The UBA2 Domain Functions As an Intrinsic Stabilization Signal That Protects Rad23 from Proteasomal Degradation

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Molecular Cell, Vol. 18, 225–235, April 15, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.03.015 The UBA2 Domain Functions as an Intrinsic Stabilization Signal that Protects Rad23 from Proteasomal Degradation

Stijn Heessen,1,2 Maria G. Masucci,1 Hershko and Ciechanover, 1998), but recent studies and Nico P. Dantuma1,3,* have revealed additional nonproteolytic functions in 1Microbiology and Tumor Biology Center DNA repair and transcription (Ferdous et al., 2001; Gon- Karolinska Institutet zalez et al., 2002; Russell et al., 1999). Nobels va¨ g 16 The presence of several characteristic structural do- S-171 77 Stockholm mains highlights the close relationship of Rad23 with Sweden the ubiquitin/proteasome system. Rad23 contains an 2 Ludwig Institute for Cancer Research N-terminal ubiquitin-like (UbL) domain and two ubiqui- Nobels va¨ g 3 tin-associated (UBA) domains: an internal UBA1 do- S-171 77 Stockholm main and a C-terminal UBA2 domain (Buchberger, Sweden 2002). The UbL domain, which can be functionally re- 3 Department of Cell and Molecular Biology placed by ubiquitin (Watkins et al., 1993), mediates Karolinska Institutet the interaction between Rad23 and the proteasome Nobels va¨ g3 (Schauber et al., 1998). The UBA domain was originally S-171 77 Stockholm identified as a sequence motif present in proteins Sweden linked to the ubiquitination system (Hofmann and Bucher, 1996). It was later reported that, notwithstand- ing their low sequence homology, all UBA domains share the ability to bind ubiquitin (Bertolaet et al., Summary 2001b; Chen et al., 2001; Wilkinson et al., 2001). Based on its ability to simultaneously bind ubiquitinated pro- The proteasome-interacting protein Rad23 is a long- teins and the proteasome, it has been proposed that lived protein. Interaction between Rad23 and the pro- Rad23 may function as a scaffold that facilitates in- teasome is required for Rad23’s functions in nu- teractions between substrates and the proteasome cleotide excision repair and ubiquitin-dependent (Chen and Madura, 2002; Kim et al., 2004). Recently, degradation. Here, we show that the ubiquitin-associ- direct biochemical evidence has been provided for a ated (UBA)-2 domain of yeast Rad23 is a cis-acting, role of Rad23 in targeting ubiquitinated substrates to transferable stabilization signal that protects Rad23 the proteasome (Elsasser et al., 2004; Saeki et al., from proteasomal degradation. Disruption of the UBA2 2002a; Verma et al., 2004). Several studies revealed that domain converts Rad23 into a short-lived protein that a concerted action of the ubiquitin/proteasome system is targeted for degradation through its N-terminal and Rad23 is important for nucleotide excision repair ubiquitin-like domain. UBA2-dependent stabilization (NER) although the precise mode of action of the pro- is required for Rad23 function because a yeast strain teasome in this event remains obscure (Russell et al., expressing a mutant Rad23 that lacks a functional 1999; Schauber et al., 1998). Interestingly, this DNA re- UBA2 domain shows increased sensitivity to UV light pair activity requires only the 19S regulatory particle of and, in the absence of Rpn10, severe growth defects. the proteasome and is independent of the proteolyti- The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and cally active 20S core particle (Gillette et al., 2001). the human Rad23 homolog hHR23A have similar pro- Substrates of the ubiquitin/proteasome system are tective activities. Thus, the UBA2 domain of Rad23 is targeted by degradation signals, which are small motifs an evolutionarily conserved stabilization signal that or domains that accommodate interaction with the allows Rad23 to interact with the proteasome without proteasome in a ubiquitin-dependent (Hershko and facing destruction. Ciechanover, 1998) or, less common, a ubiquitin-independent manner (Chen et al., 2004; Murakami et al., Introduction 1992). As a general rule, recruitment of proteins to the proteasome results in their rapid inactivation by pro- The Saccharomyces cerevisiae Rad23 protein is in- cessive degradation. However, the interaction between volved in regulation of nucleotide excision repair, pro- Rad23 and the proteasome does not lead to Rad23 de- teolysis and cell cycle progression (Clarke et al., 2001; struction (Schauber et al., 1998; Watkins et al., 1993). Lambertson et al., 1999; Watkins et al., 1993). While the We have previously described a similar phenomenon precise mode of action of Rad23 is unknown, increas- with a viral repetitive sequence that can be used for ing evidence suggests that a close interplay between cis-stabilization of proteasome substrates (Heessen et Rad23 and the ubiquitin/proteasome system is essen- al., 2002; Levitskaya et al., 1995; Sharipo et al., 1998). tial for its diverse functions (Chen and Madura, 2002; Based on these findings, we postulated that cellular Russell et al., 1999; Schauber et al., 1998). The ubiqui- proteins might contain similar protective “stabilization tin/proteasome system was originally identified as the signals” that spare them from proteolysis (Dantuma and primary degradation machinery in the cytosol and nu- Masucci, 2002). Here, we show that the C-terminal UBA cleus of eukaryotic cells (Baumeister et al., 1998; domains of Rad23, Dsk2, Ddi1, and Ede1 function as stabilization signals that can protect proteins from pro- *Correspondence: [email protected] teasomal degradation. Molecular Cell 226

Figure 1. Cis Stabilization of Proteasome Reporter Substrates by the UBA2 Domain (A) Schematic drawing of the fusion of the N-end rule reporter substrate Ub-R-GFP and Rad23 fragments. The locations of the UbL, UBA1, and UBA2 domains in Rad23 are indicated. ⌬UbL 3HA (B) Lysates of 10 OD600 units of log-phase yeast coexpressing Ub-R-GFP-Rad23 and ubiquitin were subjected to immunoprecipitation with an anti-GFP antibody, followed by Western blotting with an anti-HA antibody. Yeast cells transformed with an empty vector and 3HAubiqui- tin were used as negative control. The molecular weight markers, ubiquitinated species, and the immunoglobulin heavy chain (HC) are indicated. (C) Representative histograms of flow cytometric analysis of yeast expressing the Ub-M-GFP, Ub-R-GFP, or the Ub-R-GFP fusions shown in (A). The mean fluorescence intensity of each sample is indicated in the upper-right corner (corrected for background fluorescence of vector transformed yeast). (D) Quantification of mean fluorescence intensities of yeast cells expressing Ub-M-GFP, Ub-R-GFP, or the Ub-R-GFP fusion proteins measured by flow cytometry. Ub-M-GFP was standardized as 100%. Values are means and standard deviations of three independent experiments. Asterisks indicate values that are significantly different from Ub-R-GFP (t test, p < 0.05). (E) Representative histograms of flow cytometric analysis of yeast cells expressing UbG76V-GFP or UbG76V-GFP-Rad23⌬UbL. UBA2 Is an Intrinsic Stabilization Signal 227

Figure 2. An Intact UBA2 Domain Protects from Proteasomal Degradation and Does Not Cause General Impairment of Proteasomal Degra- dation (A) Western blot analysis of turnover of Ub-R-GFP, Ub-R-GFP-UBA1, and Ub-R-GFP-UBA2 levels in wild-type yeast and Ub-R-GFP in the ubr1D strain at 0, 10, 20, and 40 min after promoter shutoff. (B) Densitometric quantification of the Ub-R-GFP (closed circles) and Ub-R-GFP-UBA2 (open circles) signal in (A). The signal at t = 0 is standardized as 100%. (C) Mean fluorescence intensities of yeast expressing Ub-R-GFP-UBA1, Ub-R-GFP-UBA2, and Ub-R-GFP-UBA2L392A. Mean and standard deviations of three independent experiments are shown. Asterisk indicates values that are significantly different from Ub-R-GFP-UBA1 (t test, p < 0.01). (D) Western blot analysis with specific GFP antibody of Ub-R-GFP and Ub-R-GFP-Rad23⌬UbL levels at 0, 10, 20, and 40 min after switching off the GAL1 promoter in yeast expressing Ub-R-GFP alone or together with Ub-R-GFP-Rad23⌬UbL. Putative ubiquitinated species (asterisks) and molecular weight markers are indicated.

Results somal degradation. For this purpose, we analyzed whether insertion of Rad23 fragments in green fluores- Cis Stabilization of Proteasome Reporter cent protein (GFP)-based substrates of the ubiquitin/ Substrates by the UBA2 Domain proteasome system could inhibit their degradation (Fig- The remarkable stability of the proteasome-interacting ure 1A). These GFP-based substrates are targeted to protein Rad23 prompted us to investigate whether this the proteasome through the presence of well-defined protein may harbor domains that protect it from protea- degradation signals that deem the GFP for ubiquitin- Molecular Cell 228

Table 1. Yeast Strains Used in this Study proteasome system is responsible for degradation of this substrate in yeast (Bartel et al., 1990). Thus, the Strain Genotype Reference UBA2 domain increases the steady-state levels of DF5 MATa lys2-801 leu2-3, 112 ura3-52 Finley et al. Ub-R-GFP by delaying its proteasomal degradation. his3-⌬200 trp1-1 Structural analysis of the UBA2 domains of the hu- BBY47 DF5 ubr1D::LEU2 Bartel et al. man homolog of Rad23 A (hHR23A) has revealed that a SHY002 DF5 rad23D::kanMX-KlURA3 this study conserved leucine corresponding with leucine 392 in SHY003 DF5 FLAG-RAD23 this study SHY004 DF5 FLAG-rad23(L392A) this study yeast Rad23 is important for the structural integrity of SHY012 DF5 rpn10D::HIS3 this study the characteristic UBA fold (Mueller and Feigon, 2002). SHY013 DF5 rad23D::kanMX-KlURA3 this study Substitution of this single amino acid with an alanine rpn10D::HIS3 was sufficient to completely abrogate the protective ef- SHY014 DF5 FLAG-RAD23 rpn10D::HIS3 this study fect, suggesting that a proper UBA fold is important for SHY015 DF5 FLAG-rad23(L392A) rpn10D::HIS3 this study the inhibitory activity (Figure 2C).

The UBA2 domain Does Not Cause General dependent proteolysis (Dantuma et al., 2000b). Be- Impairment of the Ubiquitin/Proteasome System cause it is well established that the UbL domain of It has been reported that UBA domains can act as Rad23 mediates interaction with the proteasome, we general inhibitors of ubiquitination (Ortolan et al., 2000) omitted the UbL domain to assure that only the degra- and deubiquitination (Hartmann-Petersen et al., 2003). dation signal of the reporter substrate would target the Thus, stabilization of the GFP reporter could be the fusion to the proteasome. consequence of a general impairment of the ubiquitin/ ⌬ Introduction of the Rad23 UbL did not interfere with proteasome system caused by the overexpressed cleavage of the N-terminal ubiquitin of ubiquitin-R-GFP UBA2 domain. To probe into this possibility, we exam- (Ub-R-GFP; data not shown), which is required for acti- ined whether overexpression of the stable Ub-R-GFP- vation of the N-end rule degradation signal in this re- Rad23⌬UbL fusion protein led to a general accumulation porter substrate (Varshavsky, 1996), nor did it affect of proteasome substrates. Ub-R-GFP-Rad23⌬UbL and targeting of the Ub-R-GFP for ubiquitination because the Ub-R-GFP or UbG76V-GFP substrates were coex- we could readily detect ubiquitinated reporter sub- pressed in yeast and their degradation was followed ⌬ strates (Figure 1B). Insertion of the Rad23 UbL in the after simultaneously switching off expression of both Ub-R-GFP reporter resulted in a 14-fold increase in the proteins. Overexpression of Ub-R-GFP-Rad23⌬UbL did mean fluorescent intensity of the reporter-expressing not affect the turnover of Ub-R-GFP (Figure 2D) or yeast to approximately 50% of the level observed with UbG76V-GFP (data not shown). Furthermore, while gene- the stable Ub-M-GFP that lacks a degradation signal ral impairment of the ubiquitin/proteasome system re- (Figures 1C and 1D). To identify the region in Rad23 that sults in accumulation of ubiquitinated substrates and was responsible for this effect, we produced a set of induction of cell cycle arrest and apoptosis (Dantuma ⌬ C-terminal deletions of the Ub-R-GFP-Rad23 UbL fu- et al., 2000b), overexpression of Ub-R-GFP-Rad23⌬UbL sion protein (Figure 1A). Removal of the C-terminal did not increase the total pool of ubiquitin-conjugates UBA2 domain brought the fluorescence intensity al- nor did it affect proliferation (data not shown). We con- most back to the Ub-R-GFP level (Figures 1C and 1D). clude that insertion of the Rad23 UBA2 domain can se- Insertion of the UBA2 domain was sufficient to recapit- lectively protect proteasome substrates carrying the ⌬ ulate the effect observed with Rad23 UbL, whereas the UBA2 domain from degradation without disturbing related UBA1 domain alone had no appreciable effect overall proteasomal degradation. on the mean fluorescent intensity (Figures 1C and 1D). To test whether the stabilizing effect of UBA2 was The UBA2 Domain Functions as an Intrinsic dependent on the type of degradation signal, we ana- Stabilization Signal in Rad23 lyzed the effect of the UBA2 domain on a reporter sub- In order to explore whether the UBA2 domain plays a strate carrying an ubiquitin fusion degradation (UFD) protective role in the context of native Rad23, we took G76V signal, Ub -GFP. Of note, N-end rule and UFD sub- advantage of the single L392A amino acid substitution strates are targeted through distinct ubiquitination that abrogated the protective effect of the UBA2 do- pathways for degradation by the proteasome (Bartel et main. Introduction of this amino acid substitution con- al., 1990; Johnson et al., 1995; Koegl et al., 1999). We verted Rad23 from a very stable into a short-lived pro- found a very similar protective effect on the UFD repor- tein with a half-life of approximately 10 min (Figures 3A ter substrate, suggesting that the effect is largely inde- and 3B). Because mutations can result in rapid degra- pendent of the type of degradation signal (Figure 1E). dation due to misfolding, it was important to assess whether the Rad23L293A was targeted for degradation An Intact UBA2 Domain Is Required for Protection through a misfolded UBA2L392A domain or through an from Proteasomal Degradation endogenous targeting signal that is also functional in Promoter shutoff experiments demonstrated that the the context of native Rad23. We argued that the UbL increased steady-state levels of UBA2-containing sub- domain could fulfill this role as it has been shown to strates were due to delayed turnover of the reporter mediate the interaction of Rad23 with the proteasome while the UBA1 domain had no effect on degradation (Schauber et al., 1998). Deletion of the UbL domain re- (Figures 2A and 2B). The Ub-R-GFP reporter was stable sulted in full stabilization of the mutant Rad23L392A (Fig- in the ubr1D yeast strain, which lacks the N-end rule ures 3A and 3B), excluding the possibility that the ubiquitin ligase (Table 1), confirming that the ubiquitin/ UBA2L392A domain targets the mutant Rad23 for degra- UBA2 Is an Intrinsic Stabilization Signal 229

Figure 3. The UBA2 Domain Functions as an Intrinsic stabilization Signal in Rad23 (A) Turnover of FLAGRad23, FLAGRad23L392A, and FLAGRad23⌬UbL/L392A after promoter shutoff. Samples were collected at the indicated time points after shutting off the GAL1 promoter and analyzed in anti-FLAG Western blot analysis. (B) Densitometric quantification of Western blot shown in (A). FLAGRad23 (closed circles), FLAGRad23L392A (open circles), and FLAGRad23⌬UbL/ L392A (squares). The signal att=0foreach of the constructs was standardized as 100%. (C) Steady-state levels of FLAG-tagged Rad23 in the wild-type, rad23D, FLAGRad23, and FLAGRad23L392A yeast strains determined in an anti- FLAG Western blot on 0.25 OD600 units. FLAGRad23 and FLAGRad23L392A are expressed from the endogenous Rad23 promoter. (D) Turnover of FLAGRad23 and FLAGRad23L392A was determined by metabolic labeling with 35S-methionine/cysteine followed by chasing within the presence of an excess cold methionine and cysteine for the indicated times. FLAGRad23 and FLAGRad23L392A were immunoprecipitated with anti-FLAG antibodies. Asterisks indicate a nonspecific band. dation. This shows that UbL domain is important for FLAGRad23L392A mutant was below the detection limit targeting Rad23 to the proteasome (Schauber et al., (Figure 3C). Pulse-chase analysis revealed that this 1998) and, in the absence of the protective UBA2 do- striking difference in steady-state levels was due to ac- main, for induction of Rad23 proteolysis. celerated degradation of the Rad23L392A mutant (Figure Three yeast strains were generated to gain insight in 3D). We conclude that the UBA2 domain is important the role and functional significance of the UBA2 domain for physiological expression levels of Rad23 from its under physiological conditions: (1) a rad23D strain, endogenous promoter. which lacks the Rad23 gene, (2) a FLAGRad23 strain, and (3) a FLAGRad23L392A strain, which express FLAG- Rad23 Lacking a Protective UBA2 Domain tagged wild-type Rad23 and FLAG-tagged mutant Is Functionally Compromised Rad23L392A, respectively, from the endogenous Rad23 It has been reported that the UBA1 and UBA2 domains promoter (Table 1). The FLAGRad23 could easily be de- of Rad23 are dispensable for functional NER (Bertolaet tected in Western blot analysis, whereas the level of the et al., 2001b). Consistent with this earlier report, we Molecular Cell 230

Figure 4. Rad23 Lacking a Protective UBA2 Domain Is Functionally Compromised (A) UV sensitivity of the wild-type, rad23⌬, FLAGRad23, and FLAGRad23L392A yeast strains. Serial dilutions of yeast cultures were spotted on YPD agar and were left untreated or exposed to 300 J/m2 UV light. (B) UV sensitivity of the wild-type, rad23⌬, FLAGRad23, and FLAGRad23L392A yeast strains. Serial dilutions of yeast cultures were spotted on YPD agar and were left untreated or exposed to one, two, or three exposures of 100 J/m2 UV light with 20 min intervals between each exposure. (C) Quantification of UV sensitivity by colony survival assay. Yeast was exposed to 3 × 100 J/m2 or left untreated. The numbers were standardized to untreated controls. Mean and standards deviation of triplicate measurements are shown. Asterisks indicate that values are significantly different from wild-type strain (t test, p < 0.02). (D) Serial dilutions of the indicated yeast cultures were spotted on YPD agar and were grown at 30°C or 25°C. found that yeast cells expressing the destabilized more dramatic effect on cell survival was revealed FLAGRad23L392A were only slightly affected in their abil- when the yeast was subjected to three subsequent UV ity to survive a 300 J/m2 UV light exposure, whereas exposures of 100 J/m2 with 20 min intervals. The 1st UV rad23D cells were highly sensitive to this UV dose (Fig- exposure had a strong inhibitory effect on the growth ure 4A). This suggests that the reduced Rad23 levels in of the rad23D strain, whereas the FLAGRad23 and the FLAGRad23L392A strain are still sufficient for NER. A FLAGRad23L392A strains coped equally well (Figure 4B). UBA2 Is an Intrinsic Stabilization Signal 231

of Rad23 as multiubiquitin chain receptor engaged in the targeting of substrates to the proteasome, a notion that was recently supported by detailed biochemical studies (Verma et al., 2004). We investigated the significance of the protective UBA2 domain in relation to this function of Rad23. Deletion of Rad23 and Rpn10 resulted indeed in slow growth, in particular at 25°C (Fig- ure 4D). Whereas introduction of the FLAGRad23 re- stored the growth to wild-type levels, the destabilized FLAGRad23L392A did not rescue the growth phenotype of the rad23D/rpn10D double mutant. Thus, the presence of a protective UBA2 domain is of critical significance for Rad23 function.

The C-Terminal UBA Domains of Dsk2, Ddi1, and Ede1 Act as Stabilization Signals Dsk2 and Ddi1 share with Rad23 the presence of an N-terminal UbL domain and a C-terminal UBA domain (Figure 5A). In addition, some proteins such as Ede1, a protein engaged in membrane trafficking (Aguilar et al., 2003), carry a C-terminal UBA domain but lack an UbL domain (Figure 5A). In order to examine whether the UBA domains of these proteins share the protective ca- pacity of Rad23’s UBA2 domain, they were fused to the C terminus of the Ub-R-GFP reporter. Flow cytometric analysis clearly demonstrated a significant increase in the steady-state fluorescence levels of the chimeric re- porters (Figure 5B). Most striking was the effect of the UBA domain of Dsk2, which even exceeded the effect of the Rad23 UBA2 domain, while the Ddi1 and Ede1 had weaker effects. Analysis of the turnover of these reporter constructs confirmed that the increase in the steady-state levels was due to a delay in degradation (Figure 5C). We conclude that the stabilizing effect of the C-terminal UBA domain is not unique for Rad23 but Figure 5. C-terminal UBA Domains of Dsk2, Ddi1, and Ede1 Can is a more general phenomenon shared with at least Protect from Proteasomal Degradation three other UBA domains. (A) Schematic representation of the UbL and UBA domains in Rad23, Ddi1, Dsk2, and Ede1. The Protective Effect of the UBA2 Domain (B) Mean fluorescence intensities of yeast expressing Ub-R-GFP, Is Evolutionarily Conserved Ub-R-GFP-UBA2(Rad23), Ub-R-GFP-UBA(Dsk2), Ub-R-GFP-UBA To examine whether the protective effect of the UBA2 (Ddi1), and Ub-R-GFP-UBA(Ede1). Mean and standard deviations of three independent experiments are shown. Asterisks indicate domain has been conserved during evolution, we next values that are significantly different from Ub-R-GFP: *(t test, p < tested the activity of the UBA1 and UBA2 domains of 0.02), **(t test, p < 0.01). the human homolog hHR23A. We found that the UBA2 (C) Western blot analysis with specific GFP antibody of Ub-R-GFP, domains of Rad23 and hHR23A but not their UBA1 Ub-R-GFP-UBA(Dsk2), Ub-R-GFP-UBA(Ddi1), and Ub-R-GFP- counterparts are equally capable of stabilizing the Ub- UBA(Ede1) levels at 0, 10, 20, and 40 min after switching off the R-GFP reporter in yeast (Figure 6A). In order to assess GAL1 promoter. whether the protective effect also operates in mamma- lian cells, we used a previously described assay in which human cervix carcinoma HeLa cells are tran- However, a clear difference was observed in the sur- siently transfected with these fusions and the percen- vival of the FLAGRad23L392A strain after the second and tage of fluorescent cells is determined in the absence third UV exposures, with increased sensitivity to UV ir- or presence of a specific proteasome inhibitor (Dan- radiation with each exposure (Figures 5B and 5C). Thus, tuma et al., 2000a). Introduction of the UBA1 domain of although the short-lived FLAGRad23L392A appeared to be Rad23 or hHR23A in Ub-R-GFP did not affect the sta- sufficient for UV resistance after a single UV exposure, bility of this reporter in HeLa cells, whereas the UBA2 it was not capable of rescuing yeast from multiple domains inhibited degradation (Figures 6B and 6C). We rounds of UV exposure. conclude that the stabilizing effect of the UBA2 domain Yeast strains lacking both Rad23 and the ubiquitin in Rad23 is conserved during evolution. binding proteasome subunit Rpn10 display impaired degradation of proteasome substrates, accumulation Discussion of ubiquitinated proteins, and growth retardation that is most striking at low temperatures (Lambertson et al., We have shown that the UBA2 domain of Rad23 act as 1999). These findings are in line with a possible function an intrinsic, cis-acting, transferable stabilization signal. Molecular Cell 232

Thus, Rad23 harbors counteracting degradation and stabilization signals, which enable the protein to interact with the proteasome without being degraded. UBA2-dependent stabilization is required for Rad23 function in DNA repair and protein degradation. Our data show that protein stabilization is a characteristic shared by at least four C-terminally positioned UBA domains present in Rad23, Dsk2, Ddi1, and Ede1. Rad23, Dsk2, and Ddi1 each contain an N-terminal proteasome-interacting UbL domain and a C-terminal UBA domain (Jentsch and Pyrowolakis, 2000), whereas Ede1 lacks the UbL domain. Whether Ede1, which is the yeast homolog of eps15 and involved in ubiquitin- dependent membrane trafficking (Aguilar et al., 2003), interacts with the proteasome awaits clarification. Rad23, Dsk2, Ddi1, and Ede1 are to our knowledge the first examples of cellular proteins that harbor a specific domain that enables proteins to resist degradation. It remains to be seen how widespread this phenomenon is, but there are several findings in the literature suggesting that other proteins may contain related and un- related stabilization signals (Dantuma and Masucci, 2002). For instance, a truncated variant of the ataxin 1-interacting ubiquitin-like protein lacking the UBA domain was also found to be degraded by the ubiquitin/ proteasome system (Riley et al., 2004). How do C-terminal UBA domains protect from degradation? There are indications that UBA domains do not only bind ubiquitin but also UbL domains (Raasi and Pickart, 2003; Wang et al., 2003). Based on these findings, it has been proposed that intramolecular interactions between the UbL and UBA domains may give Rad23 a closed conformation that cannot bind to the proteasome (Madura, 2002; Wang et al., 2003). Others have proposed that UBA domains can inhibit elongation of polyubiquitin chains by capping conjugated ubiquitin (Chen et al., 2001; Ortolan et al., 2000). Al- though either UbL binding or inhibition of polyubiquiti- nation provides possible explanations for the stabilizing effect of the C-terminal UBA domain, there are several observations that do not support these models. First, Figure 6. Protective Effect of the UBA2 Domain Is Evolutionarily the ubiquitin binding activity of the UBA domains is not Conserved sufficient for the protective effect since the very similar (A) Mean fluorescence intensities of yeast expressing Ub-M-GFP, UBA1 domains of Rad23 and hHR23A, that bind ubiqui- Ub-R-GFP, or Ub-R-GFP fused to the UBA1 or UBA2 domain of tin and block ubiquitination equally well as UBA2 do- yeast Rad23 and human hHR23A. Mean fluorescent intensity of Ub- mains (Bertolaet et al., 2001b; Chen et al., 2001; Rao M-GFP-expressing yeast was standardized as 100%. Means and and Sastry, 2002), lacked this protective activity. Sec- standard deviations of three independent experiments are shown. ond, the presence of the UBA domain did not have any Asterisks indicate values significantly different from Ub-R-GFP (t test, p < 0.05). striking effects on the handling of ubiquitin on the sub- (B) Dot plots of flow cytometric analysis of HeLa cells transiently strate: the N-terminal ubiquitin was efficiently cleaved transfected with the indicated constructs. Cells were left untreated of the Ub-R-GFP to activate the N-end rule degradation ␮ or incubated with 10 M of the proteasome inhibitor Z-L3-VS for 8 signal and the reporter was found to be polyubiquiti- hr. The percentage of GFP-positive cells in each sample is indi- nated. Third, Madura and colleagues previously re- cated in the upper right corner. Values from one representative ex- ported that C-terminal tagging of Rad23 dramatically periment out of three are shown. affected its half-life and resulted in rapid proteasomal (C) Quantitative analysis of flow cytometric analysis depicted in (B). Relative fluorescence is expressed as the percentage of GFP posi- degradation (Schauber et al., 1998). We have obtained tive cells in the absence of inhibitor divided by the percentage of preliminary data suggesting that a C-terminal extension GFP positive cells in the presence of inhibitor. Means and standard disturbs the protective effect of the UBA2 domain (un- deviations from three independent experiments are shown. Aster- published data). It seems unlikely that short motifs isks indicate values significantly different from Ub-R-GFP (t test, lacking any secondary structure would interfere with p < 0.05). UBA interactions since both internal and C-terminal UBA domains were found to bind ubiquitin in the context of the native protein (Bertolaet et al., 2001b). Fourth, the interaction between Rad23 and the protea- UBA2 Is an Intrinsic Stabilization Signal 233

some has been studied in much detail (Elsasser et al., can induce DNA damage-independent responses (Bryan 2002; Kim et al., 2004; Saeki et al., 2002b; Schauber et et al., 2004), it cannot be excluded that this unique phe- al., 1998), leaving little doubt about the fact that Rad23 notype is caused by a combination of reduced NER ac- does interact with the proteasome in vivo. tivity and reduced substrate targeting activity as a con- A possibility that cannot be excluded is masking of sequence of the low Rad23 levels. polyubiquitin chains by the UBA2 domain. Thus the Our study identifies C-terminal UBA domains as cel- UBA2 domain could bind to polyubiquitin chains conju- lular stabilization signals and the primary element that gated to Rad23, thereby modifying the interaction be- enables Rad23 and hHR23A to interact with the protea- tween Rad23 to the proteasome in such a way that it some without facing destruction. An attractive hypoth- does not promote degradation. It remains unclear esis is that Rad23 by acquiring the UBA2 domain has whether Rad23 itself is a target for ubiquitination, but evolved from a proteasome substrate into a privileged the fission yeast homolog Rph23 was shown to be protected “substrate” that assists the ubiquitin/protea- polyubiquitinated (Elder et al., 2002). In disfavor of this some system as a dedicated proteasome scaffold in model, it was recently shown that the UBA1 and UBA2 DNA repair and substrate targeting. domains bind synthetic Lys48 tetraubiquitin chains equally well (Raasi and Pickart, 2003), but more de- Experimental Procedures tailed analyses on the in vivo preferences of UBA domains for different polyubiquitin chains is required to Yeast Strains and Plasmids All strains used in this work are listed in Table 1 and are derivatives conclusively address this issue. of DF5. Correct integration of gene cassettes was confirmed by Our study shows that the structural integrity of the whole-locus PCR analysis. SHY002 was constructed by replacing UBA2 domain is an essential constraint for the protec- the entire RAD23 open reading frame (ORF) by transforming DF5 tive effect. An attractive hypothesis that could explain with a PCR product encompassing the kanMX4-KlURA3 cassette these results is that the C-terminal UBA2 domain hin- from pCORE (Storici et al., 2001). Ura+ G418R clones were isolated ders unfolding of proteasome-bound Rad23. Prakash and UV sensitivity was confirmed. To obtain SHY003, the SHY002 and coworkers have recently shown that degradation strain was transformed with a FLAG-RAD23 PCR fragment from pYES3/CT-FLAG-RAD23 deleting the kanMX4-KlURA3 cassette, signals require an unstructured initiation site for effi- giving rise to 5-fluorootic acid-resistant (5-FOAR) and G418S cient unfolding and proteasomal degradation (Prakash clones. SHY004 was created similarly by transforming SHY002 with et al., 2004). Unfolding is a rate-limiting and crucial step a FLAG-RAD23L392A PCR product amplified from pYES3/CT-FLAG- in the sequence of events that leads to degradation of RAD23L392A. RPN10D strains SHY012-15 were generated by re- ubiquitinated substrates (Thrower et al., 2000), and in placement of the complete RPN10 ORF with an HIS3 containing vitro studies have shown that tightly folded C-terminal cassette that was PCR amplified from pRS313. The FLAGRad23 open reading frame and fragments were PCR am- domains can delay or block proteasomal degradation plified from pCS13-FLAGRad23 (gift from Dr. Madura, Robert Wood (Navon and Goldberg, 2001). Disturbing the UBA2 fold Johnson Medical School, Piscataway, NJ) and subcloned under the may enable the proteasome to unfold and degrade regulation of a GAL1 promoter in the yeast expression vector Rad23. This would also explain the intriguing observa- pYES2 (Invitrogen, 2 ␮ URA3) or pYES3/CT (Invitrogen, 2 ␮ TRP1). tion that simple C-terminal tagging can destabilize The UBA domains of Dsk2, Ddi1, and Ede1 were PCR amplified Rad23 (Schauber et al., 1998) because extending the from genomic yeast DNA. The hHR23A UBA1 (codons 159–200) UBA2 domain with unstructured epitope tags may pro- and UBA2 (codons 317–357) domains were PCR-amplified from total HEK293T cell cDNA. vide the proteasome with a “handle” to unwind the UBA domain. Notably, it has been hypothesized that pro- Flow Cytometry cessing domains, i.e., domains that interrupt proteaso- HeLa cells transiently transfected with lipofectamine were treated mal degradation of a precursor protein at a specific 8 hr with 10 ␮M of the proteasome inhibitor carboxybenzyl-leucyl- point (Rape et al., 2001), may mediate protein-protein leucyl-leucine vinyl sulfone (Z-L3VS, gift from Dr. Ploegh, Harvard interactions that terminate degradation by forming sta- Medical School, Boston, MA) and subjected to flow cytometric ble structures (Rape and Jentsch, 2002). UBA domains analysis on a fluorescent activated cell sorter (Beckton & Dick- inson). can interact with each other (Bertolaet et al., 2001a), and a similar mechanism may therefore apply for stabi- Western Blot Analysis and Immunoprecipitations lization by UBA domains. Western blots of total protein extracts were probed with mouse We have shown that yeast expressing destabilized monoclonal anti-FLAG (M5, Sigma-Aldrich), anti-HA antibody (Boeh- Rad23 can resist a high, single dose of UV exposure, ringer-Mannheim), anti-HA ascites, and rabbit polyclonal anti-GFP an- despite extremely low Rad23 levels. This observation is tibody (Molecular Probes Europe). For immunoprecipitations, yeast consistent with earlier reports showing that deletion of was lysed with acid-washed glass beads in HEPES lysis buffer (50 the UBA2 domain does not affect UV sensitivity after a mM HEPES, 5mM EDTA, 1% TritonX-100, 150 mM NaCl), supple- mented with 20mM NEM (Sigma-Aldrich) and 0.5 ␮M epoxomycin single UV exposure (Bertolaet et al., 2001b; Chen and (Affinity Research Products). For anti-FLAG immunoprecipitations, Madura, 2002). Apparently cells contain excess amounts sepharose beads with conjugated M2 anti-FLAG monoclonal anti- of Rad23. This may be related to the emerging role of body were used (M2-Sepharose, Sigma). Rad23 in the targeting of substrates for ubiquitin/proteasome-dependent proteolysis (Verma et al., 2004). Turnover Analysis Even more remarkable is the increased sensitivity of For GAL1 promoter shutoff experiments, transcription and transla- yeast expressing Rad23L392A to repeated UV expo- tion were arrested by adding glucose and cycloheximide (Sigma) to final concentrations of 2% and 0.5 mg/ml, respectively. For en- sures. Conceivably, NER lesions may induce Rad23- dogenously expressed proteins, only cycloheximide was added to proteasome interactions and cause a further depletion the growth media (final concentration 100 ␮g/ml). Aliquots were taken of the already low levels of Rad23 in this strain. Be- at the indicated time points, and total protein extracts were pre- cause UV exposure also causes proteotoxic stress and pared by NaOH lysis and TCA precipitation. Samples were heated Molecular Cell 234

10 min at 65°C and subjected to SDS-PAGE before Western blot Chen, L., Shinde, U., Ortolan, T.G., and Madura, K. (2001). Ubiqui- analysis with the appropriate antibodies. Pulse-chase analyses tin-associated (UBA) domains in Rad23 bind ubiquitin and promote were performed by metabolic labeling of 6 OD600 units of yeast inhibition of multi-ubiquitin chain assembly. EMBO Rep. 2, 933– with 100 ␮C 35S-methionine/35S-cysteine (Redivue ProMix, Amer- 938. sham) for 10 min and chasing in the presence of 10 mM cold methi- Chen, X., Chi, Y., Bloecher, A., Aebersold, R., Clurman, B.E., and onine and 1 mM cold cysteine for the indicated time. Yeast was Roberts, J.M. (2004). N-acetylation and ubiquitin-independent pro- washed, lysed with acid-washed glass beads, and the proteins of teasomal degradation of p21(Cip1). Mol. Cell 16, 839–847. interest were immunoprecipitated from the lysate. Clarke, D.J., Mondesert, G., Segal, M., Bertolaet, B.L., Jensen, S., Wolff, M., Henze, M., and Reed, S.I. (2001). Dosage suppressors of UV Survival Assays pds1 implicate ubiquitin-associated domains in checkpoint control. For drop assays, yeast cells were resuspended in sterile water and Mol. Cell. Biol. 21, 1997–2007. serial 10-fold dilutions were spotted as 1.5 ␮l drops on YPD plates. Plates were irradiated with a single dose of 300 J/m2 or a maximum Dantuma, N.P., Heessen, S., Lindsten, K., Jellne, M., and Masucci, of 3× 100 J/m2 254 nm UV light using the Stratalinker UV crosslinker M.G. (2000a). Inhibition of proteasomal degradation by the Gly-Ala (Stratagene). All plates were wrapped in aluminum foil and kept in repeat of Epstein-Barr virus is influenced by the length of the re- the dark both during the 20 min recovery intervals and after the peat and the strength of the degradation signal. Proc. Natl. Acad. final UV exposure. Plates were photographed after 2 days of Sci. USA 97, 8381–8385. growth at 30°C. For quantitative survival analyses, yeast cells were Dantuma, N.P., Lindsten, K., Glas, R., Jellne, M., and Masucci, M.G. diluted in a 24-well plate before serial exposures to 100 J/m2 UV (2000b). Short-lived green fluorescent proteins for quantifying with 20 min intervals. After the final exposure, the yeast was evenly ubiquitin/proteasome-dependent proteolysis in living cells. Nat. spread out over YPD plates and the number of colonies was deter- Biotechnol. 18, 538–543. mined after growth at 30°C for 2 days in the dark. Dantuma, N.P., and Masucci, M.G. (2002). Stabilization signals: a novel regulatory mechanism in the ubiquitin/proteasome system. FEBS Lett. 529, 22–26. Acknowledgments Elder, R.T., Song, X.Q., Chen, M., Hopkins, K.M., Lieberman, H.B., and Zhao, Y. (2002). Involvement of rhp23, a Schizosaccharomyces We thank Per Ljungdahl for technical advise and support; Kiran pombe homolog of the human HHR23A and Saccharomyces cere- Madura, Hidde Ploegh, Alex Varshavsky, Sigurd Braun, and Stefan visiae RAD23 nucleotide excision repair genes, in cell cycle control Jentsch for generous gifts of reagents and advice; Marianne Jellne and protein ubiquitination. Nucleic Acids Res. 30, 581–591. for excellent technical support; Kristina Lindsten, Lisette Verhoef, Victoria Menéndez-Benito, Florian Salomons, Claes Andréasson, Elsasser, S., Chandler-Militello, D., Muller, B., Hanna, J., and Finley, Steven Bergink, Wim Vermeulen, and Cecile Pickart for stimulating D. (2004). Rad23 and Rpn10 serve as alternative ubiquitin receptors discussions and useful comments. This work was supported by for the proteasome. J. Biol. Chem. 279, 26817–26822. grants awarded by the Swedish Research Council (N.P.D.), Swedish Elsasser, S., Gali, R.R., Schwickart, M., Larsen, C.N., Leggett, D.S., Cancer Society (N.P.D. and M.G.M.), Swedish Foundation for Strat- Muller, B., Feng, M.T., Tubing, F., Dittmar, G.A., and Finley, D. (2002). egy Research (M.G.M.) and the Karolinska Institute. N.P.D. is sup- Proteasome subunit Rpn1 binds ubiquitin-like protein domains. ported by a fellowship from the Swedish Research Council. S.H. Nat. Cell Biol. 4, 725–730. has been supported in part by a grant from the Ludwig Institute for Ferdous, A., Gonzalez, F., Sun, L., Kodadek, T., and Johnston, S.A. Cancer Research. (2001). The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II. Mol. Received: October 8, 2004 Cell 7, 981–991. Revised: January 11, 2005 Gillette, T.G., Huang, W., Russell, S.J., Reed, S.H., Johnston, S.A., Accepted: March 21, 2005 and Friedberg, E.C. (2001). The 19S complex of the proteasome Published: April 14, 2005 regulates nucleotide excision repair in yeast. Genes Dev. 15, 1528–1539. References Gonzalez, F., Delahodde, A., Kodadek, T., and Johnston, S.A. (2002). Recruitment of a 19S proteasome subcomplex to an acti- Aguilar, R.C., Watson, H.A., and Wendland, B. (2003). The yeast vated promoter. Science 296, 548–550. Epsin Ent1 is recruited to membranes through multiple independent interactions. J. Biol. Chem. 278, 10737–10743. Hartmann-Petersen, R., Hendil, K.B., and Gordon, C. (2003). Ubiq- uitin binding proteins protect ubiquitin conjugates from disassem- Bartel, B., Wunning, I., and Varshavsky, A. (1990). The recognition bly. FEBS Lett. 535, 77–81. component of the N-end rule pathway. EMBO J. 9, 3179–3189. Heessen, S., Leonchiks, A., Issaeva, N., Sharipo, A., Selivanova, G., Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. (1998). The pro- Masucci, M.G., and Dantuma, N.P. (2002). Functional p53 chimeras teasome: paradigm of a self-compartmentalizing protease. Cell 92, containing the Epstein-Barr virus Gly-Ala repeat are protected from 367–380. Mdm2- and HPV-E6-induced proteolysis. Proc. Natl. Acad. Sci. Bertolaet, B.L., Clarke, D.J., Wolff, M., Watson, M.H., Henze, M., USA 99, 1532–1537. Divita, G., and Reed, S.I. (2001a). UBA domains mediate protein- Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. protein interactions between two DNA damage-inducible proteins. Annu. Rev. Biochem. 67, 425–479. J. Mol. Biol. 313, 955–963. Hofmann, K., and Bucher, P. (1996). The UBA domain: a sequence Bertolaet, B.L., Clarke, D.J., Wolff, M., Watson, M.H., Henze, M., motif present in multiple enzyme classes of the ubiquitination path- Divita, G., and Reed, S.I. (2001b). UBA domains of DNA damage- way. Trends Biochem. Sci. 21, 172–173. inducible proteins interact with ubiquitin. Nat. Struct. Biol. 8, 417– 422. Jentsch, S., and Pyrowolakis, G. (2000). Ubiquitin and its kin: how Bryan, B.A., Knapp, G.S., Bowen, L.M., and Polymenis, M. (2004). close are the family ties? Trends Cell Biol. 10, 335–342. The UV response in Saccharomyces cerevisiae involves the mito- Johnson, E.S., Ma, P.C., Ota, I.M., and Varshavsky, A. (1995). A pro- gen-activated protein kinase Slt2p. Curr. Microbiol. 49, 32–34. teolytic pathway that recognizes ubiquitin as a degradation signal. Buchberger, A. (2002). From UBA to UBX: new words in the ubiqui- J. Biol. Chem. 270, 17442–17456. tin vocabulary. Trends Cell Biol. 12, 216–221. Kim, I., Mi, K., and Rao, H. (2004). Multiple interactions of Rad23 Chen, L., and Madura, K. (2002). Rad23 promotes the targeting of suggest a mechanism for ubiquitylated substrate delivery impor- proteolytic substrates to the proteasome. Mol. Cell. Biol. 22, tant in proteolysis. Mol. Biol. Cell 15, 3357–3365. 4902–4913. Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D., Mayer, T.U., and UBA2 Is an Intrinsic Stabilization Signal 235

Jentsch, S. (1999). A novel ubiquitination factor, E4, is involved in Varshavsky, A. (1996). The N-end rule: functions, mysteries, uses. multiubiquitin chain assembly. Cell 96, 635–644. Proc. Natl. Acad. Sci. USA 93, 12142–12149. Lambertson, D., Chen, L., and Madura, K. (1999). Pleiotropic de- Verma, R., Oania, R., Graumann, J., and Deshaies, R.J. (2004). fects caused by loss of the proteasome-interacting factors Rad23 Multiubiquitin Chain Receptors Define a Layer of Substrate Selec- and Rpn10 of Saccharomyces cerevisiae. Genetics 153, 69–79. tivity in the Ubiquitin-Proteasome System. Cell 118, 99–110. Levitskaya, J., Coram, M., Levitsky, V., Imreh, S., Steigerwald-Mul- Wang, Q., Goh, A.M., Howley, P.M., and Walters, K.J. (2003). Ubiqui- len, P.M., Klein, G., Kurilla, M.G., and Masucci, M.G. (1995). Inhibi- tin recognition by the DNA repair protein hHR23a. Biochemistry 42, tion of antigen processing by the internal repeat region of the Ep- 13529–13535. stein-Barr virus nuclear antigen-1. Nature 375, 685–688. Watkins, J.F., Sung, P., Prakash, L., and Prakash, S. (1993). The Madura, K. (2002). The ubiquitin-associated (UBA) domain: on the Saccharomyces cerevisiae DNA repair gene RAD23 encodes a path from prudence to prurience. Cell Cycle 1, 235–244. nuclear protein containing a ubiquitin-like domain required for bio- logical function. Mol. Cell. Biol. 13, 7757–7765. Mueller, T.D., and Feigon, J. (2002). Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein Wilkinson, C.R., Seeger, M., Hartmann-Petersen, R., Stone, M., interactions. J. Mol. Biol. 319, 1243–1255. Wallace, M., Semple, C., and Gordon, C. (2001). Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Nat. Cell Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Biol. 3, 939–943. Tamura, T., Tanaka, K., and Ichihara, A. (1992). Ornithine decarbox- ylase is degraded by the 26S proteasome without ubiquitination. Nature 360, 597–599. Navon, A., and Goldberg, A.L. (2001). Proteins are unfolded on the surface of the ATPase ring before transport into the proteasome. Mol. Cell 8, 1339–1349. Ortolan, T.G., Tongaonkar, P., Lambertson, D., Chen, L., Schauber, C., and Madura, K. (2000). The DNA repair protein rad23 is a negative regulator of multi-ubiquitin chain assembly. Nat. Cell Biol. 2, 601–608. Prakash, S., Tian, L., Ratliff, K.S., Lehotzky, R.E., and Matouschek, A. (2004). An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat. Struct. Mol. Biol. 11, 830–837. Raasi, S., and Pickart, C.M. (2003). Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by se- questering lysine 48-linked polyubiquitin chains. J. Biol. Chem. 278, 8951–8959. Rao, H., and Sastry, A. (2002). Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23. J. Biol. Chem. 277, 11691–11695. Rape, M., Hoppe, T., Gorr, I., Kalocay, M., Richly, H., and Jentsch, S. (2001). Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone. Cell 107, 667–677. Rape, M., and Jentsch, S. (2002). Taking a bite: proteasomal protein processing. Nat. Cell Biol. 4, E113–E116. Riley, B.E., Xu, Y., Zoghbi, H.Y., and Orr, H.T. (2004). The polyglu- tamine repeat protein ataxin-1 and its effects on the UbL-UBA protein, A1Up. J. Biol. Chem. 279, 42290–42301. Russell, S.J., Reed, S.H., Huang, W., Friedberg, E.C., and Johnston, S.A. (1999). The 19S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair. Mol. Cell 3, 687–695. Saeki, Y., Saitoh, A., Toh-e, A., and Yokosawa, H. (2002a). Ubiquitin- like proteins and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis. Biochem. Biophys. Res. Commun. 293, 986–992. Saeki, Y., Sone, T., Toh-e, A., and Yokosawa, H. (2002b). Identifica- tion of ubiquitin-like protein-binding subunits of the 26S proteasome. Biochem. Biophys. Res. Commun. 296, 813–819. Schauber, C., Chen, L., Tongaonkar, P., Vega, I., Lambertson, D., Potts, W., and Madura, K. (1998). Rad23 links DNA repair to the ubiquitin/proteasome pathway. Nature 391, 715–718. Sharipo, A., Imreh, M., Leonchiks, A., Imreh, S., and Masucci, M.G. (1998). A minimal glycine-alanine repeat prevents the interaction of ubiquitinated IκB-α with the proteasome: a new mechanism for selective inhibition of proteolysis. Nat. Med. 4, 939–944. Storici, F., Lewis, L.K., and Resnick, M.A. (2001). In vivo site- directed mutagenesis using oligonucleotides. Nat. Biotechnol. 19, 773–776. Thrower, J.S., Hoffman, L., Rechsteiner, M., and Pickart, C.M. (2000). Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102.