that
To validate the observations made with
Our results indicate APC/C Cdh1p and Slx5p/Slx8p are both critical for optimal fitness in the presence of hygromycin B, which is associated with increased concentrations of aberrant proteins. This is consistent with prominent roles for both ubiquitin ligases in PQC (although our data do not formally exclude non-catalytic function of these proteins in mitigating hygromycin B toxicity). Previous reports established contributions of Slx5p/Slx8p to nuclear PQC (e.g. (Wang and Prelich, 2009)). Further, Slx5p and Slx8p mitigate toxicity associated with polyglutamine-expanded huntingtin protein (Ohkuni et al., 2018).
To our knowledge, a role for APC/C
Cdh1p
in PQC has not been suggested. In addition to well-characterized roles in cell cycle-regulated degradation of nuclear and cytoplasmic substrates, our data imply APC/C
Cdh1p
may also contribute to destruction of misfolded or otherwise aberrant proteins. Consistent with a role for APC/C
Cdh1p
in PQC, negative genetic relationships between
Each of the ubiquitin ligases in this study (Doa10p, Asi complex, APC/C Cdh1p , and Slx5p/Slx8p) promotes regulatory degradation and possesses characterized or implied (based on this study) roles in PQC. To cope with the staggering number of ways protein molecules may conceivably misfold, become damaged, or behave aberrantly, we speculate many ubiquitin ligases with characterized function in regulatory protein turnover (such as APC/C Cdh1p ) moonlight in PQC.
Yeast growth experiments were performed as previously described (Watts et al., 2015). Four μl of sixfold serial dilutions were spotted onto agar plates containing yeast extract-peptone-dextrose medium (Guthrie and Fink, 2004) lacking or possessing hygromycin B (Gibco) at indicated concentrations. Plates were incubated at 30°C and imaged on the indicated days.
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VJY6 (alias MHY500) |
|
1C |
(Chen et al., 1993) |
VJY102 |
|
1A |
(Tong et al., 2001) |
VJY360 |
|
1A |
(Tong et al., 2001) |
VJY476 (alias BY4741) |
|
1A |
(Tong et al., 2001) |
VJY643 |
|
1A |
(Tong et al., 2001) |
VJY659 |
|
1A |
(Tong et al., 2001) |
VJY660 |
|
1A |
(Tong et al., 2001) |
VJY921 (alias HY5850) |
|
1B |
(Koch et al., 2019) |
VJY922 (alias HY5901) |
|
1B |
(Koch et al., 2019) |
VJY987 (alias MHY3712) |
|
1C |
(Xie et al., 2010) |
VJY988 (alias MHY3716) |
|
1C |
(Xie et al., 2010) |
VJY989 (alias MHY3861) |
|
1C |
(Xie et al., 2010) |
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This work was funded by NIH grant R15 GM111713 (EMR) and the Ball State University Department of Biology. Work in the lab of PJS is funded by NIH grant R15 G067291 and NIH grant R15 CA252996. Work in the lab of ALK is funded by the Indiana Academy of Science and Ball State University Provost Laboratory Start-up funds. This project was conceived while EMR was supported in part by a Ball State University Excellence in Teaching award (sponsored by the Ball State University Division of Online and Strategic Learning and the Office of the Provost).