Rpn4 Is a Physiological Substrate of the Ubr2 Ubiquitin Ligase*

The homeostatic abundance of the proteasome in Saccharomyces cerevisiae is controlled by a feedback circuit in which transcriptional activator Rpn4 up-regulates the proteasome genes and is destroyed by the assembled, active proteasome. Remarkably, the degradation of Rpn4 can be mediated by two independent pathways. One pathway is independent of ubiquitin, whereas the other involves ubiquitination on internal lysines. In the present study, we investigated the mechanism underlying the ubiquitin-dependent degradation of Rpn4. We demonstrated, through in vivo and in vitro assays, that Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase, which was originally identified as a sequence homolog of Ubr1, the E3 component of the N-end rule pathway. The ubiquitin-conjugating enzyme Rad6, which directly interacts with Ubr2, is also required for the ubiquitin-dependent degradation of Rpn4. Furthermore, we showed that deletion of UBR2 exhibited a strong synthetic growth defect with a mutation in the Rpt1 proteasome subunit when Rpn4 was overexpressed. This study not only identified the ubiquitination apparatus for Rpn4 but also unveiled the first physiological substrate of Ubr2. The biological significance of Ubr2-mediated degradation of Rpn4 is also discussed.

The ubiquitin (Ub) 1 proteasome system is the primary intracellular machinery responsible for elimination of abnormal proteins and selective destruction of regulatory proteins involved in a wide variety of cellular processes (1)(2)(3). Ubiquitination of a protein substrate is a consecutive process involving multiple enzymes (4). Ub is first activated by the Ub-activating enzyme (E1), forming a thioester between the C-terminal carboxyl group of Ub and a specific cysteine of the E1. The Ub moiety of the E1ϳUb thioester is thereafter transferred to one of the Ub-conjugating enzymes (E2). The Ub moiety of the E2ϳUb thioester is conjugated via an isopeptide bond to the ⑀-amino group of a lysine residue of a substrate or a preceding Ub molecule conjugated to the substrate, the latter reaction resulting in a substrate-linked multi-Ub chain. Most E2s function in complex with one of the E3 enzymes or Ub ligases. A Ub ligase also denotes an E2⅐E3 complex. Ubiquitination of a specific substrate is regulated through modulation of its degradation signal and through control of the activity of a cognate E3 (4 -9). The isopeptide bond between Ub and a substrate can be hydrolyzed by deubiquitinating enzymes, which provides yet another layer of regulation for substrate ubiquitination (10).
Most known E3s are grouped into two families (homology to E6-AP C terminus domain E3s and RING E3s) based on their catalytic modules and features of sequence and structure (4,9). A homology to E6-AP C terminus-domain E3 can accept Ub moiety from an associated E2ϳUb thioester, forming an E3ϳUb thioester and acting as a proximal Ub donor to the substrate that it selects. By contrast, formation of thioesters between RING E3s and Ub has not been detected. The precise mechanism of RING E3-mediated ubiquitination remains speculative (11)(12)(13)(14)(15). The current consensus is that a RING E3 acts as an adaptor to optimize the orientation of the ubiquitination site of a substrate to the active site of a cognate E2, which allows the transfer of the Ub moiety from an E2ϳUb thioester to the substrate. The Saccharomyces cerevisiae Ubr1, the E3 component of the N-end rule pathway, is one of the most extensively studied RING E3s (15)(16)(17). Recent studies showed that Ubr1 is conserved from yeast to humans (18). Moreover, numerous sequence homologs of Ubr1 have been revealed from the data bases, comprising a subfamily of RING E3s (18 -20). However, the functions of these UBR E3s other than Ubr1 remain unknown.
Most substrates attached with a multi-ubiquitin chain are degraded by the 26 S proteasome, an ϳ2500-kDa self-compartmentalized multisubunit protease (21,22). The 26 S proteasome consists of a barrel-shaped proteolytic core (the 20 S core) with four stacked rings of seven subunits each in an ␣ 7 ␤ 7 ␤ 7 ␣ 7 configuration. The 20 S core possesses three types of catalytic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolyzing activities, which are provided by three of the seven distinct ␤ subunits. The proteolytic active sites are located inside the chamber of the 20 S core, which is capped at one or both ends by the 19 S regulatory particle (also known as cap or PA700). The 19 S regulatory particle is composed of at least 18 different subunits that are formed into two subcomplexes, the base and the lid (23)(24)(25). The base is in contact with the 20 S core and possesses six ATPase subunits (Rpt1-Rpt6) and two non-ATPase subunits (Rpn1 and Rpn2). The lid including nine non-ATPase subunits (Rpn3, Rpn5-Rpn9, Rpn11-Rpn13) is linked to the base partly via Rpn10, another non-ATPase subunit. The 19 S regulatory particle mediates the binding and unfolding of ubiquitinated substrates before their translocation into the cavity of the 20 S core for degradation (22). Recent reports demonstrated that the 19 S regulatory particle, specifically the Rpn11 subunit, also possesses a deubiquitinating activity (26,27).
An important aspect in regard to the regulation of the proteasome is how the proteasome genes are regulated. Biochemical analysis has shown that the proteasome subunits are stoichiometrically present in vivo, suggesting that the proteasome genes are coordinately regulated (23)(24)(25)28). Recent studies revealed that Rpn4 (also named Son1 and Ufd5) is a transcriptional activator required for normal expression of the S. cerevisiae proteasome genes (29,30). Furthermore, Rpn4 was found to be extremely short lived (t1 ⁄2 Ϸ2 min) and degraded by the proteasome (30). These observations and subsequent reports demonstrated that the proteasome abundance is regulated by a negative feedback circuit in which Rpn4 up-regulates the proteasome genes and is destroyed by the proteasome (31,32). Regulated degradation of Rpn4 is apparently a key mechanism that controls proteasome homeostasis. Strikingly, the proteasomal degradation of Rpn4 can be mediated by two distinct pathways (33). One pathway involves ubiquitination on internal lysines, whereas the other is Ub-independent. The details of these two pathways, however, remain unexplored.
In the current work, we investigated the mechanism underlying the Ub-dependent degradation of Rpn4. We found that Rpn4 is a physiological substrate of the Ubr2 Ub ligase, which was originally identified as a sequence homolog of Ubr1 (19). Furthermore, we demonstrated that the Ubr2-mediated degradation of Rpn4 is critical for cell growth when the proteasome activity is compromised.
Immunoblotting, Immunoprecipitation, and Pulse-Chase-S. cerevisiae cells were grown to A 600 of 0.8 -1.0, harvested, and resus-pended in equal volume of 2ϫ SDS buffer (2% SDS, 30 mM dithiothreitol, 90 mM Na-HEPES, pH 7.5) and incubated at 100°C for 3 min. Comparable amounts of extracts (from comparable numbers of cells) were separated by SDS-PAGE followed by immunoblotting analysis with antibodies as indicated. For immunoprecipitation, the cell extracts were diluted 20-fold with buffer A (1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 50 mM Na-HEPES, pH 7.5) before incubation with anti-HA antibody and protein A-agarose. The pulse-chase procedure was described previously (33).
Pulldown and in Vitro Ubiquitination Assays-ubr2⌬ cells or ubr2⌬ transformants harboring 425GAL1FLAGUBR2 were grown in synthetic selective medium containing 2% galactose to an A 600 of 1.8. Cells were spun down and manually ground to fine powder with a pestle. The pellet being ground was kept frozen by liquid nitrogen. Crude extracts were prepared by incubation of the powder in buffer B (0.2% Triton X-100, 50 mM NaCl, 50 mM HEPES, pH 7.5) plus protease inhibitor mix (Roche Diagnostics). Approximately 0.2 mg of crude extract was used for each pulldown. The procedure for pulldown assays was described previously (30). After standard pulldown, the beads were washed three times with buffer C (25 mM HEPES, pH 7.5, 25 mM KCl, 5 mM MgCl 2 , 2 mM ATP and 0.1 mM dithiothreitol) and then subjected to ubiquitination reactions containing 100 nM Uba1, 1 M Rad6, and 7 M ubiquitin in buffer C at 30°C for 30 min. Rad6 was overexpressed from vector pET-11d in Escherichia coli and purified by the procedure described previously (17). N-terminally hexahistidine-tagged Uba1 was overexpressed from YEplac181 in S. cerevisiae and purified by nickelnitrilotriacetic acid chromatography according to the manufacturer's instructions (Qiagen).

Ub-dependent Degradation of Rpn4 Is Mediated by Ubr2-
The contribution of the Ub system in Rpn4 degradation remained unclear until very recently when we discovered that Rpn4 can be degraded by two distinct pathways, either Ub-dependent or Ub-independent (30,33). We demonstrated that the Ub-independent degradation of Rpn4 can be inhibited by deletion of its N-terminal 10 amino acids and that the Ub-dependent degradation signal of Rpn4 is located in the N-terminal domain including residues 11-229 (33). To elucidate the mechanism underlying the Ub-dependent degradation of Rpn4, we wished to identify the cognate Ub ligase for Rpn4. We first tested whether the degradation of Rpn4 11-229 is mediated by one of the known E3s. A plasmid expressing C-terminally FLAG-tagged Rpn4 11-229 from the P CUP1 promoter on a low copy vector was transformed into yeast mutants that either lack one of the non-essential E3s or are deficient in one of the essential E3s at a restrictive temperature. Immunoblotting with anti-FLAG antibody was used to compare the steady-state levels of Rpn4 11-229 FLAG in these E3 mutants. Among the 15 E3 mutants tested, Rpn4 11-229 FLAG was detected only in the ubr2⌬ mutant (Fig. 1A). Consistent with the immunoblotting results, pulse-chase analysis showed that Rpn4 11-229 FLAG was stabilized in the ubr2⌬ mutant (Fig. 1B).
We then examined whether the Ub-dependent degradation of Rpn4 is dependent on Ubr2. A low copy plasmid expressing C-terminally HA-tagged Rpn4 that lacks the first 10 residues (Rpn4 ⌬1-10 ha) from the native RPN4 promoter was introduced into ubr2⌬ and a congenic WT strain, respectively. Pulse-chase analysis was used to measure the turnover rates of Rpn4 ⌬1-10 ha in these two strains. Although it was rapidly degraded in the WT strain, Rpn4 ⌬1-10 ha was stabilized in the ubr2⌬ mutant (Fig. 1, C and E). It is highly unlikely that deletion of the first 10 residues of Rpn4 drastically changed the protein structure and created a cryptic degradation signal for Ubr2, as this small deletion did not affect the transcriptional activity of Rpn4 (Fig. 4C, compare lanes 3 and 2). Moreover, the degradation of intact Rpn4 expressed from the native RPN4 promoter in a low copy vector was also slower in the ubr2⌬ mutant than in the WT strain (Fig. 1, D and E). The incomplete stabilization of Rpn4 in ubr2⌬ indicates that Ubr2 is not involved in the Ub-independent degradation of Rpn4. Taken to-gether, these results demonstrate that Ubr2 mediates the Ub-dependent degradation of Rpn4.
Rad6 Is Required for the Ub-dependent Degradation of Rpn4 -Ubr2 was originally identified as a sequence homolog of Ubr1, the RING E3 component of the N-end rule pathway in S. cerevisiae (15,19). However, the physiological function of Ubr2 remained unknown. Early work using two-hybrid analysis showed that the basic residue-rich domain of Ubr2 interacted with Rad6 (also termed Ubc2), the E2 enzyme that acts together with Ubr1 to ubiquitinate the N-end rule substrates (15). Consistent with the two-hybrid analysis, GST pulldown assay further demonstrated that full-length Ubr2 is physically associated with Rad6 ( Fig. 2A). These observations suggested that Rad6 may function in complex with Ubr2 in mediating the Ub-dependent degradation of Rpn4. To test this possibility, we compared the stability of Rpn4 ⌬1-10 ha in rad6⌬ and WT cells by pulse-chase analysis. As shown in Fig. 2B, Rpn4 ⌬1-10 ha was indeed stabilized in the rad6⌬ mutant, whereas it was rapidly degraded in the congenic WT strain. Thus, Rad6 is the E2 enzyme required for the Ub-dependent degradation of Rpn4.
In Vitro Ubiquitination of Rpn4 by Ubr2-To examine whether Ubr2 directly binds Rpn4, which is expected for a cognate Ub ligase of Rpn4, we performed pulldown assays with Rpn4 ⌬1-10 fused to GST. Yeast extracts containing N-terminally FLAG-tagged Ubr2 were incubated with agarose beads preloaded with GST-Rpn4 ⌬1-10 , GST-Rpn2, or GST alone. GST-Rpn2 and GST alone served as controls. (Rpn2 is a subunit of the 19 S particle.) Proteins retained by the GST fusions were resolved by SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibody. As shown in Fig. 3A, Ubr2 specifically bound to GST-Rpn4 ⌬1-10 . Note that GST-Rpn4 ⌬1-10 retained Ubr2 as efficiently as GST-Rpn4 in the pulldown assay (data not shown), indicating that deletion of the N-terminal 10 residues of Rpn4 does not affect the Ubr2-Rpn4 interaction.
Taking advantage of the binding of Ubr2 to Rpn4 and the identification of Rad6 as the E2 enzyme for Rpn4 ⌬1-10 degradation, we set up a pulldown ubiquitination assay to examine whether Rpn4 ⌬1-10 could be ubiquitinated by Ubr2 in vitro (Fig. 3B). Specifically, agarose beads preloaded with GST-Rpn4 ⌬1-10 were incubated with extracts from either ubr2⌬ cells (lane 2) or ubr2⌬ cells overexpressing Ubr2 from the GAL1 promoter in a high copy plasmid (lanes 3 and 4). After a standard pulldown, the beads were further incubated with purified Rad6 and Uba1 in the presence (lanes 2 and 3) or absence (lane 4) of ubiquitin. The GST fusions were then separated by SDS-PAGE and subjected to immunoblotting with anti-GST antibody. Remarkably, a ladder of ubiquitinated species of GST-Rpn4 ⌬1-10 was formed in the presence of Ubr2 and ubiquitin (lane 3) but not in the absence of Ubr2 or ubiquitin (lanes 2 and  4). To confirm that the ubiquitination was specific to the lysine(s) on Rpn4 ⌬1-10 , we conducted a similar pulldown ubiquitination assay using a GST fusion with Rpn4 ⌬1-10/10R instead of Rpn4 ⌬1-10 . Rpn4 ⌬1-10/10R was derived from Rpn4 ⌬1-10 with the 10 N-terminal lysines mutated to arginines. (Lys-9 was already deleted in Rpn4 ⌬1-10 .) It has been shown that the Ub-dependent degradation of Rpn4 requires one or more of the 11 N-terminal lysines (33). Although its affinity to Ubr2 was not noticeably reduced as compared with Rpn4 ⌬1-10 (Fig. 3A, compare lanes 2 and 4), Rpn4 ⌬1-10/10R was not ubiquitinated by Ubr2 (Fig. 3B, lane 1). These observations confirmed that the ubiquiti-FIG. 2. Rad6 is required for the Ub-dependent degradation of Rpn4. A, Ubr2 interacts with Rad6. Extracts from cells overexpressing N-terminally FLAG-tagged Ubr2 ( F Ubr2) were incubated with agarose beads preloaded with GST or GST-Rad6. Retained F Ubr2 was separated by SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibody (upper panel). 10% of input extract was included in the immunoblotting to evaluate the pulldown efficiency. Coomassie Blue staining showed that comparable amounts of GST and GST-Rad6 were used in the pulldown assay (lower panel). F, FLAG-tagged. B, pulse-chase analysis of Rpn4 ⌬1-10 ha in WT (lanes 1-3) and rad6⌬ (lanes 4 -6). Rpn4 ⌬1-10 ha was expressed from the native RPN4 promoter in a low copy vector.
FIG. 1. Ub-dependent degradation of Rpn4 by Ubr2. A, immunoblotting analysis of C-terminally FLAG-tagged Rpn4 11-229 in E3 mutants. All E3 deletion mutants were derived from BY4741 (wild type). ts mutants rsp5-1, apc11-13, and cdc53-1 were shifted to 37°C 30 min before extract preparation. The asterisk marks a cross-reactive band with anti-FLAG antibody, which is just above the position of Rpn4 11-229 FLAG indicated by an arrow. F, FLAG-tagged. B, pulse-chase analysis of Rpn4 11-229 FLAG in WT (lanes  1-3) and ubr2⌬ (lanes 4 -6). The ubr2⌬ mutant used in pulse-chase assays was derived from JD52 (wild type) by replacing the UBR2 open reading frame with HIS3 via a PCR-mediated approach. C, the stability of C-terminally HA-tagged Rpn4 ⌬1-10 in WT (lanes 1-3) and ubr2⌬ (lanes 4 -6) was measured by pulse-chase analysis. D, pulse-chase analysis was carried out to compare the degradation of C-terminally HA-tagged Rpn4 in WT (lanes 1-3) and ubr2⌬ (lanes 4 -6). E, quantitation of the data from C and D by PhosphorImager to show the decay of Rpn4 ⌬1-10 ha in WT (E) and ubr2⌬ (•), and the decay of Rpn4ha in WT (Ⅺ) and ubr2⌬ (f). nation of GST-Rpn4 ⌬1-10 by Ubr2 is specific to the N-terminal lysine(s) of Rpn4. Taken together, these data (Figs. 1-3) allow us to conclude that Ubr2 is the cognate Ub ligase for Rpn4.
Overexpression of Rpn4 Is Tolerable in ubr2⌬ Cells-Deletion of UBR2 produced no noticeable phenotypes under normal conditions. The finding of Ubr2 as the cognate Ub ligase of Rpn4 prompted us to investigate if the proteasome abundance is higher in ubr2⌬ compared with WT cells. We deleted UBR2 from a strain that expressed C-terminally FLAG-His 6 -tagged Pre1 (Pre1 FH ), a subunit of the 20 S core, from its chromosomal locus. Immunoblotting analysis with anti-FLAG antibody demonstrated that the steady-state level of Pre1 FH was only modestly higher in ubr2⌬ than in the WT counterpart (data not shown). This observation is not entirely unexpected as Rpn4 is still degraded even with a lower turnover rate by the Ubindependent pathway in ubr2⌬. Overexpression of Rpn4 from the P CUP1 promoter on a low copy vector, however, clearly produced more Pre1 FH in ubr2⌬ compared with the WT strain (Fig. 4B, lanes 2 and 3). These observations indicate that the Ubr2-mediated degradation of Rpn4 plays a role in the regulation of proteasome expression.
We then tested whether overexpression of Rpn4 in ubr2⌬ would lead to cell growth defects. A low copy plasmid expressing Rpn4 from the P CUP1 promoter was transformed into WT and ubr2⌬. Serial dilution assays were carried out to compare the growth of these transformants in the presence or absence of Cu 2ϩ induction. As shown in Fig. 4A, overexpression of Rpn4 did not affect the growth rates of WT and ubr2⌬ cells. The lack of phenotype of Rpn4 overexpression in ubr2⌬ was not because of the degradation of Rpn4 by the Ub-independent pathway as ubr2⌬ cells overexpressing Rpn4 ⌬1-10 did not exhibit any growth defects even though Rpn4 ⌬1-10 was stabilized in ubr2⌬ (Fig. 1C and data not shown). Interestingly, the transcriptional activity of Rpn4 ⌬1-10 was comparable with that of the full-length Rpn4 (Fig. 4C, compare lanes 2  and 3). Taken together, these results indicate that accumulation of Rpn4 and a higher abundance of proteasome can be tolerated under normal conditions.
Deletion of UBR2 Exhibits a Synthetic Growth Defect with cim5-1 When Rpn4 Is Overexpressed-Our recent work and that of others demonstrated that deletion of RPN4 produces a synthetic growth defect with proteasome mutants (31,32). We wondered whether overexpression of Rpn4 is also detrimental to cell growth when the proteasome activity is compromised. Moreover, we wished to examine if deletion of UBR2 could augment the effect of Rpn4 overexpression in this setting. To this end, we transformed a low copy plasmid expressing Rpn4 from the P CUP1 promoter into WT, cim5-1, and a cim5-1 ubr2⌬ double mutant. cim5-1 is a temperature-sensitive mutant bearing a mutated Rpt1 subunit of the proteasome (38). Although overexpression of Rpn4 did not affect the WT cells, the growth of cim5-1 cells was slower when Rpn4 was overexpressed (Fig. 4A). Remarkably, the cim5-1 ubr2⌬ double mutant was much more sensitive to Rpn4 overexpression than cim5-1. Indeed, a slower growth of the cim5-1 ubr2⌬ transformants was readily observed even without Cu 2ϩ induction. Thus, proteasome mutant cells are sensitive to Rpn4 overexpression, and deletions of UBR2 and the cim5-1 mutation produce a strong synthetic growth defect when Rpn4 is overexpressed. The toxicity of Rpn4 overexpression relied on the transcriptional activity of Rpn4 in that overexpression of Rpn4 C447A , an Rpn4 mutant the transcriptional activity of which is abolished because of a Cys 3 Ala mutation at Cys-477 of the C 2 H 2 DNA binding motif (Fig. 4C), did not impair the growth of the cim5-1 ubr2⌬ double mutant (Fig. 4A).
It has been shown that Rpn4 is partially stabilized in cim5-1 at a permissive temperature (30°C) (31). To determine whether the synthetic effect of deletion of UBR2 and cim5-1 correlates with a slower degradation of Rpn4 in the cim5-1 ubr2⌬ double mutant, we wanted to compare the turnover rates of Rpn4 in cim5-1 and the cim5-1 ubr2⌬ double mutant. Because expression of Rpn4 from the induced P CUP1 promoter severely impaired the growth of the cim5-1 ubr2⌬ double mutant, and Rpn4 C447A had a turnover rate similar to Rpn4 in WT cells (Fig. 4D), we decided to compare the stability of Rpn4 C447A instead of Rpn4 in cim5-1 and the cim5-1 ubr2⌬ double mutant. Pulse-chase analysis demonstrated that the degradation of Rpn4 C447A was indeed slower in the cim5-1 ubr2⌬ double mutant than in cim5-1 (Fig. 4, E and F). Consistent with the pulse-chase analysis, the steady-state level of Rpn4 C447A was higher in the cim5-1 ubr2⌬ double mutant than in cim5-1 (data not shown). Therefore, the synthetic growth defect of cim5-1 and ubr2⌬ does correlate to slower turnover of Rpn4 in the cim5-1 ubr2⌬ double mutant. DISCUSSION The identification of Ubr2/Rad6 as the cognate Ub ligase for Rpn4 through in vivo and in vitro assays revealed for the first time the physiological function of Ubr2, which has been considered a member of the UBR E3s family (19). Although the overall sequence homology between Ubr2 and Ubr1 is statistically significant (ϳ22% identity and ϳ46% similarity), and the featured structures including the RING finger and Rad6 bind- The same amounts of extracts were used in these two immunoblots. Cell extract from an untagged strain was used as a negative control (lane 1). D, pulse-chase analysis of C-terminally HA-tagged Rpn4 (lanes 1-3) and Rpn4 C447A (lanes 4 -6) expressed from the P CUP1 promoter on a low copy vector in JD52 in the presence of 100 M CuSO 4 . E, pulse-chase analysis of C-terminally HA-tagged Rpn4 C447A expressed from the P CUP1 promoter on a low copy vector in cim5-1 (lanes 1-3) and cim5-1 ubr2⌬ (lanes 4 -6) with 100 M CuSO 4 . Pulse-chase was conducted at a permissive temperature (30°C). F, quantitation of the data from (E) by PhosphorImager to show the decay curves of Rpn4 C447A in cim5-1 (•) and cim5-1 ubr2⌬ (E).  (ScUbr2) and E. gossypii Ubr2 (EgUbr2). The amino acids essential for the degradation of type 1 substrates (*) and type 2 substrates (ϩ) are marked. White-on-black and gray shading highlight the conserved residues with ScUbr1 and ScUbr2, respectively. ing site appear to be similar, Ubr2 lacks the residues essential for the degradation of N-end rule model substrates, which are conserved in Ubr1 enzymes from yeast to mammals (Fig. 5 and Ref. 18). These sequence variations suggest that Ubr2 may function outside the N-end rule pathway. Consistent with the sequence analysis, we found that the turnover rates of N-end rule model substrates were not decreased in ubr2⌬ when compared with WT cells (data not shown). The demonstration of Rpn4 as a substrate of Ubr2 but not Ubr1 clearly separated Ubr2 from the N-end rule pathway. It is likely that Ubr1 and Ubr2 were derived from the same ancestral gene via duplication and diverged afterward through mutations, generating two different branches of UBR E3s. Interestingly, Ubr2 and Ubr1 use the same E2 enzyme (Rad6) even though they target different substrates. Similarly, several S. cerevisiae Skp1-Cullin-F-box E3s utilize a common E2, Cdc34, in ubiquitinating different substrates (4). There appears to be a rule in the cell that individual E3s of the same type share one E2. This rule is in line with the observations that there are more E3s than E2s in the data bases and that the substrate specificity is mainly determined by E3s.
Ubr2-mediated degradation of Rpn4 is one of the two mechanisms that regulate the steady-state level of Rpn4 (33). (The other one is a Ub-independent degradation pathway that remains to be further characterized.) We demonstrated that overexpression of Rpn4 impairs the growth of the cim5-1 proteasome mutant. Interestingly, deletion of UBR2 augmented this growth defect. The synthetic growth defect of UBR2 deletion and cim5-1 appeared to correlate to a slower degradation of Rpn4 in the cim5-1 ubr2⌬ double mutant. However, it is noteworthy that accumulation of Rpn4 per se is not toxic because overexpression of a stable version of Rpn4 in WT did not impair cell growth (data not shown). Similarly, ubr2⌬ cells overexpressing Rpn4 ⌬1-10 did not exhibit any growth defects even though Rpn4 ⌬1-10 is stabilized in ubr2⌬ and is as active as full-length Rpn4 in regard to transcriptional activity. The exact cause of the toxicity of overexpressed Rpn4 in proteasome mutants is currently unclear. It is likely that overproduction of defective proteasomes by a higher steady-state level of Rpn4 is detrimental to the cell. For instance, excessive amounts of inefficient proteasomes may delay the releasing and recycling of multi-Ub chains from ubiquitinated substrates, which is important to maintain the free Ub pool in the cell. Alternatively, overproduction of defective proteasomes may sequester proteasome-associated proteins such as Rad23 and Dsk2 that are required to target certain ubiquitinated substrates to the proteasome (40). It is also possible that overexpression of Rpn4 results in up-regulation of other Rpn4 target genes (non-proteasome genes) the protein products of which are normally short lived in WT cells but stabilized in the proteasome mutants; accumulation of such proteins may lead to growth defects. It will be of interest to identify the downstream targets of Rpn4 that are responsible for the cell toxicity. Nonetheless, Ubr2-mediated degradation of Rpn4 plays an important role in controlling the steady-state level of Rpn4, which is critical for cell growth when the proteasome activity is compromised.