The SCFMet30 ubiquitin ligase senses cellular redox state to regulate the transcription of sulfur metabolism gene The SCFMet30 ubiquitin ligase senses cellular redox state to regulate the 1 transcription of sulfur metabolism genes 2 3 Zane Johnson1, Yun Wang1, Benjamin M. Sutter1, Benjamin P. Tu1* 4 5 1 Department of Biochemistry, University of Texas Southwestern Medical Center, 6 Dallas, TX 75390-9038 7 8 *Correspondence and Lead Contact: benjamin.tu@utsouthwestern.edu 9 10 11 12 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 2 SUMMARY 13 14 In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor which 15 activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and 16 methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by 17 the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses 18 sulfur status to tune its E3 ligase activity remains unresolved. Here, using a combination of 19 genetics and biochemistry, we show that Met30 utilizes exquisitely redox-sensitive cysteine 20 residues in its WD-40 repeat region to sense the availability of sulfur metabolites in the cell. 21 Oxidation of these cysteine residues in response to sulfur starvation inhibits binding and 22 ubiquitination of Met4, leading to induction of sulfur metabolism genes. Our findings reveal how 23 SCFMet30 dynamically senses redox cues to regulate synthesis of these special amino acids, and 24 further highlight the mechanistic diversity in E3 ligase-substrate relationships. 25 26 INTRODUCTION 27 28 The biosynthesis of sulfur-containing amino acids supplies cells with increased levels of cysteine 29 and methionine, as well as their downstream metabolites glutathione and S-adenosylmethionine 30 (SAM). Glutathione serves as a redox buffer to maintain the reducing environment of the cell and 31 provide protection against oxidative stress, while SAM serves as the methyl donor for nearly all 32 methyltransferase enzymes (Ljungdahl and Daignan-Fornier, 2012, Cantoni, 1975). In the yeast 33 Saccharomyces cerevisiae, biosynthesis of all sulfur metabolites can be performed de novo via 34 enzymes encoded in the gene transcriptional network known as the MET regulon. Activation of 35 the MET gene transcriptional program under conditions of sulfur starvation relies on the 36 transcription factor Met4 and additional transcriptional co-activators that allow Met4 to be 37 recruited to the MET genes (Kuras et al., 1996, Blaiseau and Thomas, 1998). 38 39 When yeast cells sense sufficiently high levels of sulfur in the environment, the MET gene 40 transcriptional program is negatively regulated by the activity of the SCF E3 ligase Met30 41 (SCFMet30) through ubiquitination of the master transcription factor Met4 (Kaiser et al., 2000). 42 Met4 is unique as an E3 ligase substrate as it contains an internal ubiquitin interacting motif (UIM) 43 which folds in and caps the growing ubiquitin chain generated by SCFMet30, resulting in a 44 proteolytically stable but transcriptionally inactive oligo-ubiquitinated state (Flick et al., 2006). 45 Upon sulfur starvation, SCFMet30 ceases to ubiquitinate Met4, allowing Met4 to become 46 deubiquitinated and transcriptionally active. 47 48 Since its discovery, much effort has gone into understanding how Met30 senses the sulfur status 49 of the cell. Several mechanisms have been attributed to Met30 to describe how Met4 and itself 50 work together to regulate levels of MET gene transcripts in response to the availability of sulfur or 51 the presence of toxic heavy metals (Thomas et al., 1995). After the discovery that Met30 is an E3 52 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 3 ligase that negatively regulates Met4 through ubiquitin-dependent and both proteolysis-dependent 53 and independent mechanisms (Rouillon et al., 2000, Flick et al., 2004, Kuras et al., 2002), it was 54 found that Met30 dissociates from SCF complexes upon cadmium addition, resulting in the 55 disruption of the aforementioned ubiquitin-dependent regulatory mechanisms (Barbey et al., 56 2005). It was later reported that this cadmium-specific dissociation of Met30 from SCF complexes 57 is mediated by the Cdc48/p97 AAA+ ATPase complex, and that Met30 ubiquitination is required 58 for Cdc48 to strip Met30 from these complexes (Yen et al., 2012). In parallel, attempts to identify 59 the sulfur metabolic cue sensed by Met30 suggested that cysteine, or possibly some downstream 60 metabolite, was required for the degradation of Met4 by SCFMet30, although glutathione was 61 reportedly not involved in this mechanism (Hansen and Johannesen, 2000, Menant et al., 2006). 62 A genetic screen for mutants that fail to repress MET gene expression found that cho2D cells, 63 which are defective in the synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine 64 (PE), results in elevated SAM levels and deficiency in cysteine levels (Sadhu et al., 2014). 65 However, while Met30 and Met4 have been studied extensively for over two decades, the 66 biochemical mechanisms by which Met30 senses and responds to the presence or absence of sulfur 67 remains incomplete (Sadhu et al., 2014). 68 69 Herein, we utilize prototrophic yeast strains grown in sulfur-rich and sulfur-free respiratory 70 conditions to elucidate the mechanism by which Met30 senses sulfur. Using a combination of in 71 vivo and in vitro experiments, we find that instead of sensing any single sulfur-containing 72 metabolite, Met30 indirectly senses the levels of sulfur metabolites in the cell by acting as a sensor 73 of redox state. We describe a novel mechanism by which an F-box protein can be regulated through 74 the use of multiple cysteine residues as redox sensors that, upon oxidation, disrupt binding of the 75 E3 ligase to its target to enable the activation of a coordinated transcriptional response. 76 77 RESULTS 78 79 SYNTHESIS OF CYSTEINE IS MORE IMPORTANT THAN METHIONINE FOR MET4 80 UBIQUITINATION 81 82 Previous work in our lab has characterized the metabolic and cellular response of yeast cells 83 following switch from rich lactate media (YPL) to minimal lactate media (SL) (Wu and Tu, 2011, 84 Sutter et al., 2013, Laxman et al., 2013, Kato et al., 2019, Yang et al., 2019, Ye et al., 2017, Ye et 85 al., 2019). Under such respiratory conditions, yeast cells engage regulatory mechanisms that might 86 otherwise be subject to glucose repression. Among other phenotypes, this switch results in the 87 acute depletion of sulfur metabolites and the activation of the MET gene regulon (Sutter et al., 88 2013, Ye et al., 2019). To better study the response of yeast cells to sulfur starvation, we 89 reformulated our minimal lactate media to contain no sulfate, as prototrophic yeast can assimilate 90 sulfur in the form of inorganic sulfate into reduced sulfur metabolites. After switching cells from 91 YP lactate media (Rich) to the new minimal sulfur-free lactate media (−Sulfur), we found that 92 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 4 Met30 and Met4 quickly respond to sulfur starvation through the extensively studied ubiquitin-93 dependent mechanisms regulating Met4 activity (Figure 1A) (Yen et al., 2005, Flick et al., 2006, 94 Barbey et al., 2005, Kaiser et al., 2000, Flick et al., 2004). As previously observed, the 95 deubiquitination of Met4 resulted in the activation of the MET genes (Figure 1B) and corresponded 96 well with changes in observed sulfur metabolite levels (Figure 1C). Addition of sulfur metabolites 97 quickly rescued Met30 activity and resulted in the re-ubiquitination of Met4 and the repression of 98 the MET genes. 99 100 As previously noted, Met4 activation in response to sulfur starvation results in the emergence of a 101 second, faster-migrating proteoform of Met30, which disappears after rescuing yeast cells with 102 sulfur metabolites (Sadhu et al., 2014). We found that the appearance of this proteoform is 103 dependent on both MET4 and new translation, as it was not observed in either met4D cells or cells 104 treated with cycloheximide during sulfur starvation (Figure S1A and C). Additionally, this 105 proteoform persists after rescue with a sulfur source in the presence of a proteasome inhibitor 106 (Figure S1B). 107 108 We hypothesized that this faster-migrating proteoform of Met30 might be the result of translation 109 initiation at an internal methionine residue. In support of this possibility, mutation of methionine 110 residues 30, 35, and 36 to alanine blocked the appearance of a lower form during sulfur starvation 111 (Figure S1D). Conversely, deletion of the first 20 amino acids containing the first three methionine 112 residues of Met30 resulted in expression of a Met30 proteoform that migrated at the apparent 113 molecular weight of the wild type short form and did not generate a new, even-faster migrating 114 proteoform under sulfur starvation (Figure S1D). Moreover, the Met30M30/35/36A and Met30D1-20 115 strains expressing either solely the long or short form of the Met30 protein had no obvious 116 phenotype with respect to Met4 ubiquitination or growth in high or low sulfur media (Figure S1E). 117 We conclude that the faster-migrating proteoform of Met30 that is produced during sulfur 118 starvation has no discernible effect on sulfur metabolic regulation under these conditions. 119 120 The sulfur amino acid biosynthetic pathway is bifurcated into two branches at the central 121 metabolite homocysteine, where this precursor metabolite commits either to the production of 122 cysteine or methionine (Figure 1E). After confirming Met30 and Met4 were responding to sulfur 123 starvation as expected, we sought to determine whether the cysteine or methionine branch of the 124 sulfur metabolic pathway was sufficient to rescue Met30 E3 ligase activity and re-ubiquitinate 125 Met4 after sulfur starvation. To determine whether the synthesis of methionine is necessary to 126 rescue Met30 activity, cells lacking methionine synthase (met6D) were fed either homocysteine or 127 methionine after switching to sulfur-free lactate (−Sulfur) media. Interestingly, cells fed 128 homocysteine were still able to ubiquitinate and degrade Met4, while methionine-fed cells 129 appeared to oligo-ubiquitinate and stabilize Met4 (Figure 1D). These observations are consistent 130 with previous reports and suggest Met30 and Met4 interpret sulfur sufficiency through both 131 branches of sulfur metabolism to a degree (Hansen and Johannesen, 2000, Kaiser et al., 2000, 132 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 5 Kuras et al., 2002, Flick et al., 2004, Menant et al., 2006, Sadhu et al., 2014), with the stability of 133 Met4, but not the E3 ligase activity of Met30, apparently dependent on the methionine branch. 134 135 To determine whether Met30 specifically responds to cysteine, cells lacking cystathionine beta-136 lyase (str3D), the enzyme responsible for the conversion of cystathionine to homocysteine, were 137 starved of sulfur and fed either cysteine or methionine. This mutant is incapable of synthesizing 138 methionine from cysteine via the two-step conversion of cysteine into the common precursor 139 metabolite homocysteine. Our results show cysteine was able to rescue Met30 activity even in a 140 str3D mutant, further suggesting cysteine or a downstream metabolite, and not methionine, as the 141 signal of sulfur sufficiency for Met30 (Figure 1D). 142 143 CYSTEINE RESIDUES IN MET30 ARE OXIDIZED DURING SULFUR STARVATION 144 145 The synthesis of cysteine from homocysteine contributes to the production of the downstream 146 tripeptide metabolite glutathione (GSH), which exists at millimolar concentrations in cells and is 147 the major cellular reductant for buffering against oxidative stress (Cuozzo and Kaiser, 1999, Wu 148 et al., 2004). Specifically, glutathione serves to neutralize reactive oxygen species such as 149 peroxides and free radicals, detoxify heavy metals, and preserve the reduced state of protein thiols 150 (Pompella et al., 2003, Penninckx, 2000). Considering the relatively high number of cysteine 151 residues in Met30 (Figure 2A), we sought to determine if these residues might become oxidized 152 during acute sulfur starvation. Utilizing the thiol-modifying agent methoxy-PEG-maleimide 153 (mPEG2K-mal), which adds ~2 kDa per reduced cysteine residue, we assessed Met30 cysteine 154 oxidation in vivo by Western blot. Theoretically, full modification of the 23 cysteines in Met30 by 155 mPEG2K-mal should significantly shift the apparent molecular weight of Met30 by ~45-50 kDa. 156 As expected, Met30 in sulfur-replete rich media migrates at ~140 kDa (Figure 2B, first lane), 157 nicely corresponding to the modification of most if not all of its 23 cysteine residues, suggesting 158 they are all in the reduced state while sulfur levels are high and Met4 is being negatively regulated. 159 However, after shifting into sulfur-free minimal lactate media, Met30 migrates at ~80 kDa — 160 suggesting the majority of its cysteine residues are rapidly becoming oxidized in vivo following 161 acute sulfur starvation (Figure 2B, second and third lane). In contrast, the loading control Rpn10 162 contains a single cysteine residue, and did not exhibit significant oxidation within the same time 163 period of sulfur starvation. As expected, repletion of sulfur metabolites led to the reduction and 164 modification of Met30’s cysteine residues by mPEG2K-mal to the extent seen in the rich media 165 condition. Such oxidation and re-reduction of Met30 cysteines corresponds well with Met4 166 ubiquitination status (Figure 2B). Additionally, when cells were grown in sulfur-free media 167 containing glucose (SFD) as the carbon source, Met30 also becomes oxidized, although on a 168 slower timescale — suggesting this mechanism is not specific to yeast grown under non-169 fermentable conditions (Figure 2C). 170 171 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 6 Considering the link between sulfur starvation and oxidative stress, we next assessed whether 172 simply changing the redox state of sulfur-starved cells could mimic sulfur repletion with respect 173 to Met30 E3 ligase activity. Addition of the potent, membrane-permeable reducing agent DTT to 174 yeast cells starved of sulfur readily reversed Met30 cysteine oxidation. DTT also resulted in the 175 partial re-ubiquitination of Met4, suggesting that Met30 cysteine redox status influences its 176 ubiquitination activity against Met4 (Figure 2D). Taken together, these data strongly suggest 177 cysteine residues within Met30 are poised to become rapidly oxidized in response to sulfur 178 starvation, which is correlated with the deubiquitination of its substrate Met4. 179 180 MET30 CYSTEINE POINT MUTANTS EXHIBIT DYSREGULATED SULFUR SENSING 181 IN VIVO 182 183 After establishing Met30 cysteine redox status as an important factor in sensing sulfur starvation, 184 we sought to determine whether specific residues played key roles in the sensing mechanism. 185 Through site-directed mutagenesis of Met30 cysteines individually and in clusters (Figure S2A 186 and B), we observed that mutation of cysteines in the WD-40 repeat regions of Met30 with the 187 highest concentration of cysteine residues (WD-40 repeat regions 4 and 8) resulted in dysregulated 188 Met4 ubiquitination status (Figure 3A) and MET gene expression (Figure 3B). Specifically, 189 conservatively mutating these cysteines to serine residues mimics the reduced state of the Met30 190 protein, resulting in constitutive ubiquitination of Met4 by Met30 even when cells are starved of 191 sulfur. The mixed population of ubiquitinated and deubiquitinated Met4 in the mutant strains 192 resulted in reduced induction of SAM1 and GSH1, while MET17 appears to be upregulated in the 193 mutants but is largely insensitive to the changes in the sulfur status of the cell. Interestingly, a 194 single cysteine to serine mutant, C414S, phenocopies the grouped cysteine to serine mutants 195 C414/426/436/439S (data not shown) and C614/616/622/630S. These mutants also exhibit slight 196 growth phenotypes when cultured in both rich and −sulfur lactate media supplemented with 197 homocysteine (Figure 3C). Furthermore, these point mutants only effect Met4 ubiquitination in 198 the context of sulfur starvation, as strains expressing these mutants exhibited a normal response to 199 cadmium as evidenced by rapid deubiquitination of Met4 (Figure S2C). 200 201 MET30 CYSTEINE OXIDATION DISRUPTS UBIQUITINATION AND BINDING OF 202 MET4 IN VITRO 203 204 Having observed that Met30 cysteine redox status is correlated with Met4 ubiquitination status in 205 vivo, we next sought to determine whether the sulfur/redox-sensing ability of SCFMet30 E3 ligase 206 activity could be reconstituted in vitro. To this end, we performed large scale immuno-purifications 207 of SCFMet30-Flag to pull down Met30 and its interacting partners in both high and low sulfur 208 conditions for in vitro ubiquitination assays with recombinantly purified E1, E2, and Met4 (Figure 209 4A). Initial in vitro ubiquitination experiments showed little difference in activity between the two 210 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 7 conditions, mirroring prior efforts to demonstrate differential activity of the Met30 E3 ligase in 211 response to stimuli that effect its activity in vivo (Figure S3A) (Barbey et al., 2005). 212 213 Since the cysteine residues within Met30 became rapidly oxidized in sulfur-free conditions, the 214 addition of DTT as a standard component in our IP buffer and in in vitro ubiquitination reactions 215 could potentially reduce oxidized Met30 cysteines and alter its ubiquitination activity towards 216 Met4. To test this possibility, we next performed the Met30 IP and in vitro assay in the complete 217 absence of reducing agent. Strikingly, we observed little to no ubiquitination activity in these 218 conditions (Fig. S3B), suggesting that oxidized Met30 exhibits significantly reduced 219 ubiquitination activity. 220 221 To more rigorously test the effect of reducing agents on the activity of immunopurified SCFMet30, 222 we performed in parallel the Met30-Flag IP with cells grown in both high and low sulfur 223 conditions, with and without reducing agent in the IP. Silver stains of the eluted co-IP Met30 224 complexes showed similar levels of total protein overall and little difference in the abundance of 225 major binding partners between the four conditions (Figure S3C). Western blots of the co-IP 226 samples for the Cdc53/cullin scaffold showed similar binding between the samples with the 227 exception of the −sulfur, −DTT sample which had approximately a third of the amount of Cdc53 228 bound to Met30 (Figure S3D). We suspect this difference is due to the canonical regulation of SCF 229 E3 ligases, which uses cyclic changes in the affinity of Skp1/F-box protein heterodimers to the 230 cullin scaffold based on binding between the F-box protein and its substrate (Reitsma et al., 2017). 231 After performing the initial IP and washing the beads in buffer with and without reducing agent, 232 the final wash step and Flag peptide elution were done without reducing agent in the buffer for all 233 four IP conditions in order to remove any residual reducing agent from the final ubiquitination 234 reaction, which was also performed without reducing agent. A small aliquot of the rich and −sulfur 235 “−DTT” immunopurified SCFMet30 was transferred to a new tube and treated with 5 mM TCEP, a 236 non-thiol, phosphine-based reducing agent, for approximately 30 min while the in vitro 237 ubiquitination assays were set up to test if the low activity of the oxidized SCFMet30 complex could 238 be rescued by treating with another reducing agent before addition to the final reaction. The data 239 clearly demonstrate that the presence of reducing agent in the IP and wash buffer, but not in the 240 elution or final reaction, significantly increased the E3 ligase activity of SCFMet30 in vitro regardless 241 of whether the cells were grown in high (Figure 4C) or low sulfur media (Figure 4D). Further 242 supporting our hypothesis, brief treatment of the oxidized −DTT IP complex with TCEP 243 (−DTT/+TCEP) rescued the activity of the E3 complex in vitro (Figures 4B and C). The same +/ 244 − DTT in vitro ubiquitination experiment done with the C414S and C614/616/622/630S Met30 245 mutants showed lower E3 ligase activity overall relative to wild type Met30, but smaller 246 differences between the plus and minus reducing agent condition (Figure S4A). 247 248 As SCFMet30 E3 ligase activity in vitro is independent of the sulfur-replete or -starved state of the 249 cells from which the co-IP concentrate is produced, and that the activity of the SCFMet30 co-IP 250 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 8 concentrate purified in the absence of reducing agent can be rescued by treatment with another 251 reducing agent, we hypothesized that the low E3 ligase activity of SCFMet30 purified in the absence 252 of reducing agent is due to decreased binding between Met30 and Met4, and not decreased binding 253 between Met30 and the other core SCF components. To test this possibility, lysate for “rich” and 254 “−sulfur” cells was prepared and each was split into three groups, with either reducing agent 255 (+DTT), the thiol-specific oxidizing agent tetramethylazodicarboxamide (+Diamide), or control 256 (−DTT) (Figure 4A). Met30-Flag IPs were performed as previously described for the in vitro 257 ubiquitination assay, except instead of eluting Met30 off of the beads, the +DTT, −DTT, and 258 +Diamide beads were each split into two tubes containing IP buffer ±DTT and bacterially purified 259 Met4. The beads were incubated with purified Met4 prior to washing with IP buffer with or without 260 DTT. We observed a clear, DTT-dependent increase in the fraction of Met4 bound to the Met30-261 Flag beads, with the “+DTT” Met30 IP showing a larger initial amount of bound Met4 compared 262 to the “−DTT” Met30 IP, with even less Met4 bound to the “+Diamide” Met30-Flag beads. 263 Consistent with our hypothesis, the addition of DTT to the Met4 co-IP with “−DTT” or 264 “+Diamide” Met30-Flag beads restored the Met30/Met4 interaction to the degree seen in the 265 “+DTT” Met30-Flag beads. We then performed the same experiment with our Met30 cysteine 266 point mutants. The amount of Met4 bound to these mutants was less sensitive to the presence or 267 absence of reducing agent (Figure S4B). Collectively, these data suggest that the reduced form of 268 key cysteine residues in Met30 enables it to engage its Met4 substrate and facilitate ubiquitination. 269 270 DISCUSSION 271 272 The unique redox chemistry offered by sulfur and sulfur-containing metabolites renders many of 273 the biochemical reactions required for life possible. The ability to carefully regulate the levels of 274 these sulfur-containing metabolites is of critical importance to cells as evidenced by an exquisite 275 sulfur-sparing response. Sulfur starvation induces the transcription of MET genes and specific 276 isozymes, which themselves contain few methionine and cysteine residues (Fauchon et al., 2002). 277 Furthermore, along with the dedicated cell cycle F-box protein Cdc4, Met30 is the only other 278 essential F-box protein in yeast, linking sulfur metabolite levels to cell cycle progression (Su et 279 al., 2005, Su et al., 2008). Our findings highlight the intimate relationship between sulfur 280 metabolism and redox chemistry in cellular biology, revealing that the key sensor of sulfur 281 metabolite levels in yeast, Met30, is regulated by reversible cysteine oxidation. Such oxidation of 282 Met30 cysteines in turn influences the ubiquitination status and transcriptional activity of the 283 master sulfur metabolism transcription factor Met4. While much work has been done to 284 characterize the molecular basis of sulfur metabolic regulation in yeast between Met30 and Met4, 285 this work describes the biochemical basis for sulfur sensing by the Met30 E3 ligase (Figure 5). 286 287 The ability of Met30 to act as a cysteine redox-responsive E3 ligase is unique in Saccharomyces 288 cerevisiae, but is reminiscent of the redox-responsive Keap1 E3 ligase in humans. In humans, 289 Keap1 ubiquitinates and degrades its Nrf2 substrate to regulate the cellular response to oxidative 290 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 9 stress. When cells are exposed to electrophilic metabolites or oxidative stress, key cysteine 291 residues are either alkylated or oxidized into disulfides, resulting in conformational changes that, 292 in turn, either disrupt Keap1 association with Cul3 or Nrf2, both leading to Nrf2 activation 293 (Yamamoto et al., 2018). Our data suggest that in response to sulfur starvation, Met30 can still 294 maintain its association with the SCF E3 ligase cullin scaffold, but that treatment of the oxidized 295 complex with reducing agent is sufficient to stimulate ubiquitination of Met4 in vitro. This, along 296 with the in vivo and in vitro Met30 cysteine point mutant data, leads us to conclude that it is the 297 ability of Met30 to bind its substrate Met4 that is being disrupted by cysteine oxidation. 298 299 Previous work on the yeast response to cadmium toxicity demonstrated that Met30 is stripped from 300 SCF complexes by the p97/Cdc48 segregase upon treatment with cadmium, suggesting that like 301 Keap1, Met30 can utilize both dissociation from SCF complexes and disrupted interaction with 302 Met4 to modulate Met4 transcriptional activation (Barbey et al., 2005, Yen et al., 2012). Recent 303 work on the sensing of oxidative stress by Keap1 has found that multiple cysteines in Keap1 can 304 act cooperatively to form disulfides, and that the use of multiples cysteines to form different 305 disulfide bridges creates an “elaborate fail-safe mechanism” to sense oxidative stress (Suzuki et 306 al., 2019). In light of our findings, we suspect Met30 might similarly use multiple cysteine residues 307 in a cooperative disulfide formation mechanism to disrupt the binding interface between Met30 308 and Met4, but more work will be needed to demonstrate this definitively. It is worth noting the 309 curious spacing and clustering of cysteine residues in Met30, with the highest density and closest 310 spacing of cysteines found in two WD-40 repeats that are expected to be directly across from each 311 other in the 3D structure (Figure 2A). That the mutation of these cysteine clusters to serine have 312 the largest in vivo effect, but mutation of any one cysteine to serine (with the notable exception of 313 Cys414) has no effect, implies some built-in redundancy in the cysteine-based redox-sensing 314 mechanism (Figure S2B). We speculate that the oxidation of the cysteines in the WD-40 repeat 315 region of Met30 work cooperatively to produce structural changes that position Cys414 to make a 316 key disulfide linkage that disrupts the interaction with Met4. 317 318 It was previously hypothesized that an observed, faster-migrating proteoform of Met30 might be 319 involved in the regulation of sulfur metabolism (Sadhu et al., 2014). We deduced that the lower 320 form of Met30 does appear to be the result of transcriptionally-guided, alternative translational 321 initiation. However, this faster-migrating proteoform appears dispensable for sulfur metabolic 322 regulation under the conditions we examined. It is curious that such an ostensibly obvious feedback 323 loop between Met30 and Met4 would appear to have little to no effect on sulfur metabolic 324 regulation. However, during sulfur starvation, a decrease in global translation coincides with an 325 increase in ribosomes containing one, instead of two, methyl groups at universally conserved, 326 tandem adenosines near the 3’end of 18S rRNA (Liu et al.) We speculate that these ribosomes 327 might preferentially translate MET gene mRNAs, as well as preferentially initiate translation at the 328 internal 30, 35, and 36th methionine residues of Met30. 329 330 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 10 The utilization of a redox mechanism for Met30 draws interesting comparisons to the regulation 331 of Met4 via ubiquitination in that both mechanisms are rapid and readily reversible, require no 332 new RNA or protein synthesis, and there is no requirement for the consumption of sulfur 333 equivalents so as to spare them for use in MET gene translation under conditions of sulfur scarcity. 334 It is also striking that while Met30 contains many cysteine residues, Met4 contains none – which 335 has the consequence that as Met30 cysteines are oxidized, there is no possibility that Met4 can 336 make an intermolecular disulfide linkage that might interfere with its release and recruitment to 337 the promoters of MET genes. Upon repletion of sulfur metabolites, cellular reducing capacity is 338 restored, and Met30 cysteine reduction couples the regulation of MET gene activation to sulfur 339 assimilation, both of which require significant reducing equivalents. 340 341 Lastly, we highlight the observation that nearly all of the Met30 protein becomes rapidly oxidized 342 within 15 min of sulfur starvation, in contrast to other nucleocytosolic proteins (Fig. 2B). Bulk 343 levels of oxidized versus reduced glutathione are also minimally changed within this timeframe. 344 These considerations suggest that Met30 is either located in a redox-responsive microenvironment 345 within cells, or that key cysteine residues such as Cys414 are predisposed to becoming oxidized 346 to subsequently inhibit binding and ubiquitination of Met4. Future structural characterization of 347 SCFMet30 in its reduced and oxidized states may reveal the underlying basis of its exquisite 348 sensitivity to, and regulation by, oxidation. Nonetheless, along with SoxR and OxyR transcription 349 factors in E. coli (Imlay, 2013) the Yap1 transcription factor in yeast (Herrero et al., 2008), and 350 Keap1 in mammalian cells, our studies add the F-box protein Met30 to the exclusive list of bona 351 fide cellular redox sensors that can initiate a transcriptional response. 352 353 ACKNOWLEDGMENTS 354 355 We thank members of the Tu lab, Deepak Nijhawan, Hongtao Yu, and George DeMartino for 356 helpful discussions. This work was supported by NIH R01GM094314, R35GM136370, and an 357 HHMI-Simons Faculty Scholars Award to B.P.T. 358 359 AUTHOR CONTRIBUTIONS 360 361 This study was conceived by Z.J. and B.P.T. B.M.S. performed Met30 cysteine point mutant strain 362 construction, Y.W. performed cysteine point mutant cloning and Cdc34 protein purification, and 363 all remaining experiments were directed and performed by Z.J. The paper was written by Z.J. and 364 B.P.T. and has been approved by all authors. 365 366 DECLARATION OF INTERESTS 367 368 The authors declare no competing interests. 369 370 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 11 EXPERIMENTAL PROCEDURES 371 372 Yeast strains, construction, and growth media 373 The prototrophic CEN.PK strain background (van Dijken et al., 2000) was used in all experiments. 374 Strains used in this study are listed in Table S1. Gene deletions were carried out using either tetrad 375 dissection or standard PCR-based strategies to amplify resistance cassettes with appropriate 376 flanking sequences, and replacing the target gene by homologous recombination (Longtine et al., 377 1998). C-terminal epitope tagged strains were similarly made with the PCR-based method to 378 amplify resistance cassettes with flanking sequences. Point mutations were made by cloning the 379 gene into the tagging plasmids, making the specific point mutation(s) by PCR, and amplifying and 380 transforming the entire gene locus and resistance markers with appropriate flanking sequences 381 using the lithium acetate method. 382 383 Media used in this study: YPL (1% yeast extract, 2% peptone and 2% lactate); sulfur-free glucose 384 and lactate media (SFD/L) media composition is detailed in Table S2, with glucose or lactate 385 diluted to 2% each; YPD (1% yeast extract, 2% peptone and 2% glucose). 386 387 Whole cell lysate Western blot preparation 388 Five OD600 units of yeast culture were quenched in 15% TCA for 15 min, pelleted, washed with 389 100% EtOH, and stored at −20°C. Cell pellets were resuspended in 325 µL EtOH containing 1 390 mM PMSF and lysed by bead beating. The lysate was separated from beads by inverting the 391 screwcap tubes, puncturing the bottom with a 23G needle, and spinning the lysate at 2,500xg into 392 an Eppendorf for 1 min. Beads were washed with 200 µL of EtOH and spun again before 393 discarding the bead-containing screwcap tube and pelleting protein extract at 21,000xg for 10 min 394 in the new Eppendorf tube. The EtOH was aspirated and EtOH precipitated protein pellets were 395 resuspended in 150 µL of sample buffer (200 mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.2 mg/ml 396 bromophenol blue), heated at 42°C for 45 min, and debris was pelleted at 16,000xg for 3 min. DTT 397 was added to a final concentration of 25 mM and incubated at RT for 30 min before equivalent 398 amounts of protein were loaded onto NuPAGE 4-12% bis-tris or 3-8% tris-acetate gels. For protein 399 samples modified with mPEG2K-mal, an aliquot of the sample buffer resuspended protein pellets 400 was moved to a fresh Eppendorf and sample buffer containing 15 mM mPEG2K-mal was added 401 for a final concentration of 5 mM mPEG2K-mal before heating at 42°C for 45 min, pelleting 402 debris, and adding DTT. 403 404 Western blots 405 Western blots were carried out by transferring whole cell lysate extracts or in vitro ubiquitination 406 or binding assay samples onto 0.45 micron nitrocellulose membranes and wet transfers were 407 carried out at 300 mA constant for 90 min at 4°C. Membranes were incubated with ponceau S, 408 washed with TBST, blocked with 5% milk in TBST for 1 h, and incubated with 1:5000 Mouse 409 anti-FLAG M2 antibody (Sigma, Cat#F3165), 1:5000 Mouse anti-HA(12CA5) (Roche, 410 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 12 Ref#11583816001), 1:50,000 Rabbit anti-RPN10 (Abcam, ab98843), or 1:3000 Goat anti-Cdc53 411 (Santa Cruz, yC-17) in 5% milk in TBST overnight at 4°C. After discarding primary antibody, 412 membranes were washed 3 times for 5 min each before incubation with appropriate HRP-413 conjugated secondary antibody for 1 h in 5% milk/TBST. Membranes were then washed 3 times 414 for 5 min each before incubating with Pierce ECL western blotting substrate and exposing to film. 415 416 RNA Extraction and Real Time Quantitative PCR (RT-qPCR) Analysis 417 RNA isolation of five OD600 units of cells under different growth conditions was carried out 418 following the manufacture manual using MasterPure yeast RNA purification kit (epicentre). RNA 419 concentration was determined by absorption spectrometer. 5 μg RNA was reverse transcribed to 420 cDNA using Superscript III Reverse Transcriptase from Invitrogen. cDNA was diluted 1:100 and 421 real-time PCR was performed in triplicate with iQ SYBR Green Supermix from BioRad. 422 Transcripts levels of genes were normalized to ACT1. All the primers used in RT-qPCR have 423 efficiency close to 100%, and their sequences are listed below. 424 425 ACT1_RT_F TCCGGTGATGGTGTTACTCA 426 ACT1_RT_R GGCCAAATCGATTCTCAAAA 427 MET17_RT_F CGGTTTCGGTGGTGTCTTAT 428 MET17_RT_R CAACAACTTGAGCACCAGAAAG 429 GSH1_RT_F CACCGATGTGGAAACTGAAGA 430 GSH1_RT_R GGCATAGGATTGGCGTAACA 431 SAM1_RT_F CAGAGGGTTTGCCTTTGACTA 432 SAM1_RT_R CTGGTCTCAACCACGCTAAA 433 434 Metabolite extraction and quantitation 435 Intracellular metabolites were extracted from yeast using a previous established method (Tu et al., 436 2007). Briefly, at each time point, ~12.5 OD600 units of cells were rapidly quenched to stop 437 metabolism by addition into 37.5 mL quenching buffer containing 60% methanol and 10 mM 438 Tricine, pH 7.4. After holding at -40°C for at least 3 min, cells were spun at 5,000xg for 2 min at 439 0°C, washed with 1 mL of the same buffer, and then resuspended in 1 mL extraction buffer 440 containing 75% ethanol and 0.1% formic acid. Intracellular metabolites were extracted by 441 incubating at 75°C for 3 min, followed by incubation at 4°C for 5 min. Samples were spun at 442 20,000xg for 1 min to pellet cell debris, and 0.9 mL of the supernatant was transferred to a new 443 tube. After a second spin at 20,000xg for 10 min, 0.8 mL of the supernatant was transferred to a 444 new tube. Metabolites in the extraction buffer were dried using SpeedVac and stored at −80°C 445 until analysis. Methionine, SAM, SAH, cysteine, GSH and other cellular metabolites were 446 quantitated by LC-MS/MS with a triple quadrupole mass spectrometer (3200 QTRAP, AB SCIEX) 447 using previously established methods (Tu et al., 2007). Briefly, metabolites were separated 448 chromatographically on a C18-based column with polar embedded groups (Synergi Fusion-RP, 449 150 3 2.00 mm 4 micron, Phenomenex), using a Shimadzu Prominence LC20/SIL-20AC HPLC-450 autosampler coupled to the mass spectrometer. Flow rate was 0.5 ml/min using the following 451 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 13 method: Buffer A: 99.9% H2O/0.1% formic acid, Buffer B: 99.9% methanol /0.1% formic acid. T 452 = 0 min, 0% B; T = 4 min, 0% B; T = 11 min, 50% B; T = 13 min, 100% B; T = 15 min, 100% B, 453 T = 16 min, 0% B; T = 20 min, stop. For each metabolite, a 1 mM standard solution was infused 454 into a Applied Biosystems 3200 QTRAP triple quadrupole-linear ion trap mass spectrometer for 455 quantitative optimization detection of daughter ions upon collision-induced fragmentation of the 456 parent ion [multiple reaction monitoring (MRM)]. The parent ion mass was scanned for first in 457 positive mode (usually MW + 1). For each metabolite, the optimized parameters for quantitation 458 of the two most abundant daughter ions (i.e., two MRMs per metabolite) were selected for 459 inclusion in further method development. For running samples, dried extracts (typically 12.5 OD 460 units) were resuspended in 150 mL 0.1% formic acid, spun at 21,000xg for 5 min at 4°C, and 125 461 µL was moved to a fresh Eppendorf. The 125 µL was spun again at 21,000xg for 5 min at 4°C, 462 and 100 µL was moved to mass-spec vials for injection (typically 50 µL injection volume). The 463 retention time for each MRM peak was compared to an appropriate standard. The area under each 464 peak was then quantitated by using Analyst® 1.6.3, and were re-inspected for accuracy. 465 Normalization was done by normalizing total spectral counts of a given metabolite by OD600 units 466 of the sample. Data represents the average of two biological replicates. 467 468 Protein purification 469 6xHis-Uba1 (E1) was purified as previously described (Petroski and Deshaies, 2005), with the 470 exception that the strain was made in the cen.pk background and the His6-tag was appended to the 471 N-terminus of Uba1. Additionally, lysis was performed by cryomilling frozen yeast pellets by 472 adding the pellet to a pre-cooled 50 ml milling jar containing a 20 mm stainless steel ball. Yeast 473 cell lysis was performed by milling in 3 cycles at 25 Hrz for 3 min and chilling in liquid nitrogen 474 for 1 min. Lysate was made by adding 4 ml of buffer for every gram of cryomilled yeast powder, 475 and clarification was performed at 35,000xg instead of 50,000xg. 476 477 Cdc34-6xHis (E2) similarly was purified according to previously described protocols (Petroski 478 and Deshaies, 2005), with the following exceptions; the CDC34 ORF was cloned into pHIS 479 parallel vector such that the N-terminal His tag was eliminated from the vector while incorporating 480 a C-terminal 6xHis tag by PCR. BL21 transformants were grown in LB medium and expression 481 was induced by addition of 0.1 mM IPTG. Cells were lysed by sonication and clarification was 482 done by spinning at 35,000xg for 20 min at 4°C before the Ni-NTA purification was performed as 483 previously described (Petroski and Deshaies, 2005). 484 485 His-SUMO-Met4-Strep-tagII-HA was purified by cloning the MET4 ORF into pET His6 Sumo 486 vector while incorporating a C-terminal Strep-tagII and a single HA tag by PCR. BL21 487 transformants were grown in 2 liters LB medium and induced by addition of 0.1 mM IPTG O/N 488 at 16°C at 200 rpm. Cell pellets were collected and lysed by sonication in buffer containing 50 489 mM Tris pH 7.5, 300 mM NaCl, 10% glycerol, 20 mM imidazole, 1 mM PMSF, 10 µM leupeptin, 490 50 mM NaF, 5 µM pepstatin, 0.5% NP-40, and 2x roche EDTA-free protease inhibitor cocktail 491 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 14 tablet. Lysate was clarified by centrifugation at 35,000xg for 20 min at 4°C and the supernatant 492 was transferred to a 50 ml conical and Met4 was batch purified with 1.5 ml of Ni-NTA agarose by 493 incubating for 30 min at 4°C. After spinning down the Ni-NTA agarose, the supernatant was 494 removed and the agarose was resuspended in the same buffer and moved to a gravity flow column 495 and washed 3 times with 50 mM Tris pH 7.5, 300 mM NaCl, 10% glycerol, and 20 mM imidazole 496 before elution with the same buffer containing 200 mM imidazole. Eluted Met4 was then run over 497 2 ml of Strep-Tactin Sepharose in a 10 ml gravity flow column, washed with 5 CVs Strep-Tactin 498 wash buffer (100 mM Tris pH 8.0, 150 mM NaCl), and eluted by diluting 1 ml 10X Strep-Tactin 499 Elution buffer in 9 ml Strep-Tactin wash buffer and collecting 1.5 ml fractions. Fractions 500 containing pure, full-length Met4 were pooled and concentrated while exchanging the buffer with 501 buffer containing 30 mM Tris pH 7.6, 100 mM NaCl, 5 mM MgCl2, 15% glycerol, and 2 mM 502 DTT. Protein concentration was measured and 1 mg/ml aliquots were made and stored at −80°C. 503 504 SCFMet30-Flag IP and in vitro ubiquitination assay 505 Strains containing Flag-tagged Met30 were grown in rich YPL media overnight to mid-late log 506 phase before dilution with more YPL and grown for 3 h before half of the culture was separated 507 and switched −sulfur SFL media for 15 min. Subsequently, approximately 3000 OD600 units each 508 of YPL and SFL cultured yeast were spun down and frozen in liquid nitrogen. Frozen yeast pellets 509 were cryomilled by adding the pellet to a pre-cooled 50 ml milling jar containing a 20 mm stainless 510 steel ball. Yeast cell lysis was performed by milling in 3 cycles at 25 Hrz for 3 min and chilling in 511 liquid nitrogen for 1 min. Cryomilled yeast powder (~ 4 grams) was moved to a 50 ml conical and 512 resuspended in 16 ml SCF IP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM NaF, 1% NP-40, 513 1 mM EDTA, 5% glycerol) containing 10 µM leupeptin, 1 mM PMSF, 5 µM pepstatin, 100 µM 514 sodium orthovanadate, 2 mM 1, 10-phenanthroline, 1 µM MLN4924, 1X Roche EDTA-free 515 protease inhibitor cocktail tablet, and 1 mM DTT when specified. Small molecule inhibitors of 516 neddylation and deneddylation were included, and along with a short IP time, intended to minimize 517 exchange and preserve F-box protein/Skp1 substrate recognition modules (Reitsma et al., 2017). 518 The lysate was then briefly sonicated to sheer DNA and subsequently clarified at 35,000xg for 20 519 min and the supernatant was incubated with with 50 µL of Thermo Fisher protein G dynabeads 520 (Cat# 10004D) DMP crosslinked to 25 µL of Mouse anti-FLAG M2 antibody (Sigma, Cat#F3165) 521 for 30 min at 4°C. The agarose was pelleted at 500xg for 5 min, the supernatant was aspirated, and 522 the magnetic beads transferred to an Eppendorf tube. The beads were washed 5 times with 1 ml 523 SCF IP buffer with or without DTT before elution with 1 mg/ml Flag peptide in PBS. The eluent 524 was concentrated in Amicon Ultra-0.5 centrifugal filter units with 10 kDa MW cutoffs to a final 525 volume of ~ 40 µL. Silver stains of the IPs were carried out using the Pierce Silver Stain for Mass 526 Spectrometry kit (Cat#24600) according to the manufacturers protocol. The in vitro ubiquitination 527 assay was performed by placing a PCR tube on ice and adding to it 29 µL of water, 8 µL of 5X 528 ubiquitination assay buffer (250 mM Tris pH 7.5, 5 mM ATP, 25 mM MgCl2, 25% glycerol), 1.2 529 µL Uba1 (FC = 220 nM), 1.2 µL Cdc34 (FC = 880 nM), 0.5 µL yeast ubiquitin (Boston Biochem, 530 FC = 15.5 µM) and incubating at RT for 20 min. The PCR tubes were then placed back on ice and 531 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 15 20 µL of water, 8 µL of 5X ubiquitination assay buffer, 10 µL of concentrated SCFMet30-Flag IP, 532 and 2 µL of purified Met4 (FC = 200 nM) were added, the tubes were moved back to RT, and 20 533 µL aliquots of the reaction were removed, mixed with 2X sample buffer, and frozen in liquid 534 nitrogen over the time course. 535 536 SCFMet30-Flag IP and in vitro Met4 binding assay 537 For the Met4 binding assay, yeast cell lysate was prepared as described for the ubiquitination 538 experiment, except that the lysate was split three ways, with 1 mM DTT, 1 mM 539 tetramethylazodicarboxamide (Diamide) (Sigma, Cat#D3648), or nothing added to the lysate prior 540 to centrifugation at 21,000xg for 30 min at 4°C. The supernatant was transferred to new tubes and 541 100 µL of Thermo Fisher protein G dynabeads (Cat# 10004D) DMP crosslinked to 50 µL of 542 Mouse anti-FLAG M2 antibody (Sigma, Cat#F3165) was divided evenly between the six Met30-543 Flag IP conditions and incubated for 2 h at 4°C while rotating end over end. After incubation, the 544 beads were washed with IP buffer containing 1 mM DTT, 1 mM Diamide, or nothing twice before 545 a final wash with plain IP buffer. Each set of Met30-Flag bound beads prepared in the different IP 546 conditions was brought up to 80 µL with plain IP buffer, and 40 µL was dispensed to new tubes 547 containing 1 mL of IP buffer ± 1 mM DTT and 1 µg of purified recombinant Met4, and were 548 incubated for 2 h at 4°C while rotating end over end for a total of twelve Met4 co-IP conditions. 549 The beads were then collected, washed 3 times with IP buffer ± 1 mM DTT, resuspended in 60 µL 550 2X sample buffer, and heated at 70°C for 10 min before Western blotting for both Met4 and Met30. 551 552 553 554 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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M., WANG, Y., KUANG, Z. & TU, B. P. 2017. A Metabolic Function for 663 Phospholipid and Histone Methylation. Mol Cell, 66, 180-193 e8. 664 YE, C., SUTTER, B. M., WANG, Y., KUANG, Z., ZHAO, X., YU, Y. & TU, B. P. 2019. 665 Demethylation of the Protein Phosphatase PP2A Promotes Demethylation of Histones to 666 Enable Their Function as a Methyl Group Sink. Mol Cell, 73, 1115-1126 e6. 667 YEN, J. L., FLICK, K., PAPAGIANNIS, C. V., MATHUR, R., TYRRELL, A., OUNI, I., 668 KAAKE, R. M., HUANG, L. & KAISER, P. 2012. Signal-induced disassembly of the 669 SCF ubiquitin ligase complex by Cdc48/p97. Mol Cell, 48, 288-97. 670 YEN, J. L., SU, N. Y. & KAISER, P. 2005. The yeast ubiquitin ligase SCFMet30 regulates 671 heavy metal response. Mol Biol Cell, 16, 1872-82. 672 673 674 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 20 FIGURE LEGENDS 675 676 Figure 1. Met30 and Met4 response to sulfur starvation and repletion under respiratory 677 growth conditions. 678 (A) Western blot analysis of a time course performed with yeast containing endogenously tagged 679 Met30 and Met4 that were cultured in rich lactate media (Rich) overnight to mid log phase before 680 switching cells to sulfur-free lactate media (−sulfur) for 1 h, followed by the addition of a mix of 681 the sulfur containing metabolites methionine, homocysteine, and cysteine at 0.5 mM each 682 (+Met/Cys/Hcy). 683 (B) Expression of MET gene transcript levels was assessed by qPCR over the time course shown 684 in (A). Data are presented as mean and SEM of technical triplicates. 685 (C) Levels of key sulfur metabolites were measured over the same time course as in (A) and (B), 686 as determined by LC-MS/MS. Data represent the mean and SD of two biological replicates. 687 (D) met6∆ or str3∆ strains were grown in “Rich” YPL and switched to “−sulfur” SFL for 1 h to 688 induce sulfur starvation before the addition of either 0.5 mM homocysteine (+HCY), 0.5 mM 689 methionine (+MET), or 0.5 mM cysteine (+CYS). 690 (E) Simplified diagram of the sulfur metabolic pathway in yeast. 691 692 Figure 2. Met30 cysteine residues become oxidized during sulfur starvation. 693 (A) Schematic of Met30 protein architecture and cysteine residue location. 694 (B) Western blot analysis of Met30 cysteine redox state in lactate media as determined by 695 methoxy-PEG-maleimide (mPEG2K-mal) modification of reduced protein thiols. For every 696 reduced cysteine thiol in a protein, mPEG2K-mal adds ~ 2 kDa in apparent molecular weight. 697 (C) Same Western blot analysis as in (B), except that yeast were cultured in sulfur-free glucose 698 media (SFD) for 3 h before the addition of 0.5 mM each of the sulfur metabolites homocysteine, 699 methionine, and cysteine (+Met/Cys/Hcy). 700 (D) Yeast were subjected to the same rich to −sulfur media switch as in (B), except that following 701 the 15 min time point, 5 mM DTT was added to the culture for 15 min and Met30 cysteine residue 702 redox state and Met4 ubiquitination were assessed by Western blot. 703 704 Figure 3. Met30 cysteine point mutants display dysregulated sulfur sensing. 705 (A) Western blot analysis of Met30 cysteine redox state and Met4 ubiquitination status in WT and 706 two cysteine to serine mutants, C414S and C614/616/622/630S. 707 (B) MET gene transcript levels over the same time course as (A) for the three strains, as assessed 708 by qPCR. Data are presented as mean and SEM of technical triplicates. 709 (C) Growth curves of the three yeast strains used in (A) and (B) in sulfur-rich YPL media or −sulfur 710 SFL media supplemented with 0.2 mM homocysteine. Cells were grown to mid-log phase in YPL 711 media before pelleting, washing with water, and back-diluting yeast into the two media conditions. 712 Data represent mean and SD of technical triplicates. 713 714 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 21 Figure 4. Met30 cysteine oxidation disrupts ubiquitination and reduces binding to Met4 in 715 vitro. 716 (A) Schematic for the large-scale SCFMet30-Flag immunopurification from rich high sulfur (YPL) 717 and −sulfur (SFL) conditions for use in in vitro ubiquitination or binding assays with recombinant 718 Met4 protein. 719 (B) Western blot analysis of Met4 in vitro ubiquitination by SCFMet30-Flag immunopurifications 720 from cells cultured in sulfur-replete rich media. Cryomilled YPL yeast powder was divided evenly 721 for two Flag IPs performed identically with the exception that one was done in the presence of 1 722 mM DTT (+DTT) and the other was performed without reducing agent present (−DTT). To test if 723 the addition of reducing agent could rescue the activity of the “−DTT” IP, a small aliquot of the 724 “−DTT” SCFMet30-Flag complex was transferred to a new tube and was treated briefly with 5 mM 725 TCEP while the in vitro ubiquitination reaction was set up (−DTT/+TCEP). The first three lanes 726 are negative control reactions performed either without SCFMet30-Flag IP, recombinant Met4, or 727 ubiquitin. 728 (C) The same Western blot analysis of Met4 in vitro ubiquitination as in (B), except that the 729 SCFMet30-Flag complex was produced from −sulfur SFL cells. 730 (D) Western blot analysis of the Met4 binding assay illustrated in (A). Rich and −sulfur lysate 731 were both split three ways, and lysate with 1 mM DTT (+DTT), 1 mM diamide (+Diamide), or 732 control (−DTT) were incubated with anti-Flag magnetic beads to isolate Met30-Flag complex. The 733 Met30-Flag bound beads from each condition were then split in half and distributed into tubes 734 containing IP buffer ± 1 mM DTT and purified recombinant Met4. The mixture was allowed to 735 incubate for 2 h before the beads were washed, boiled in sample buffer, and bound proteins were 736 separated on SDS-PAGE gels and Western blots were performed for both Met30 and Met4. 737 738 Figure 5. Model for sulfur-sensing and MET gene regulation by the SCFMet30 E3 ligase. 739 In conditions of high sulfur metabolite levels, cysteine residues in the WD-40 repeat region of 740 Met30 are reduced, allowing Met30 to bind and facilitate ubiquitination of Met4 in order to 741 negatively regulate the transcriptional activation of the MET regulon. Upon sulfur starvation, 742 Met30 cysteine residues become oxidized, resulting in conformational changes in Met30 that allow 743 Met4 to be released from the SCFMet30 complex, deubiquitinated, and transcriptionally active. 744 745 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 22 SUPPLEMENTAL FIGURE LEGENDS 746 747 Figure S1. Characterization of the faster-migrating proteoform of Met30. 748 (A) Western blot of yeast treated with 200 µg/ml cycloheximide during sulfur starvation 749 demonstrates that production of the faster-migrating proteoform is dependent on new translation. 750 (B) The faster-migrating proteoform persists after rescue from sulfur starvation when treated with 751 a proteasome inhibitor. Cells were starved of sulfur for 3 h to accumulate the faster-migrating 752 proteoform, and then sulfur metabolites were added back concomitantly with MG132 (50 µM). 753 (C) The faster-migrating proteoform of Met30 is dependent on Met4. The met4∆ yeast strain does 754 not produce the second proteoform of Met30 when starved of sulfur. 755 (D) Western blot analysis of strains expressing either wild type Met30, Met30 D1-20aa, or Met30 756 M30/35/36A. Yeast cells harboring the N-terminal deletion of the first twenty amino acids of 757 Met30 (which contain the first three methionine residues) or have the subsequent three methionine 758 residues (M30/35/36) mutated to alanine do not create faster-migrating proteoforms. 759 (E) Met30(D1-20aa) and Met30(M30/35/36A) strains do not exhibit any growth phenotypes in 760 −sulfur glucose media with or without supplemented methionine. There are also no defects in 761 growth rate following repletion of methionine. Data represent mean and SD of technical triplicates. 762 763 Figure S2. Identification of key cysteine residues in Met30 involved specifically in sulfur 764 amino acid sensing. 765 (A) Schematic of Met30 protein architecture and cysteine residue location. 766 (B) Western blot analysis of various Met30 cysteine point mutants and Met4 ubiquitination status 767 in rich and −sulfur media. 768 (C) Western blot analysis of Met30 cysteine redox state and Met4 ubiquitination status in WT and 769 two cysteine to serine mutants, C414S and C614/616/622/630S, following treatment with 500 µM 770 CdCl2. 771 772 Figure S3. SCFMet30-Flag IP/in vitro ubiquitination assay demonstrating the dependence of 773 reducing agent in the IP on SCFMet30 E3 ligase activity. 774 (A) Initial IPs for SCFMet30-Flag complex were performed in the presence of 1 mM DTT prior to 775 Flag peptide elution and concentration. No DTT was used in the in vitro ubiquitination assay itself, 776 yet the E3 ligase activities for the E3 complex were indistinguishable between complex isolated 777 from high sulfur versus low sulfur cells. 778 (B) The same IP/in vitro assay as in (A), with the sole exception that DTT was not included during 779 the IP and wash steps. 780 (C) Silver stains of immunopurified SCFMet30-Flag complex isolated from rich and −sulfur cells 781 prepared in the presence or absence of DTT used in Figures 4B and C. 782 (D) Western blot of Cdc53 amounts from immunopurified SCFMet30-Flag complex shown in S2C 783 and used in Figures 4B and C. We speculate the reduced Cdc53 abundance in the −sulfur, −DTT 784 IP is the result of the canonical regulation of SCF E3 ligases, which causes reduced association 785 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 23 between Skp1/F-box heterodimers to the Cdc53 scaffold when binding between the F-box and its 786 substrate is reduced. 787 788 Figure S4. SCFMet30-Flag IP/in vitro ubiquitination assay using Met30 cysteine point mutants 789 (A) In vitro ubiquitination assays were carried out as described in Figure 4B with cell lysate 790 powder from WT, C414S, and C614/616/622/630S Met30 strains grown in rich media. The heavier 791 loading of the C414S mutant is likely due to a difference in cryomill lysis efficiency, and is not a 792 difference in the amount of starting material used. 793 (B) Met4 binding was assessed in the C414S and C614/616/622/630S mutants as described in 794 Figure 4D using cell lysate powder from cells grown in rich media. The fold change in Met4 795 binding in the presence and absence of DTT was quantified for each strain and for each Met30 796 immunopurification condition using ImageJ (version 1.53). 797 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 24 Table S1. Strains used in this study. 798 BACKGROUND GENOTYPE SOURCE CEN.PK MATa (van Dijken et al., 2000) CEN.PK MATa (van Dijken et al., 2000) CEN.PK MATa; MET30-FLAG::KanMX This study CEN.PK MATa; MET30-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; MET30-FLAG::KanMX MET4-HA::Hyg met6D::Nat This study CEN.PK MATa; MET30-FLAG::KanMX MET4-HA::Hyg str3D::Nat This study CEN.PK MATa; met30::MET30-C414S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C614/616/622/630S- FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30D::Phleo HO::MET30-FLAG::Nat MET4-HA::Hyg This study CEN.PK MATa; met30D::Phleo HO::MET30Daa1-20- FLAG::Nat Met4-HA::Hyg This study CEN.PK MATa; met30D::Phleo HO::MET30-M30/35/36A- FLAG::Nat Met4-HA::Hyg This study CEN.PK MATa; MET30-FLAG::KanMX MET4-HA::Hyg pdr5D::Nat This study CEN.PK MATa; met4D::KanMX MET30-FLAG::Hyg This study CEN.PK MATa; cup1p-6xHis-TEV-UBA1::Hyg This study CEN.PK MATa; met30::MET30-C201S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C374S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C426S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C436S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C439S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C455S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C528S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C544S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C584S-FLAG::KanMX MET4-HA::Hyg This study .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 25 CEN.PK MATa; met30::MET30-C614S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C616S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C584/622S-FLAG::KanMX MET4-HA::Hyg This study CEN.PK MATa; met30::MET30-C630S-FLAG::KanMX MET4-HA::Hyg This study 799 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 26 Table S2. Recipe for sulfur-free media. 800 salts (g L-1) CaCl2•2H2O 0.1 NaCl 0.1 MgCl2•6H2O 0.412 NH4Cl 4.05 KH2PO4 1 metals (mg L-1) boric acid 0.5 CuCl2•2H2O 0.0273 KI 0.1 FeCl3•6H2O 0.2 MnCl2•4H2O 0.4684 Na2MoO4•2H2O 0.2 ZnCl2•H2O 0.1895 vitamins (mg L-1) biotin 0.002 calcium pantothenate 0.4 folic acid 0.002 inositol 2 niacin 0.4 4-aminobenzoic acid 0.2 pyridoxine HCl 0.4 riboflavin 0.2 thiamine-HCl 0.4 801 Recipes are derived from (Miller et al., 2013). 802 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ Met4-HA Rich Met30-Flag Rpn10 75 kDa 100 kDa 150 kDa −Sulfur +Met/Cys/Hcy Time (min) 0 15 60 15 60 A Lactate (respiratory) 75 kDa 100 kDa 150 kDa Met4-HA Met30-Flag Rpn10 Time (min) Rich −Sulfur 0 15 60 15 60 15 60 0 15 60 15 60 15 60 met6∆ str3∆ D Lactate (respiratory) +Hcy +Met Rich −Sulfur +Cys +Met R -S 15 -S 60 +M CH 15 +M CH 60 0.01 0.1 1 10 100 R el at iv e ab un da nc e Methionine R -S 15 -S 60 +M CH 15 +M CH 60 0.1 1 10 R el at iv e ab un da nc e GSH R -S 15 -S 60 +M CH 15 +M CH 60 0.1 1 10 100 R el at iv e ab un da nc e Cysteine R -S 15 -S 60 +M CH 15 +M CH 60 0.1 1 10 R el at iv e ab un da nc e GSSG R -S 15 -S 60 +M CH 15 +M CH 60 0.1 1 10 100 R el at iv e ab un da nc e Cystathionine R -S 15 -S 60 +M CH 15 +M CH 60 0.01 0.1 1 10 R el at iv e ab un da nc e SAM R -S 15 -S 60 +M CH 15 +M CH 60 0.1 1 10 100 R el at iv e ab un da nc e SAH C SO4 2- homocysteine methionine SAM GSH SAH cystathionine cysteine MET6 STR3 CYS4 STR2CYS3 GSH1 GSH2 SAH1 SAM1 SAM2 E B Figure 1 Ub-Met4-HA Ub-Met4-HA R -S 15 -S 60 +M CH 15 +M CH 60 0 5 10 15 20 25 R el at iv e m R N A E xp re ss io n MET17 R -S 15 -S 60 +M CH 15 +M CH 60 0 1 2 3 SAM1 R el at iv e m R N A E xp re ss io n R -S 15 -S 60 +M CH 15 +M CH 60 0 5 10 15 GSH1 R el at iv e m R N A E xp re ss io n .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ WD 8 WD 7 WD 6 WD 5 WD 4 WD 3 WD 2 WD 1F-Box 95111 164 201 205 211 228 614 616 622 630 236 239 293 374 414 426 436 439 455 528 544 584 640607-635550-578509-538461-499419-449380-408340-368300-328180-227a.a. 1 SCF-Binding Met4-BindingA Met30-Flag B 75 kDa 100 kDa 100 kDa 150 kDa 75 kDa mPEG2K-mal Met30-Flag mPEG2K-mal Rpn10 Met4-HA Rich Rpn10 150 kDa −Sulfur +Met/Cys/Hcy Time (min) 0 15 60 15 60 Lactate (respiratory) Met30-Flag C 75 kDa 100 kDa 100 kDa 150 kDa 75 kDa mPEG2K-mal Met30-Flag mPEG2K-mal Rpn10 Met4-HA +Met Rpn10 150 kDa −Sulfur +Met/Cys/Hcy Time (min) 0 90 180 15 60 Glucose (glycolytic) Met30-Flag D 75 kDa 100 kDa 100 kDa 150 kDa 75 kDa mPEG2K-mal Rpn10 Met4-HA Rich Rpn10 150 kDa −Sulfur +DTT Time (min) 0 15 15 Lactate (respiratory) Figure 2 Ub-Met4-HA Red-Met30 Ox-Met30 Ub-Met4-HA Red-Met30 Ox-Met30 mPEG2K-mal Met30-Flag Red-Met30 Ox-Met30 Ub-Met4-HA Ox Red .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ Met4-HA Met30-Flag Rpn10 75 kDa 100 kDa 150 kDa 100 kDa 150 kDa 75 kDa Lactate (respiratory) mPEG2K-mal Rpn10 Rich −Sulfur +Met/Cys/Hcy Time (min) 0 15 60 15 60 WT Rich −Sulfur +Met/Cys/Hcy 0 15 60 15 60 C414S Rich −Sulfur +Met/Cys/Hcy 0 15 60 15 60 C614/616/622/630S A R -S 15 -S 60 +M CH 15 +M CH 60 R -S 15 -S 60 +M CH 15 +M CH 60 R -S 15 -S 60 +M CH 15 +M CH 60 0 2 4 6 R el at iv e m R N A E xp re ss io n MET17 R -S 15 -S 60 +M CH 15 +M CH 60 R -S 15 -S 60 +M CH 15 +M CH 60 R -S 15 -S 60 +M CH 15 +M CH 60 0 10 20 30 R el at iv e m R N A E xp re ss io n SAM1 R -S 15 -S 60 +M CH 15 +M CH 60 R -S 15 -S 60 +M CH 15 +M CH 60 R -S 15 -S 60 +M CH 15 +M CH 60 0 5 10 15 GSH1 R el at iv e m R N A E xp re ss io n WT C414S C614/616/ 622/630S B 0.0 1.5 3.0 4.5 6.0 7.5 9.0 0.0 0.5 1.0 1.5 Time (h) in YPL O D 60 0 WT C414S C614/616/622/630S C 0 3 6 9 12 15 18 21 24 0.0 0.1 0.2 0.3 0.4 0.5 Time (h) in SFL + 0.2 mM Hcy after switch O D 60 0 WT C414S C614/616/622/630S Figure 3 Ub-Met4-HA mPEG2K-mal Met30-Flag Red-Met30 Ox-Met30 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 75 kDa 100 kDa 150 kDa Met4-HA Met30-Flag Time (min) 60 60 60 0 30 60 180 60 60 60 0 30 60 180 0 30 60 180 + + − + + + + + + − + + + + + + + + + − + + + + + + − + + + + + + + + + − + + + + + + − + + + + + + + + + + Flag purification Rich SCFMet30-Flag Ubiquitin Met4 +DTT −DTT −DTT/ +TCEP B 75 kDa 100 kDa 150 kDa Met4-HA Met30-Flag Time (min) 60 60 60 0 30 60 180 60 60 60 0 30 60 180 0 30 60 180 + + − + + + + + + − + + + + + + + + + − + + + + + + − + + + + + + + + + − + + + + + + − + + + + + + + + + + Flag purification −Sulfur SCFMet30-Flag Ubiquitin Met4 +DTT −DTT −DTT/ +TCEP C A Rich −SulfurRich Rich Switch 50% of cells to −Sulfur media Collect and cryomill cell pellets "Rich" cell lysate powder "−Sulfur" cell lysate powder Met30 IP and in vitro Met4 ubiquitination assay Add IP buffer to Rich and −Sulfur powder Split lysate, IP Met30 and SCF core components +/− DTT +DTT −DTT +DTT −DTT Wash Met30-bound beads, elute and concentrate the Met30 E3 complex, and perform in vitro ubiquitination assays with purified E1 (Uba1), E2 (Cdc34), ubiquitin, and Met4 Met30 IP and in vitro Met4 binding assay Prepare Rich and −Sulfur lysate identically as for the ubiquitination experiment Split lysate, IP Met30 in the presence of DTT, Diamide, or control −DTT+DTT +Diamide Wash Met30-bound beads of unbound Met4, boil beads in sample buffer, and Western blot for Met4 to assess binding Wash Met30-bound beads, split each Met30 IP in half, and incubate beads with purified Met4 +/− DTT +/−DTT +/−DTT +/−DTT −DTT+DTT +Diamide +/−DTT +/−DTT +/−DTT Rich −Sulfur Met30-Flag Met4-HA Met30-Flag IP Met4-HA co-IP +DTT +DTT −DTT +DTT −DTT −DTT +DTT −DTT +Diamide +DTT +DTT −DTT +DTT −DTT −DTT +DTT −DTT +DiamideInput Rich −Sulfur Met4-HA D Figure 4 Ub-Met4-HA Ub-Met4-HA .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ A C dc 53 Low sulfur metabolite levels Hrt1 N8 Ub Ub Skp1 Met4 Met30 Met30 S——S High sulfur metabolite levels E2 Ub Ub Ub SH HS Met31/32 Met genes OFF C dc 53 Hrt1 N8 Skp1 Met4 E2 Ub Met31/32 Met genes ON Met4 Figure 5 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ Time (min) 0 60 120 180 15 45 90 0 60 120 180 15 45 90 0 60 120 180 15 45 90 Time (min) 0 30 60 60 120 120 180 180 Met4-HA Met30-Flag Rpn10 100 kDa 150 kDa − − − + − + − +CHX Met4-HA Met30-Flag Rpn10 100 kDa 150 kDa −Sulfur+Met Time (min) 0 30 60 120 180 15 45 90 Met30-Flag Rpn10 +Met −Sulfur+Met +Met WT 0 30 60 120 180 15 45 90 met4∆ Met4-HA Met30-Flag Rpn10 A C D E 0 2 4 6 8 0 1 2 3 4 Time (h) in +Met Glucose O D 60 0 0 2 4 6 8 0.0 0.5 1.0 1.5 2.0 Time (h) in −Sulfur Glucose O D 60 0 +Met −Sulfur Glucose (glycolytic) Time (min) 0 180 15 15 30 30 60 60 − − − + − + − +MG132 B +Met +Met Glucose (glycolytic) −Sulfur Glucose (glycolytic) Glucose (glycolytic) −Sulfur+Met +Met WT −Sulfur+Met +Met ∆1-20 −Sulfur+Met +Met M30/35/36A Glucose (glycolytic) Figure S1 100 kDa 150 kDa 0 2 4 6 8 0.0 0.5 1.0 1.5 2.0 Time (h) in +Met Glucose after switch from −Sulfur Glucose (3h) O D 60 0 WT Δ1-20 M30/35/36A Ub-Met4-HA Ub-Met4-HA .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ WD 8 WD 7 WD 6 WD 5 WD 4 WD 3 WD 2 WD 1F-Box 95111 164 201 205 211 228 614 616 622 630 236 239 293 374 414 426 436 439 455 528 544 584 640607-635550-578509-538461-499419-449380-408340-368300-328180-227a.a. 1 SCF-Binding Met4-BindingA Met30-Flag B 75 kDa 100 kDa 100 kDa 150 kDa 75 kDa Met4-HA R Rpn10 150 kDa −S Time (min) 0 15 15 0 15 0 15 0 15 0 15 0 15 0 15 0 15 WT Ub-Met4-HA Lactate (respiratory) +DTT R −S C201S R −S C374S R −S C414S R −S C426S R −S C436S R −S C439S R −S C455S Met30-Flag Met4-HA R Rpn10 −S Time (min) 0 15 0 15 0 15 0 15 0 15 0 15 0 15 0 15 WT Ub-Met4-HA R −S C528S R −S C544S R −S C584S R −S C614S R −S C616S R −S C584/622S R −S C630S Figure S2 C Met4-HA Met30-Flag Rpn10 75 kDa 100 kDa 150 kDa 100 kDa 150 kDa 75 kDa Lactate (respiratory) mPEG2K-mal Rpn10 Rich +Cd Time (min) 0 15 45 90 0 15 45 90 0 15 45 90 WT Rich +Cd C414S Rich +Cd C614/616/622/630S Ub-Met4-HA mPEG2K-mal Met30-Flag Red-Met30 Ox-Met30 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ 75 kDa 100 kDa 150 kDa Met4-HA Met30-Flag Time (min) 60 60 60 0 15 30 60 180 60 60 60 0 15 30 60 180 + + − + + + + + + + − + + + + + + − + + + + + + + − + + + + + + − + + + + + + + − + + + + + + + Flag purification +DTT SCFMet30-Flag Ubiquitin Met4 Rich −Sulfur A 75 kDa 100 kDa 150 kDa Met4-HA Met30-Flag Time (min) 60 60 60 0 15 30 60 180 60 60 60 0 15 30 60 180 + + − + + + + + + + − + + + + + + − + + + + + + + − + + + + + + − + + + + + + + − + + + + + + + Flag purification −DTT SCFMet30-Flag Ubiquitin Met4 Rich −Sulfur B C +DTT −DTT +DTT −DTT Rich −Sulfur 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa Cdc53 Met30 Skp1 Cdc53 100 kDa +DTT −DTT Rich +DTT −DTT −SulfurD Figure S3 Ub-Met4-HA Ub-Met4-HA .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/ Figure S4 75 kDa 100 kDa 150 kDa Met4-HA Met30-Flag Time (min) 0 60 180 0 60 180 0 60 180 0 60 180 0 60 180 0 60 180 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Flag purification Rich SCFMet30-Flag Ubiquitin Met4 +DTT A Ub-Met4-HA +D TT −D TT +D iam id e +D TT −D TT +D iam id e +D TT −D TT +D iam id e 0 2 4 6 M et 4 pu lld ow n (+ D TT /– D TT ) WT C414S C614/616/622/630S Input +DTT −DTT Met4-HA Met30-Flag Met4-HA Met30-Flag IP Met4-HA co-IP WT C414S +DTT +DTT −DTT +DTT −DTT −DTT +DTT −DTT +Diamide +DTT +DTT −DTT +DTT −DTT −DTT +DTT −DTT +Diamide C614/616/622/630S +DTT +DTT −DTT +DTT −DTT −DTT +DTT −DTT +Diamide WT C414S C614/616/622/630S −DTT +DTT −DTT +DTT −DTT B .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 7, 2021. ; https://doi.org/10.1101/2021.01.06.425657doi: bioRxiv preprint https://doi.org/10.1101/2021.01.06.425657 http://creativecommons.org/licenses/by/4.0/