Quantitative analysis of the ubiquitin-proteasome system under proteolytic and folding stressors

Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increasedRPN4transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.


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The ubiquitin-proteasome system (UPS) is the primary route for the disposal of defective 26 proteins in eukaryotic cells (Hershko et al. 1983(Hershko et al. , 1984Lecker, Goldberg, and Mitch 2006). 27 Aging, genetic mutations, and environmental changes all challenge the UPS and can lead to 28 accumulation of defective proteins ("proteotoxic stress"), which is a hallmark of many 29 neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's 30 disease, and amyotrophic lateral sclerosis (Labbadia and Morimoto 2015; Sweeney et al. 2017;31 Klaips, Jayaraj, and Hartl 2018). Characterizing the performance and adaptability of the UPS in 32 clearing defective proteins under proteotoxic stressors is thus likely to aid in understanding 33 numerous diseases. 34 In the UPS, ubiquitin ligases modify selected proteins with polyubiquitin chains that 35 target them for degradation by the 26S proteasome, a 2.5 MDa protein complex composed of 36 33 unique subunits (Voges, Zwickl, and Baumeister 1999 To adapt the expression of UPS components based on cellular needs, cells regulate 44 Rpn4 levels via multiple stress-sensitive mechanisms. These include proteasomal degradation 45 of Rpn4 via two encoded degradation signals (degrons) that target it to the proteasome: one 46 ubiquitin-independent signal at the N-terminus, and one signal recognized by the E3 ubiquitin 47 ligase Ubr2 (Ha, Ju, and Xie 2012; L. Wang et al. 2004). Due to these degrons, Rpn4 has a 48 short half-life of 2 minutes and will therefore quickly accumulate if the proteasome is impaired 49 (Xie and Varshavsky 2001). Additionally, RPN4 is transcriptionally regulated by several stress-50 sensitive transcription factors, including Yap1, a responder to oxidative stress; Pdr1/3, the 51 drivers of the pleiotropic drug resistance response; and Hsf1, the driver of the heat shock 52 response (HSR) ( have not been investigated in diverse proteotoxic conditions. Two ways stressors may increase 60 levels of misfolded proteins are to 1) cause proteins to misfold or obstruct their folding ("folding 61 stress"), or 2) impair degradation rates of misfolded proteins ("proteolytic stress") ( Figure 1A). A 62 naive expectation is that folding and proteolytic stressors have overlapping effects on the 63 proteome and UPS. For example, misfolded proteins generated by a folding stressor may 64 become targeted to the proteasome, increasing competition between proteasome substrates 65 and thereby lowering degradation rates for each substrate (i.e. a folding stressor leading 66 indirectly to proteolytic stress). Conversely, UPS substrates that are stabilized by a proteolytic 67 stressor may potentiate the misfolding of other proteins (i.e. a proteolytic stressor indirectly 68 leading to folding stress), as has been observed when expression of one misfolded protein 69 causes others to misfold (Satyal et al. 2000; Gidalevitz et al. 2006Gidalevitz et al. , 2009 and escapes UPS targeting. The sfGFP/mCherry ratio is therefore proportional to the stability of 114 the degron-fused protein, where a high ratio indicates high stability and a low ratio indicates low 115 stability. Cyto-Deg and ERm-Deg were both capable of reporting on UPS performance, as 116 evidenced by an increase in the sfGFP/mCherry ratio upon treatment with a 40 µM dose of the 117 proteasome inhibiting drug bortezomib ( Figure 1C). 118 To measure the PSR, we built a synthetic promoter specifically sensitive to changes in 119 the PSR. The PSR is driven by the binding of Rpn4 to a DNA motif called the proteasome-120 associated control element (PACE), which is found in the promoters of all proteasomal subunits 121 and many proteasome-associated factors (Mannhaupt et al. 1999; Shirozu, Yashiroda, and 122 Murata 2015). The PSR reporter features four tandem copies of the PACE sequence along with 123 a minimal promoter to drive expression of sfGFP ( Figure 1D). We validated the reporter's 124 sensitivity by blocking Rpn4 degradation with bortezomib and observing a monotonic increase in 125 GFP expression in response ( Figure 1D). This increase corresponded to increased levels of 126 hemagglutinin-tagged endogenous Rpn4 ( Figure 1D). 127 We demonstrating that PSR activity is tightly coupled to UPS performance ( Figure 1E). 134 135 Proteotoxic stressors elicit multiple adaptive regimes 136 To understand the adaptive potential of the UPS, we investigated the role of the PSR in clearing 137 defective proteins during proteotoxic stress. We achieved this by measuring the PSR and 138 degron stability in cells after 5 hour treatment with three proteotoxic compounds: bortezomib, 139 canavanine (an arginine analog), and azetidine-2-carboxylic acid ("AZC"; a proline analog). 140 Bortezomib directly inhibits the proteasome to cause proteolytic stress. Canavanine and AZC 141 directly disrupt protein folding when incorporated into newly synthesized proteins, causing 142 folding stress. By measuring cellular responses after 5 hours, we aimed to capture the system 143 after adaptive mechanisms had taken effect. At the highest concentrations tested, all three 144 stressors increased the stability of both Cyto-Deg and ERm-Deg and induced the PSR, 145 consistent with their proteotoxicity (Figure 2A). We were concerned that high doses of 146 canavanine and AZC would directly disrupt PSR reporter inducibility, so we limited the 147 concentrations of canavanine and AZC to a range in which the PSR reporter remained 148 comparably inducible by 5 µM bortezomib and could therefore reliably report on PSR activity 149 ( Figure 2B). 150 To determine how the UPS adapts to each stressor, we compared the relationship 151 between UPS performance and PSR activation ( Figure 2C). Perfect adaptation, a regime where 152 cells respond to a stressor without any loss of UPS performance, would be observed as 153 activation of the PSR without any change in UPS performance. Non-adaptation would manifest 154 as a lack of PSR activation with concurrent loss of UPS performance. Partial adaptation would 155 present as an intermediate between these two regimes, where the PSR activates but UPS 156 performance still declines. Finally, overadaptation would be evidenced by PSR activation 157 coupled to an increase in UPS performance. Strikingly, cells exhibited near-perfect adaptation in 158 response to low doses of bortezomib (≤2.5 µM), as the PSR was activated but stability 159 remained the same or nominally increased for both degrons (Figure 2A,2D). At higher doses 160 (>2.5 µM), the response to bortezomib resulted in partial adaptation, showing decreasing UPS 161 performance as bortezomib dose increased despite PSR activation. Responses to canavanine 162 and AZC caused a divergent response: UPS adaptation was perfect or overadaptive up to 200 163 µM and 1 mM respectively for Cyto-Deg but partial for ERm-Deg. These observations suggest 164 UPS adaptation is highly effective but becomes less so under severe stressors, as noted by an 165 increase in stability of both degrons at high doses of bortezomib and ERm-Deg at several doses 166 of canavanine and AZC. Furthermore, the divergence of adaptive response between proteolytic 167 stressors (partial adaptation regardless of degron monitored) and folding stressors (degron-168 specific adaptation regimes) suggests that these stressors cause distinct effects on cells.

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Activating the PSR improves UPS performance under stress 171 To understand the limitations of the PSR, we next investigated why adaptation was imperfect for 172 ERm-Deg under AZC and canavanine treatment and for both degrons at high doses of 173 bortezomib. One model to explain these results is that the PSR is insufficiently activated in 174 . CC-BY 4.0 International license not certified by peer review) is the author/funder. It is made available under a As expected, expressing a second copy of RPN4 in the absence of stress increased the PSR 181 and destabilized Cyto-Deg and ERm-Deg relative to an empty vector control ( Figure 1E and 3). 182 Under stress conditions, the addition of a second RPN4 copy lowered degron stability for nearly 183 all concentrations of bortezomib, canavanine, or AZC tested ( Figure 3). We conclude that 184 activating the PSR is sufficient to improve UPS performance under all stressors and that Rpn4 185 is insufficiently activated to clear specific substrates in partially adaptive regimes. 186 187

Folding stressors activate the PSR predominantly via transcription of RPN4
188 Because amino acid analogs caused a degron-specific adaptive regime while bortezomib did 189 not ( Figure 2D), we reasoned that these stressors may be sensed differently by cells. presence of canavanine and AZC (up to 6-and 7-fold activation at their highest concentrations, 200 respectively), with comparatively weak (up to 2-fold) maximal activation by bortezomib ( Figure  201 4A). Because Hsf1 targets the RPN4 promoter (Hahn, Neef, and Thiele 2006), we predicted that 202 the promoter of RPN4 (pRPN4) should be upregulated in canavanine and AZC stress. We 203 measured the activity of pRPN4 using a pRPN4:GFP plasmid reporter and found that 204 bortezomib did not activate pRPN4, while canavanine and AZC modestly increased the 205 promoter's activity (1.6-and 1.3-fold)( Figure 4B). This is consistent with a model that folding 206 stressors but not proteolytic stressors upregulate the PSR via transcriptional regulation of 207 RPN4. 208 209 210 Given the similarity in magnitude of PSR activation and pRPN4 induction in canavanine and 211 AZC (Figure 2A and 4B), we hypothesized that PSR activation by these two stressors is fully 212 accounted for by transcriptional targeting of pRPN4, with little or no contribution through 213 stabilization of Rpn4. By contrast, bortezomib does not activate pRPN4 ( Figure 4B), and 214

Folding stressors do not increase UPS substrate load
presumably induces the PSR through stabilization of Rpn4 alone. To determine the contribution 215 of Rpn4 stabilization to the PSR under canavanine and AZC treatments, we disabled 216 transcriptional regulation by engineering strains in which the genomic copy of RPN4 is under the 217 control of the YEF3 promoter (pYEF3), which is not targeted by Hsf1 but retains the same 218 . CC-BY 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 22, 2019. . https://doi.org/10.1101/780676 doi: bioRxiv preprint approximate basal expression (1.23 fold basal, s.e.=.027). In the absence of stress-sensitive 219 transcriptional regulation of RPN4, PSR induction was retained in bortezomib treatment, but 220 partially lost in canavanine and fully lost in AZC treatment ( Figure 4C). We therefore predicted 221 that loss of promoter-mediated PSR activation will impair adaptation to folding stressors and 222 minimally affect proteolytic stressors. Indeed, Cyto-Deg and ERm-Deg stability was sensitized 223 to canavanine and AZC in the pYEF3:RPN4 background, evidenced by greater degron 224 stabilization relative to wildtype ( Figure 4D). Furthermore, degron stabilization was greater 225 under AZC treatment than canavanine treatment, which correspondingly had a greater loss of 226 PSR activation in the pYEF3:RPN4 background. We conclude that protein folding stress caused 227 by canavanine and AZC does not lead to stabilization of Rpn4, and instead UPS adaptation 228 under these stressors occurs predominantly via increased RPN4 transcription. 229 Transcriptional activation of the PSR by canavanine and AZC ( Figure 4C) could be 230 specific to these treatments or it could be the general response to folding stressors. To test the 231 generality of this phenomenon, we increased the likelihood of protein misfolding by raising the 232 steady state temperature of wildtype yeast from 30°C to 37°C. This was sufficient to activate the 233 HSR, but caused no change in PSR activation ( Figure 4E). Consistent with a model in which 234 folding stress does not increase the burden of substrates on the proteasome, deletion of protein 235 chaperone genes (HSC82, SSA2, HSP104) increased the HSR but not the PSR ( Figure 4F). 236 These results suggest that proteins that misfold due to folding stressors do not create proteolytic 237 stress. 238 239 Folding stressors cause aggregation and result in failure to target 240 aggregation-prone substrates to the proteasome 241 Given that the misfolded proteins generated by folding stressors did not appear to increase 242 proteasome burden, we explored the possibility that the misfolded proteins are instead 243 sequestered into aggregates in which they are protected from proteasomal degradation. Indeed, 244 it has been previously reported that AZC can cause aggregation of endogenous proteins (Weids 245 and Grant 2014), and that aggregation can be a mechanism for avoiding degradation ( We expressed an extra copy of Hsf1 with an N-terminal truncation that renders it constitutively 252 active (Hsf1 Δ1-147 ) (Sorger 1990) in a strain expressing RPN4 from the CYC1 promoter (to 253 eliminate the confounding effect that Hsf1 activates pRPN4). Hsf1 Δ1-147 reduced Cyto-Deg and 254 ERm-Deg levels in response to canavanine and AZC but not bortezomib ( Figure 5A). These 255 results suggest that the decrease in UPS performance due to canavanine and AZC is resolvable 256 by increasing chaperone levels, supporting that they cause protein folding stress, while 257 bortezomib does not. This chaperone dependence for degradation is consistent with the 258 hypothesis that folding stressors result in sequestration of UPS substrates into aggregates. 259 To directly assess the presence of aggregates in canavanine and AZC treated cells, we 260 performed fluorescence microscopy on cells expressing Cyto-Deg or ERm-Deg. Canavanine 261 and AZC caused GFP in both Cyto-Deg and ERm-Deg to form inclusions ( Figure 5B). 262 of Hsp104 and relocalized it into foci ( Figure 5C) in cells without a degron reporter. A similar 264 response was observed with 800 µM canavanine. This change in Hsp104 localization suggests 265 that endogenous proteins (not just our synthetic reporters) are sequestered into aggregates in 266 the presence of canavanine and AZC. By contrast, even a high dose (40 µM) of bortezomib that 267 strongly increased degron levels did not alter the localization of the degrons or induce Hsp104 268 expression. These observations suggest that canavanine and AZC cause protein folding stress 269 that drives misfolded proteins into aggregates rather than targeting them to the proteasome. 270 Under this model, folding stress fails to stabilize Rpn4 because it does not produce proteasome 271 substrates that compete with Rpn4 for degradation. 272 If aggregation interferes with the degradation of misfolded proteins by the UPS, we 273 predicted that highly soluble proteasome substrates that escape aggregation during folding 274 stress would continue to be degraded normally. To test this prediction, we built a third degron 275 reporter, ATA-Deg (a "CAT tail" degron), which was demonstrated in previous work to be poly-276 ubiquitylated and targeted for proteasomal degradation, but whose six-peptide degron sequence 277 ("ATAATA") is soluble (Sitron and Brandman, 2019). Accordingly, ATA-Deg was diffuse 278 throughout the cytosol even under high doses of canavanine, AZC, or bortezomib ( Figure 6A). 279 Bortezomib treatment increased the stability of ATA-Deg, indicating sensitivity to proteolytic 280 stress. Conversely, ATA-Deg levels were constant or decreased under all concentrations of 281 canavanine and AZC treatment ( Figure 6B), suggesting that folding stress does not impair 282 degradation of ATA-Deg. Consistent with this and in contrast to Cyto-Deg and ERm-Deg ( Figure  283 5A), ATA-Deg stability was not affected by expression of Hsf1 Δ1-147 under folding stressors. We 284 conclude that folding stressors sequester aggregation-prone UPS substrates without disrupting 285 degradation of soluble substrates ( Figure 6D). 286 287

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Here we assessed the performance and adaptability of the UPS in yeast under stress conditions 289 using quantitative measurements of UPS performance and the adaptive transcriptional 290 response of the UPS (the "proteasome stress response," or PSR). We found that proteolytic and 291 protein folding stressors stabilized misfolded proteins through separate, non-overlapping 292 mechanisms, with the former blocking degradation of misfolded proteins and the latter resulting 293 in their aggregation rather than their targeting to the proteasome. Despite a difference in the 294 underlying proteostasis defect, the UPS productively responded to both proteolytic and folding 295 stressors, and in both cases, this included perfect or near-perfect adaptation (no loss in 296 degradation performance) for some substrates ( Figure 2D). 297 The perfect and near-perfect adaptation we observed for the UPS implies the existence 298 of an underlying network that can mechanistically achieve this (Ferrell 2016). In the case of 299 folding stress, RPN4 is activated transcriptionally ( Figure 4B and 4C), likely by Hsf1, to achieve 300 perfect adaptation for proteins that aggregate in the cytosol. This intervention may cause 301 increased degradation rates of soluble proteasome substrates, an intriguing consequence of a 302 system that tunes the UPS to "problem" proteins that are poor UPS substrates (aggregated 303 proteins). This substrate-specific adaptation likely occurs to some degree in all stress responses 304 that use concerted transcriptional regulation to address substrates with distinct adaptive needs. 305 near-perfect adaptation occurs and its underlying mechanisms is an important topic for future 314 study. 315 Our data suggest that protein folding stressors do not burden the proteasome with 316 increased overall substrate load ( Figure 4C, 4F, and 4G

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All experiments were performed with at least three biological replicates measured on different 382 days. For experiments testing the effects of drug stressors, stock solutions of the drugs were 383 prepared in advance (5 mM bortezomib in ethanol, 0.5 M canavanine in water, 0.5 M AZC in 384 water). Yeast were inoculated into selective SD media such that after overnight growth (>12 385 hours) in aerated culture tubes, their OD600 was between 0.05 and 0.3. Yeast were then diluted 386 to 0.05 in a 96-well plate and incubated for 30 minutes. The drug stressors were serial diluted to 387 50x concentrations, then added to cells 1:50 to reach 1x concentrations. The yeast were grown 388 for 5 hrs while shaking at 1050 rpm. Fluorescence was measured on a BD Accuri C6 flow 389 cytometer (BD Biosciences). 390 Measurements of cells treated with multiple drugs ( Figure 2C) differed in that drug 391 stressors were added at two distinct timepoints according to the schematic in Figure 2c. 392 Measurements of the knockout strains ( Figure 4G) differed in that upon overnight growth, cells 393 . CC-BY 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 22, 2019. . https://doi.org/10.1101/780676 doi: bioRxiv preprint with OD600 between 0.05 and 0.3 were immediately measured. Comparative measurements of 394 cells in 30 °C or 37 °C growth ( Figure 4F) differed in that they were grown overnight to 395 saturation at 30 °C, diluted to log phase and grown for 5 hours at 30 °C, then split for growth at 396 30 °C or 37 °C at dilutions such that they were in log phase after overnight growth, then 397 immediately measured. 398 399 Reporter quantification 400 All quantitative analysis was performed using MATLAB v8.6 (MathWorks). For the PSR, HSR, 401 and pRPN4:GFP reporters, the GFP fluorescence measurements were normalized to forward 402 scatter for each cell. For ERm-Deg, Cyto-Deg, and ATA-Deg, GFP fluorescence measurements 403 were normalized to RFP. In experiments comparing genetic backgrounds or growth 404 temperatures ( Figure 1E, 4F, and 4G), samples were normalized to a corresponding wildtype 405 control, which was set to 1. In titration experiments, samples were normalized to a 406 corresponding no-treatment control that was set to 1. For titrations in backgrounds being 407 compared to wildtype (the nonblack or broken lines in Figure 3a, 4cd, 5a), the no-treatment 408 control was set to its mean fold value relative to the no-treatment control in solid black. 409 410

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Cells expressing Cyto-Deg, ERm-Deg, ATA-Deg, or Hsp104-mKate2 were inoculated into 412 selective SD media such that after overnight growth (>12 hours) in aerated culture tubes, their 413 OD600 was between 0.1 and 0.4. Yeast were diluted to 0.1 and incubated for 30 minutes. Drug 414 stressors were then added and the cells were incubated for 5 hours. Cells were concentrated 415 through pelleting and resuspension, then immobilized on glass slides pre-treated with 416 concanavalin A. 417 Imaging was performed on an Eclipse 80i microscope (Nikon) with an X-Cite 120LED 418 light source (Excelitas Technologies) and using a 100x