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. 2020 May 1;31(10):1015-1031.
doi: 10.1091/mbc.E19-11-0622. Epub 2020 Mar 11.

Ubiquitin-proteasome-mediated cyclin C degradation promotes cell survival following nitrogen starvation

Stephen D Willis  1 , Sara E Hanley  1 , Thomas Beishke  1 , Prasanna D Tati  2 , Katrina F Cooper  1
Affiliations

Ubiquitin-proteasome-mediated cyclin C degradation promotes cell survival following nitrogen starvation

Stephen D Willis et al. Mol Biol Cell. .

Abstract

Environmental stress elicits well-orchestrated programs that either restore cellular homeostasis or induce cell death depending on the insult. Nutrient starvation triggers the autophagic pathway that requires the induction of several Autophagy (ATG) genes. Cyclin C-cyclin-dependent kinase (Cdk8) is a component of the RNA polymerase II Mediator complex that predominantly represses the transcription of stress-responsive genes in yeast. To relieve this repression following oxidative stress, cyclin C translocates to the mitochondria where it induces organelle fragmentation and promotes cell death prior to its destruction by the ubiquitin-proteasome system (UPS). Here we report that cyclin C-Cdk8, together with the Ume6-Rpd3 histone deacetylase complex, represses the essential autophagy gene ATG8. Similar to oxidative stress, cyclin C is destroyed by the UPS following nitrogen starvation. Removing this repression is important as deleting CNC1 allows enhanced cell growth under mild starvation. However, unlike oxidative stress, cyclin C is destroyed prior to its cytoplasmic translocation. This is important as targeting cyclin C to the mitochondria induces both mitochondrial fragmentation and cell death following nitrogen starvation. These results indicate that cyclin C destruction pathways are fine tuned depending on the stress and that its terminal subcellular address influences the decision between initiating cell death or cell survival pathways.

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Figures

FIGURE 1:
FIGURE 1:
The UPS is required for cyclin C destruction following nitrogen starvation. (A) Model showing the two roles coined “day job” and “night job” that cyclin C plays to regulate the oxidative stress response (see text for details and reviewed in Jezek et al., 2019b; Strich and Cooper, 2014). Importantly, the night job is independent of Cdk8 and both roles are conserved (Wang et al., 2015). (B) Western blot analyses of extracts prepared from mid-log cultures with the indicated genotypes expressing cyclin C-myc resuspended nitrogen starvation medium (SD-N) for the indicated times. (C) Degradation kinetics of cyclin C signals obtained in B. Error bars indicate SD, N = 3. (D) Western blot analyses of extracts prepared from a wild-type mid-log culture were treated with 200 ng/ml rapamycin for the indicated times. Pgk1 levels were used as a loading control for all Western blot studies.
FIGURE 2:
FIGURE 2:
The UPS is required for cyclin C degradation following nitrogen starvation. (A) Western blot analyses of extracts prepared from mid-log ubc4/5∆ (MHY508) and wild-type (MHY414) cultures expressing cyclin C-myc resuspended in nitrogen starvation medium (SD-N) for the indicated times. (B) Quantification of the results obtained in A. N = 3. (C) As in A except that cyclin C levels were monitored in wild type (SUB62), the “quint∆” Ub receptor mutant (YSS781a, dsk2∆, rad23∆, ddi1∆ rpn13-pru rpn10-uim), and the triple intrinsic receptor mutant (YSS786a, rpn13-pru rpn10-uim rpn1-ARR). (D) Quantification of the results obtained in C. N = 2. (E) Cyclin C-YFP was monitored by Western blot analysis following nitrogen starvation in wild type (MHY414), ubc4/5∆ (MHY508), and not4∆. (F) The Rsp5-HA strain (RSY2301) harboring the Tet operator plasmid (pCM1888) and cyclin C-myc were grown to mid-log phase and a sample removed for Western analysis to visualize Rsp5-HA (far right, top panel). The remaining culture was treated with doxycycline for 5 h before being subjected to nitrogen starvation. Thereafter, cyclin C-myc was monitored by Western blot analysis. For all blots, Pgk1 levels were used as loading controls.
FIGURE 3:
FIGURE 3:
Cyclin C does not induce mitochondrial fragmentation following rapamycin stress. (A) Wild-type cells (RSY10) harboring expression plasmids for Mito-TFP (mitochondrial marker) and cyclin C-YFP were untreated (top panels) treated with 0.8 mM H2O2 (middle panel) or rapamycin (200 ng/ml) for the times indicated. Merged panels of cyclin C-YFP and Mito-TFP are shown. The stippled box indicates enlarged panels. (B) Wild-type cells harboring cyclin C-YFP and Nup49-mCherry grown to mid-log were treated with rapamycin (200 ng/ml) as indicated. Merged and enlarged panels are indicated. (C) As in A except that the cells also expressed Vph1-mCherry to mark the vacuoles were then treated with either 200 ng/ml or 2.5 ng/ml rapamycin for 3 h. (D) Mutant pep4∆ prb1∆-1 (BJ5459) cells harboring cyclin C-YFP and Vph1-mCherry were treated with 200 ng/ml rapamycin for 4 h. Representative fluorescence microscopy images are shown.
FIGURE 4:
FIGURE 4:
Cyclin C does not induce mitochondrial fragmentation following rapamycin stress. (A) Wild-type cells (RSY10) harboring expression plasmids for cyclin C-YFP and Nup49-mCherry were washed and resuspended in SD-N. Representative fluorescence microscopy images of the results are shown. (B) As in A except that the cells also express Mt-Ds-Red to mark the mitochondria. For all panels, Nom. indicates Nomarski imaging. (C) Degradation kinetics of cyclin C after exposure to 1.2 and 0.4 mM H2O2 or SD-N as indicated. The data were obtained from previously published experiments (Jin et al., 2015) and Figure 1C. (D) Mitochondrial morphology of cells 2 h after the stress. The data for the H2O2 experiments were obtained from (Jin et al., 2015). (E) cnc1∆ cells (RSY1696) harboring a wild (pKC337) or cyclin CA110V-myc expression plasmid (pKC354) were grown to mid-log phase and then washed and resuspended in nitrogen starvation medium (SD-N) for the indicated times. Cyclin C-myc levels were monitored by Western blot analysis. Pgk1 levels were used as a loading control.
FIGURE 5:
FIGURE 5:
Cyclin C-Cdk8 negatively regulates ATG8 mRNA expression. (A) RT-qPCR assays probing for ATG8 mRNA expression in the mutant shown before and after 1 h 200 ng/ml rapamycin treatment. Transcript levels are given relative to the internal ACT1 mRNA control. (B) Fold increase in GFP-Atg8 levels in strains shown before and after 6 h 200 ng/ml rapamycin treatment. (C) GFP-Atg8 cleavage assays before and after 200 ng/ml rapamycin in wild-type (YC7) and cdk8 kinase dead (YC17) strains. The asterisk indicates a previously reported background band (Huang et al., 2014). (D) As in A for the ume6∆ (RSY431) and ume6 cdk8∆ (RSY2128) strains as indicated. (E, F) RT-qPCR assays probing for ATG7 and ATG14 mRNA expression respectively in wild-type (RSY10) and cdk8∆ (RSY1726) unstressed cells. Transcript levels are given relative to the internal ACT1 mRNA control. For all RT-qPCR assays, the error bars indicate the SD from the mean of two technical replicates from three independent cultures. ***p < 0.001 and NS represents no significance.
FIGURE 6:
FIGURE 6:
Deletion of either CDK8 or CNC1 promotes cell survival following low-dose rapamycin treatment. (A) Quantification of GFP-Atg8 flux depicted in Supplemental Supplemental Figure S4A for wild-type (RSY10), cdk8∆ (RSY1726) and cnc1∆ (RSY1696) cultures. N = 3. (B) Representative Nomarski and fluorescence microscopy images of GFP-Atg8 foci are shown for unstressed wild-type, cnc1∆, and cdk8 cells as in A. Quantification of GFP-Atg8 foci depicted as a percentage of population displaying 0, 1, 2, or 3 foci (percentage of mean ± SD) grown under unstressed conditions. N = 3, for all assays: **p < 0.01, ***p < 0.001. ****p < 0.0001. (C) Wild-type, cdk8∆, and cnc1∆ cells were grown to mid log in SD-complete medium and 10-fold dilutions plated on SD-complete medium containing 0, 200, or 2.5 ng/ml rapamycin. (D) Identical exposures of GFP-Atg8 cleavage assays in wild-type and cdk8∆ cells after treatment with 2.5 ng/ml rapamycin for the timepoints indicated. The asterisk indicates a previously reported background band (Huang et al., 2014). (E) Wild-type cells (RSY10) harboring expression plasmids for cyclin C-YFP, Vph1-mCherry, and MT. CFP were washed and resuspended in 2.5 ng/ml rapamycin for 3 h. Representative fluorescence microscopy images of the results are shown. Arrows point to detectable cyclin C-YFP after 3 h rapamycin stress. (F) Doubling times of wild type (RSY10), cnc1∆ (RSY1696) and atg8∆ (RSY2144) cells in YPDA with and without 2.5 ng/ml rapamycin. N = 3. (E) For all blots, Pgk1 was used as a loading control.
FIGURE 7:
FIGURE 7:
Rapamycin treatment protects mitochondria from H2O2-mediated fragmentation. (A) Wild-type cells harboring the plasmids shown were grown to mid-log and treated with 50 ng/ml rapamycin and thereafter 0.8 mM H2O2 for the times indicated. Representative fluorescence microscopy images of the results are shown. Bar = 5 µm. (B) Representative images of cells from A treated with 50 ng/ml rapamycin. Bar = 5 µm. (C, D) The percentage of cells from A that had cytoplasmic cyclin C and fragmented mitochondria respectively. N = 3. (E) GFP-Atg8 cleavage assays following treatment with 50 ng/ml rapamycin (left panel) or 0.8 mM H2O2 (middle panel) for the timepoints indicated. In the right-hand panel, H2O2 was added for 1 h after rapamycin addition. (F) Mid-log wild-type cells were subjected to the conditions shown (50 ng/ml rapamycin and 2 mM H2O2) and thereafter cell viability determined by growth (10-fold dilutions) on rich medium (YPDA). (G) Mid-log wild-type cells were treated as described in F except that except that the % of live cells was monitored by FDA staining and FACs analysis after 24 h. The % of dead cells (FDA negative) was graphed for each condition. N = 3. ****p < 0.0001.
FIGURE 8:
FIGURE 8:
Redirecting cyclin C to the mitochondria promotes cell death in response to survival signals. (A) Fluorescence microscopy of mid-log phase cnc1∆ cells harboring expression plasmids for either cyclin C-YFP or the cyclin C-YFP-Fis chimeric protein plus either the mitochondria or the vacuole markers (Mt. Ds-Red and Vph1-mCherry, respectfully). Bar = 5 µm. The top panels are Nomarski images. (B) Quantification of mitochondrial morphology in cnc1∆ cells harboring either cyclin C-YFP or cyclin C-YFP-Fis expression plasmids. At least 200 cells were counted from three individual isolates. The percentage of cells (mean ± SD) within the population displaying the different mitochondrial morphologies is given. (C) Representative fluorescence microscopy images of the mitochondrial morphologies scored in B. Bar = 5 µm. (D) Survival assays as described in Figure 6C in cnc1∆ cells harboring either cyclin C-YFP or cyclin C-YFP-Fis expression plasmids. (E) Mid-log cells with the genotypes shown were washed then switched to SD-N medium. The percent of inviable cells within the population was determined using phloxine B staining and FACs analysis after 3 days. N = 3. For all experiments **p < 0.01, ***p < 0.001.
FIGURE 9:
FIGURE 9:
Model for regulator network controlling the final subcellular address for cyclin C and subsequent cell fate decisions. In unstressed cells growing in replete media (left-hand panel), cyclin C-Cdk8 repress the expression of a subset of stress-responsive genes; 80–90% of the mitochondria show a reticular fused phenotype. After treatment with H2O2 (middle panel), cyclin repression on cyclin C–Cdk8-controlled genes is relieved by cyclin C translocation to the cytoplasm (1). There it interacts with the fission machinery (and Bax in mammalian cells) to induce stress-induced mitochondrial fission and promote cell death (2). Thereafter, cyclin C is destroyed by the UPS dependent on the Not4 E3 ligase. Following nitrogen depletion (right panel), the cyclin C-Cdk8 repression of the autophagy gene ATG8 is relieved by destroying cyclin C before it can affect mitochondrial morphology (possibly in the nucleus or at the NPC) by the UPS system. The 26S proteasome and an unknown E3 ligase(s) are required. As a result, the mitochondria remain reticular or may even become hyper-elongated to promote ATP production under starvation conditions. N = nucleus, C = cytoplasm.
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