Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation

The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington’s disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A) and phospho-mimetic (rpt6-S119D) mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

Introduction larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D mutants. Furthermore, rpt6-S119A mutants expressing Htt103Q displayed altered senescence profiles compared to wild-type or rpt6-S119D mutants. Together, these data suggest that dynamic phosphorylation of Rpt6 at S119 in yeast (S120 in mammals) may play an important, conserved function in the ability of the proteasome to counteract proteotoxic stress and protein aggregate formation.

Yeast strains and knock-in strategy
We constructed an N-terminal 9xMyc-tagged RPT6 strain as previously described [30]. Since RPT6 is essential, chromosomal gene replacement was performed in a wild-type diploid strain (LPY15916, see S1 Table). To direct integration, a PCR product was prepared using oligonucleotides oLP1751 and 1752 (S3 Table) and pLP1956 (POM20) plasmid DNA as a template (S2 Table). The strain generated was LPY17011. To allow expression of the tagged protein, the kanMX marker was removed by Cre recombinase expression, induced from plasmid pLP192 (Life Technologies pBS39; [31]) by growth in 1% galactose. Myc-Rpt6 expression was confirmed by protein immunoblotting. The myc-RPT6 heterozygous diploid (LPY17208) strain was sporulated and dissected to obtain LPY18040, the haploid myc-RPT6 strain (S1 Table).

Viability assays
Assays were performed as previously described [38]. Freshly transformed strains (with pLP3012, pLP3013 or pLP3014) were grown to saturation and diluted to A 600 0.2 in ura-selection medium. Duplicates of 400 cells were plated on ura-plates from the same starting cultures after 1, 5, 7, 10, 12 and 15 days of growth at 30˚C. Cells were counted before plating using a hemocytometer. After 3 days at 30˚C, colony forming units (CFU) were counted from three independent experiments. Results were normalized to vector transformed myc-RPT6-wildtype strain.

Flow cytometry
As described in [39], cells were ethanol fixed (A 600 0.8) during logarithmic growth. A total of 30,000 propidium iodide-stained cells of each strain were analyzed by flow cytometry (BD Accuri C6) after sonication.

Bud profiling
Morphology was assessed by light microscopy for 2500-3000 cells per mutant strain from three independent experiments. Either single cells or cells with one bud were registered as normal. Two or more fused cells, cells with two or more buds or increased size cells when compared to wild type were registered as abnormal. Statistical analysis consisted of average and one-way ANOVA p < 0.1.

Native-PAGE peptidase and 26S fluorogenic peptidase activity assays
Strains were grown to both A 600 5.0 and 0.6, spun down and freeze-dried with liquid nitrogen as previously described [40]. Briefly, cells were rehydrated using lysis buffer (25mM HEPES--KOH pH 7.4, 5mM MgCl 2 , 10% Glycerol, 1mM DTT, 2mM ATP), and ultracentrifuged at 100,000xg 30 min to obtain whole cell lysates. Native PAGE was performed as previously described [41]. 3-12% gradient gels run at constant voltage for 4 hr at 4˚C were soaked in developing buffer (50mM Tris-HCl pH 7.4, 5mM MgCl 2 , 0.5mM EDTA, 1mM ATP) containing 50μM Suc-LLVY-AMC substrate. Stimulation of CP gate opening was performed using 0.02% SDS for 30 min. All images were obtained using Protein Simple FluorChem E imaging system. Gels were then transferred to nitrocellulose and probed with anti-myc and anti-20S antibodies. Chymotrypsin-like activity of 26S proteasomes from whole cell lysates was monitored over time (20 sec/ 2 hr) using Suc-LLVY-AMC substrate (Enzo) as previously described [42]. The peptidase assay was run in triplicate using a normalized protein concentration monitored on a Perkin-Elmer HTS7000 BioAssay Microplate reader. Kinetic rates from each mutant strain were plotted from the averaged triplicate data.

Results
Rpt6 is phosphorylated in S. cerevisiae in response to stress Several subunits of the proteasome have been reported to be phosphorylated in vivo [43,44], however, relatively few studies have addressed how phosphorylation may regulate proteasome function. Phosphorylation might regulate proteasome subunit assembly, disassembly, or distinct proteasome activities, such as peptidase activity, ATPase activity, ubiquitin binding, and/ or deubiquitinating activities. Further, phosphorylation might regulate the cellular distribution and trafficking of proteasomes and association with proteasome-interacting proteins. In mammalian neurons, we found that Rpt6 is phosphorylated at serine 120 (S120) in a neuronal activity-dependent fashion [22]. It is clear that Rpt6 S120 phosphorylation increases proteasome activity and promotes the redistribution of proteasomes to synapses [22,23,45], although further mechanistic details remain under study.
In S. cerevisiae RPT6 has been previously studied with conditional alleles in which variable phenotypes were observed, depending on the strength of each mutation [46,47]. These studies were important for defining Rpt6 function in RP's base assembly and its interaction with the core particle, which is indispensable for 26S proteasome assembly [48]. Similar mutants of other RP ATPases had distinct phenotypes [49], showing specialized roles in protein degradation, chromatin regulation, gene expression and DNA damage repair, among other cellular processes [50][51][52][53][54][55]. To date however, little is known about the role of phosphorylation in the regulation of RP ATPase subunits.
The sequence of proteasomal subunits is conserved from yeast to humans. The sequence identities of the RP base ATPase subunits range from 66%-76%, with Rpt6 74% identical to its human ortholog [56]. Furthermore, the sequence surrounding S120 in mammals and S119 in yeast is 81% identical (Fig 1A). To address the functional relevance of Rpt6 phosphorylation on proteasome function in vivo, we performed studies in yeast. Two strains with chromosomally integrated mutations were constructed. The first substituted Ser119 of Rpt6 with alanine (rpt6-S119A) creating a non-modifiable phospho-dead residue; the second mutant strain was generated with an aspartic acid (rpt6-S119D) substitution as a phosphomimetic residue (S1 Table). Because the carboxy-terminus of Rpt6 contributes to proteasome assembly by establishing direct contacts with the CP [40,57] and with protein chaperone Rpn14 [58], we constructed an N-terminal Myc-epitope tagged Rpt6 to facilitate immuno-precipitation while retaining C-terminal function. Wild-type (myc-RPT6) and both mutants (myc-rpt6-S119A and -S119D) were expressed from the endogenous chromosomal locus at similar levels ( Fig 1B). To determine if Rpt6 was phosphorylated at S119 in yeast, Myc-Rpt6 was immunoprecipitated using a mouse anti-Myc antibody, and probed with an anti-phospho S120 Rpt6 pAb (pS120) by immunoblotting. We observed little signal to no signal for the myc-rpt6-S119A mutant ( Fig  1C). In contrast, strong immunoreactivity was detected for the Myc-Rpt6 protein ( Fig 1C) indicating that Rpt6 is phosphorylated in yeast at S119 (Fig 1C). The rpt6-S119A mutants had increased susceptibility to proteotoxic stress and displayed abnormal morphology. (A) Alignment of Rpt6 sequences from rat, human and yeast indicate conservation of the S120 phosphorylation site. Note the presence of a serine at position 117 in yeast. This residue may contribute to some cross-immunoreactivity for the pS120 antibody in the myc-rpt6-S119A strain (see below). (B) Qualitative analysis of myc-tagged Rpt6 (myc-Rpt6) expression. Immunoblot depicts similar levels of expression in strains tested. (C) Representative immunoblot of myc-RPT6 and myc-rpt6-S119A lysates immunoprecipitated with α-myc antibodies and resolved on SDS-PAGE. The anti-Rpt6 pS120 antibody recognizes wild-type myc-Rpt6, whereas little to no signal is observed for the myc-rpt6-S119A mutant, suggesting that Rpt6 is phosphorylated in yeast. (D) Increased sensitivity to proteotoxic stress is observed in the myc-rpt6-S119A mutant. The fitness of the myc-rpt6-S119D strain was comparable to myc-RPT6. The myc-rpt6-S119A strain was sensitive to elevated temperature, ethanol and canavanine. Arg-medium serves as a control for canavanine, which is a toxic analog of arginine. (E) Phosphorylation of endogenous Rpt6 is increased upon stress. Protein lysates from cells grown under normal and stress conditions (37˚and canavanine (1ug/ml;2h)) were probed with the anti-pS120 antibody. (F) Representative micrographs of myc-RPT6 strains (DIC and DAPI) during log phase indicate abnormal morphology in the myc-rpt6-S119A mutant. (G) Quantitative analysis of morphological studies in (F). Graphs denote 3 independent experiments. Abnormal morphology is increased to 10% of the total population in myc-rpt6-S119A.
The rpt6-S119A phospho-dead mutant is sensitive to conditions that induce protein misfolding Proteasome function is required for maintaining protein homeostasis and cell survival during protein stress. To characterize the importance of Rpt6 phosphorylation on proteasome function, we evaluated growth of the RPT6 phospho-mutant strains under proteotoxic stress. As shown in Fig 1D, the growth and fitness of myc-RPT6 and the phosphomimetic mutant, myc-rpt6-S119D, was comparable to wild-type RPT6 (See also S1 Fig). We did, however, find that the expression of the phosphomimetic rpt6-S120D on a 2μ plasmid in the RPT6 null background promoted greater resistance to proteotoxic stress (ethanol 8%) when compared to RPT6 or rpt6-S120A (also expressed on a 2μ plasmid in the RPT6 null background) (S1 Fig). In contrast, the chromosomal myc-rpt6-S119A phospho-dead mutant was sensitive to growth at 37˚C, to ethanol and to canavanine. The sensitivity phenotypes of the myc-rpt6-S119A mutant are similar to those previously found in strains with mutations in the ATPase domain of Rpt6 [40,59]. Since growth was comparable between myc-RPT6 and myc-rpt6-S119D, we suggest that phosphorylation of Rpt6 at S119 is important for maintaining proteasome function during cellular protein stress. In addition, when myc-rpt6-S119A was evaluated as a heterozygote, no sensitivity to proteotoxic stress was observed (S2B Fig), demonstrating that the S119A mutation in RPT6 was recessive.
The myc-rpt6-S119A mutant proved to be sensitive to other agents that disrupt protein structure, including formamide and cadmium chloride (S2A Fig). Furthermore, this mutant was sensitive to the DNA damage inducing agent camptothecin (CPT), an inhibitor of topoisomerase I (S2A Fig). Collectively, these data indicate that alanine substitution at S119, preventing its posttranslational modification, results in growth defects in stress conditions similar to those of null alleles of non-essential proteasome subunits and to strains carrying thermosensitive alleles of CP or RP subunits [48,50,54,55,60].
To confirm the biological relevance of the findings above, we evaluated phosphorylation of myc-Rpt6-S119 in response to stress. When cells were grown at either high temperatures or in the presence of proteotoxic stress agents, we observed an increase in S119 phosphorylation relative to cells grown under normal conditions (Fig 1E). This suggests that phosphorylation of Rpt6 at S119 may have a role in regulating proteasome function as a response to stress mechanisms.
Because of established roles for protein turnover in cell cycle regulation [61,62] the cell cycle profiles of both rpt6 phospho-mutants were also analyzed. As shown in S3 Fig, a defect in progression through the G2/M phase of the cell cycle was observed in all myc-RPT6 strains tested, which was in agreement with previous reports [46]. This shows that the N-terminal Myc-epitope may modestly interfere with Rpt6 function in the G2/M transition. This effect appears independent of any effects on proteasome function in proteotoxic and cellular stress conditions, and may thus point to a previously unsuspected role for the N-terminus. Notably, the delay in the G2/M transition was independent of S119 mutations ( S3 Fig). Typically, a G2/ M block is characterized by a high percentage of budded cells. All three myc-RPT6, myc-rpt6-S119A and myc-rpt6-S119D strains had between 40-55% budded cells (Fig 1F). However, we observed a significant increase in the multi-budded phenotype in myc-rpt6-S119A mutants (Fig 1G) suggesting additional defects in the control of cell division. Together, these data indicate that the myc-rpt6-S119D mutant strain had similar growth phenotypes to the myc-RPT6 strain, whereas the myc-rpt6-S119A mutant was sensitive to proteotoxic and other cellular stressors and had an exacerbated budding defect. Decreased proteasome activity in the myc-rpt6 S119A mutant It was important to determine if proteasome function was altered in the myc-rpt6-S119A mutant. We first evaluated proteasome activity from lysates of wild-type and mutant strains by native gel electrophoresis followed by an in-gel activity assay with the fluorogenic proteasome substrate Suc-LLVY-AMC. The myc-rpt6-S119A mutant had significantly decreased proteasome activity associated with both singly and doubly capped proteasomes (20S CP associated with one and two 19S RP, respectively), although the total level of proteasomes was comparable (Fig 2A and 2B). We additionally monitored the chymotrypsin-like activity of 26S proteasomes in whole cell lysates by Suc-LLVY-AMC cleavage over time. We found that proteasome activity was significantly decreased in lysates of myc-rpt6-S119A mutant when compared to myc-RPT6 or myc-rpt6-S119D mutant strains (Fig 2C). We did not see enhanced proteasome activity in myc-rpt6-S119D mutant strain compared to myc-RPT6. Thus, preventing Rpt6 phosphorylation at S119 decreased proteasome function and increased susceptibility to proteotoxic stress.

Altered Rpt6 phosphorylation exacerbates Htt polyQ aggregation and proteotoxicity
Since the cloning of the Huntington's disease (HD) gene in 1993, transgenic models of HD have been constructed in multiple model organisms including yeast, nematodes, flies and mice [63]. S. cerevisiae has proven to be an excellent organism in which to study molecular mechanistic aspects of human neurodegenerative disease [28,64]. In particular, HD which is caused by the expansion of a tri-nucleotide CAG repeat (poly-Q expansion) at the N-terminus of exon 1 of mutant Huntingtin (Htt), has been well-characterized [27,65,66]. The poly-Q expansion leads to misfolding of Htt and formation of mutant Htt-containing protein aggregates [27,64]. Several studies have shown that proteasome activity is decreased in the affected brain regions of HD patients [67], whereas in yeast, expression of a mutant Htt fragment (Htt72Q or higher) causes aggregate formation, transcriptional dysregulation, cellular toxicity, perturbations in kynurenine pathway metabolites, increased reactive oxygen species (ROS), mitochondrial dysfunction, and defects in endocytosis and apoptotic events [27,[64][65][66]. Importantly, some models of HD recapitulate the relationship of mutant Htt-containing protein aggregates and decreased proteasome activity.
To determine if altered Rpt6 phosphorylation influences mutant Htt-induced defects in growth and the extent of protein aggregate formation, we transformed myc-RPT6, myc-rpt6-S119A and myc-rpt6-S119D strains with plasmids to induce expression of a fragment of Htt with differing polyQ lengths (Htt25Q, Htt72Q and Htt103Q) (Fig 3A). The growth of myc-RPT6 and myc-rpt6-S119D strains expressing all mutant Htt polyQ variants upon challenge with canavanine was comparable to cells transformed with the vector control ( Fig 3B). There was a slight decrease in growth with increasing length of the polyQ repeat length (Fig 3B). In contrast, the myc-rpt6-S119A strain displayed significantly reduced growth with increasing polyQ repeat length (Fig 3B). We subsequently evaluated protein aggregate formation of mutant GFP-Htt in all RPT6 strains. As shown in Fig 3C, expression of Htt25Q was evenly distributed throughout the cell, whereas Htt72Q formed small protein aggregates in all strains, as previously described in a wild-type strain [27]. Htt103Q expression in the myc-rpt6-S119A mutant formed considerably larger mHtt aggregates compared to myc-RPT6 and myc-rpt6-S1119D (Fig 3C and 3D). Together these data demonstrate that clearance of mutant Htt103Q protein aggregates is compromised in the myc-rpt6-S119A mutant with decreased proteasome function. Compromised senescence in myc-rpt6-S119A cells expressing Htt103Q Yeast stationary phase growth or chronological life span (CLS) assays measure the survival of cell populations in post-mitotic or non-dividing phases. These metrics appear similar to those for mammalian cells such as neurons that do not divide or that have long non-mitotic rest phases [68,69]. Because we found differences in protein aggregate size among RPT6 strains, we sought to simulate conditions analogous to the neuronal non-dividing state by analyzing how cell survival upon extended chronological age might be affected. The viability of all myc-  RPT6 strains expressing vector control or mutant Htt103Q was measured to assess the influence of mutant Htt protein aggregation and senescence profiles. We grew all transformed strains in glucose-containing medium for up to 15 continuous days, plating on fresh medium after 1, 5, 7, 10, 12 and 15 days of growth. The colony forming units (CFU), corresponding to cells able to exit senescence and resume growth were quantified after three days and the results were normalized to myc-RPT6 cultures transformed with vector (plated after one day of growth). The relative number of colonies indicated the survival of the strains, where the CFU number is proportional to the fitness of the strains after varying times in stationary phase. Vector-transformed myc-RPT6, myc-rpt6-S119D and myc-rpt6-S119A strains had a similar loss in viability after 5 days of growth, also comparable to that obtained for Rpt6 mutants transformed with Htt-25Q (S4 Fig). However, when GFP-Htt103Q was expressed, the initial viability of all tested strains after one day of growth was 30% lower compared to vector transformants. We propose that this phenotype could reflect a problem in establishing a senescent state, delaying cell death as the cells age. This phenotype is stronger in the myc-rpt6-S119A mutant (Fig 4B), suggesting that normal proteasomal function may be relevant in establishing a cell's death programming during chronological aging.

Discussion
The UPS, which controls the majority of protein degradation in eukaryotic cells, is particularly important for counteracting altered protein homeostasis observed in age-related and neurodegenerative diseases. Indeed, the inability to rid cells of damaged proteins is believed to be key to the pathogenesis of many neurodegenerative diseases including AD, PD and HD. In some cases these proteins form heavily ubiquitinated protein aggregates. One hypothesis is that an increased proteotoxic load impairs proteasome function, thereby making cells more susceptible to stress. Alternatively, the degradation of non-toxic proteins may become impaired, which then alters normal cellular function. Historically, proteasome inhibitors have been used to understand how proteasome dysfunction contributes to disease pathogenesis. However, these have a limited utility because they are toxic to cells and cause death by apoptosis, perhaps due to cell cycle arrest [42,[70][71][72][73]. Several recent studies have reported the phosphorylation of multiple proteasome subunits. We and others have shown that the ATPase RP subunit, Rpt6, is phosphorylated at serine 120 by the CaMKIIα plasticity kinase in the mammalian CNS [22,45]. We reported that Rpt6 phosphorylation is important for synaptic strength and proteasome-dependent remodeling of dendritic spines [23,24]. Yet, the physiological significance of how proteasome phosphorylation regulates the rate and selectivity of substrate degradation remains an important question that is the topic of ongoing investigation. The budding yeast S. cerevisiae has been used successfully to model neurodegenerative diseases, not only to understand the molecular mechanisms involved but also to provide insight into potential therapeutic approaches for these diseases [74]. In this study, we created phospho-dead and phospho-mimetic mutants of Rpt6 serine 119 in yeast (serine 120 in mammals) and report phosphorylation of the 26S proteasome to be an important cis-regulatory posttranslational modification for the proteasome in counteracting proteotoxic stress. Using phosphospecific Rpt6 S120 antibodies we showed that Rpt6 is phosphorylated at serine 119. This posttranslational modification is increased upon temperature and proteotoxic stress, underscoring a possible role in regulation of Rpt6 and proteasome function. In addition, rpt6-S119A mutants that prevent Rpt6 phosphorylation at this site have reduced proteasome activity.
In mammalian cell lines and primary neurons we found that the overexpression of CaM-KIIα induced a dramatic increase in the chymotryptic peptidase activity of the proteasome [22]. Furthermore, purified 26S proteasomes from Rpt6 S120A mutant mouse brains have both decreased chymotryptic activity in addition to decreased ATPase activity (F.Gonzales and G.Patrick, unpublished findings). CaMKIIα activity promotes the active sequestration of 26S proteasomes in dendritic spines [23,45], however in this case, CaMKIIα kinase activity is dispensable which indicates that interactions of CaMKIIα with the 26S proteasomes can be sufficient to drive proteasomes into dendritic spines [45]. It is therefore plausible that in yeast the S119A mutation alters a variety of 19S proteasome activities (e.g. assembly, gating of 20S CP, substrate translocation) as well as interactions with the many proteasome interacting proteins (PIPs) [57,58,[75][76][77][78][79][80][81].
Although it is yet to be determined mechanistically how the S119A mutant alters 26S proteasome function, we find that it renders the cells more susceptible to various proteotoxic stresses. One might predict that the rpt6-S119D mutation would confer enhanced ability to counteract proteotoxic stress in comparison to the wild-type strain. We found this to be the case when RPT6 null strains overexpressing rpt6-S119D were grown in 8% ethanol (S1 Fig). When rpt6-S119D was expressed from the endogenous locus, the susceptibility to proteotoxic stress was similar to wild type RPT6. Nonetheless, while the rpt6-S119A strain grew similar to wild type and rpt6-S119D in normal growth media, growth was significantly perturbed in stress-inducing conditions. As we demonstrated that the myc-Rpt6-S119A protein (the only cellular source of the Rpt6 protein in the null background) is clearly incorporated into functional proteasomes allowing viability of the cells in normal growth media, we suggest that Rpt6 phosphorylation has a role in the cellular response to proteotoxic stress.
We also found protein aggregates of mutant Huntingtin protein with expanded CAG repeats (mHtt103Q) to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D mutants. Intriguingly, Krobitsch and Lindquist did not find any effects on aggregate size in various partial loss-of-function mutations of the UPS, including mutations in genes encoding 20S (DOA3) and 26S (SEN3/RPN2) proteasome subunits [27]. In this work, we showed that Htt aggregates did not appear to be toxic for cells unless sensitized by proteotoxic stress, such as growth in canavanine. Furthermore, Lin et al. (2013) showed that expressing Rpt6 S120A in mouse brain blocked the ability of a cAMP-elevating reagent to enhance proteasome activity, whereas the phosphomimetic Rpt6 mutant (Rpt6 S120D) increased proteasome activity, reduced HTT aggregates, and ameliorated motor impairment [82]. This further supports the idea that Rpt6 S120 phosphorylation is important for proteasome function under various stress conditions including protein aggregation.
We expected myc-rpt6-S120A mutants, which are significantly more sensitive to proteotoxic stress and have larger toxic Htt103Q aggregates (Figs 1 thru 3), to be more susceptible to chronological aging. However, viability was instead increased in myc-rpt6-S120A mutant strain compared to myc-rpt6 and myc-rpt6-S119D strains (Fig 4B). In part, this is reminiscent of the findings in neurodegenerative disease models, where persistence of a non-functional cell state can exhaust brain resources, causing longer time in a senescent state, until apoptosis is activated [83][84][85]. This result will be of interest to pursue as the possibility of an adaptive response involving activation of chaperones or autophagy remains. It is also possible that the senescence phenotypes observed here are distinct from the response to stress discussed above.
Together, our findings support the notion that Rpt6 phosphorylation at S119/S120 plays an important, conserved function in the ability of the proteasome to counteract proteotoxic stress and protein aggregate formation. Interestingly, the proteasome has been shown to be modified by other post-translational modifications [86-89, 89, 90]. Future studies will be important to help determine the mechanisms of how Rtp6 phosphorylation or other modifications alter 26S proteasome function in both normal and pathological states.
Supporting information S1 Fig. Overexpression of phosphomimetic rpt6-S120D promotes resistance to proteotoxic stress in the RPT6 null background. rpt6Δ null strains overexpressing RPT6 or rpt6-S120A were sensitive to ethanol 8%. All plasmids transformed in the experiment were 2μ. (PDF) S2 Fig. The rpt6-S119A mutant had increased susceptibility to proteotoxic stress. (A) Growth assays for all mutants challenged with elevated temperature or protein instability and DNA repair blocking agents. (B) WT and mutant strains were mated to WT yeast to produce diploids. Heterozygous diploid strains ( Ã ) had comparable growth to WT diploid yeast indicating that the rpt6 mutants are not dominant. (PDF) S3 Fig. Cell cycle profiles of myc-RPT6, myc-rpt6-S119A, and myc-rpt6-S119D strains were comparable. Flow cytometry analysis performed during logarithmic phase A 600 0.6 (30˚C and 34˚C) and stationary phase A 600 >5 (30˚C) revealed a similar delay in cell cycle progression in all myc-tagged strains. (PDF) S4 Fig. Strains myc-rpt6-S119A and myc-rpt6-S119D expressing Htt25Q had a similar loss of viability upon chronological aging. Strains were grown in liquid ura-medium at 30˚C and plated to asses viability after 1, 5, 7, 10, 12 and 15 days of growth. The graph is the average of three independent experiments performed with duplicate samples. Values were plotted relative to myc-RPT6 transformed with vector. (PDF) S1