- Email: [email protected]
Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome
FEBS Letters 581 (2007) 2567–2573
Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome Ki Moon Seonga,1, Je-Hyun Baeka,b,1, Myeong-Hee Yub, Joon Kima,* b
a Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea Functional Proteomics Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Received 12 March 2007; revised 6 April 2007; accepted 20 April 2007 Available online 30 April 2007 Edited by Horst Feldmann
Abstract The 26S proteasome, composed of the 20S core and 19S regulatory complexes, is important for the turnover of polyubiquitinated proteins. Each subunit of the complex plays a special role in proteolytic function, including substrate recruitment, deubiquitination, and structural contribution. To assess the function of some non-essential subunits in the 26S proteasome, we isolated the 26S proteasome from deletion strains of RPN13 and RPN14 using TAP affinity purification. The stability of Gcn4p and the accumulation of ubiquitinated Gcn4p were significantly increased, but the affinity in the recognition of proteasome was decreased. In addition, the subcomplexes of the isolated 26S proteasomes from deletion mutants were less stable than that of the wild type. Taken together, our findings indicate that Rpn13p and Rpn14p are involved in the efficient recognition of 26S proteasome for the proteolysis of ubiquitinated Gcn4p. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: 26S proteasome; 19S regulatory particle; Ubiquitinated Gcn4p; Rpn13p; Rpn14p
1. Introduction In eukaryotic cells, the breakdown of proteins by the ubiquitin-proteasome system (UPS) is a central regulatory mechanism for various cellular processes including the cell cycle, apoptosis, signal transduction, and gene expression [1,2]. In this pathway, substrate proteins are polyubiquitinated by ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. The 26S proteasome then recognizes and degrades the ubiquitin-conjugated proteins in an ATP-dependent manner [3]. The 26S proteasome is a multicatalytic protein complex that is composed of two subcomplexes, the 670 kDa 20S core particle (CP), which contains proteolytic active sites, and the 900 kDa 19S regulatory particle (RP), which binds to either or both ends of the 20S CP [3]. The 19S complex can be divided into lid and base subcomplexes. The lid consists of at least eight non-ATPase subunits (Rpn3p, Rpn5-9p, Rpn11p, and Rpn12p), while the base is * Corresponding author. Fax: +82 2 927 9028. E-mail address: [email protected] (J. Kim). 1
These authors contributed equally to this work.
made up of six AAA-ATPase subunits (Rpt1-6p) and two non-ATPase subunits (Rpn1-2p) [4]. Several components of the 19S RP play unique roles in the 26S proteasome. For example, Rpn1p acts as a receptor for ubiquitin-like proteins such as Rad23p [5]. Rpn10p is responsible for the binding of polyubiquitinated substrates with both the free form and the incorporated form in the 26S proteasome, and strengthens the interaction between the lid and base [6,7]. Rpn11p functions as a metallo-isopeptidase during proteasomal degradation [8,9]. Ubiquitin-mediated degradation ensures that key cellular factors are regulated at the appropriate time and location. Gcn4p is a typical eukaryotic transcriptional activator that is degraded by the 26S proteasome [10,11]. Under normal growth conditions, Gcn4p is rapidly degraded with a very short half-life of only 2–3 min [10]. This efficient degradation is mediated through the UPS pathway depending on the phosphorylation of specific residues, which leads to the ubiquitination and breakdown of Gcn4p [12,13]. The degradation of Gcn4p by UPS is also required for the recruitment of RNA polymerase II in the promoter of the target gene [14]. Therefore, Gcn4p seems to be a remarkable candidate molecule for use in investigating the function of the 26S proteasome in proteolysis. Considering the unique function of each subunit in the 19S complex, it is likely that each individual subunit of the 19S RP will be responsible for a specific function in the regulation of the Gcn4p level. In this study, we focused on the two non-ATPase subunits (Rpn13p and Rpn14p) of the 19S RP in the stability of Gcn4p. These are non-essential genes related to cell viability. However, non-essential subunits of 19S RP might also play a role in the fine regulation of proteasome. Rpn13p was shown to be a stoichiometric subunit of the proteasome and affected the proteasomal degradation of ubiquitinated substrates [15]. ADRM1, a human ortholog of Rpn13p, was recently reported to recruit and activate the deubiquitinating enzyme Uch37 [16– 19]. However, it seems likely that Rpn13p plays a different role considering the absence of ortholog of Uch37 in budding yeast. Rpn14p, which is reported only through high-throughput mass spectrometric protein complex identification (HMSPCI), has not yet been characterized in terms of its function [20]. We verified the normal proteolytic function of the 26S proteasome that was purified from these two deleted strains, and showed that their deletion is related to the degradation of Gcn4p. The data presented here suggested that the Rpn13p and Rpn14p were involved in the stable formation of 26S proteasome and the recognition of the ubiquitinated Gcn4p in proteasomal degradation.
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.04.064
2568
2. Materials and methods 2.1. Strains, plasmids, genetic manipulation, and growth media Yeast strains, plasmids and primers used in this study are described in Supplementary Table S1, S2. The YJK411 and YJK421 strains were generated by introducing the PCR product containing the kanMX module that was recovered from the genomic DNA of YJK302 (Drpn13) and YJK303 (Drpn14) strains with RPN13KO and RPN14KO primers, respectively. Standard genetic manipulation and transformation were used and standard yeast media such as YPD and synthetic complete media were prepared as previously described [21]. 2.2. TAP affinity purification of 26S proteasome The 26S proteasome was isolated by a modification of the previously described method [22]. The TAP-tagged and untagged strains of Saccharomyces cerevisiae were grown to an absorbance of 3.0 at 600 nm in YPD medium at 30 C. Cells were harvested and suspended in 5 ml of buffer A (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol, 5 mM ATP, 10 mM MgCl2, and 10· ATP regeneration system), and lysed with glass beads using a FastPrep-24 (MP Biomedicals Products, CA). The purified 26S proteasome was dialyzed with an excess of appropriate buffer for other experiments. Each subcomplex was verified by Western blotting with antibodies for TAP epitope (Open Biosystems, AL), Rpn11p (Santa Cruz Biotechnology, CA), Rpt5p (BIOMOL, UK), and 20S CP (BIOMOL, UK). 2.3. Identification of purified proteasome subunits by mass spectrometry The isolated 26S proteasome subunits were separated by SDS–polyacrylamide gel with a gradient of 10–20%, visualized by silver staining, digested with trypsin, and subjected to liquid chromatography and tandem mass spectrometry. Tandem mass spectra were assigned using SEQUEST searching against the Stanford yeast database (6948 entries) and validated with Trans-Proteomic Pipeline provided from the Institute for Systems Biology. 2.4. In-gel peptidase assay Non-denaturing PAGE on 4% polyacrylamide gel and the subsequent in-gel peptidase assay for the proteasome using a fluorogenic substrate, Suc-LLVY-MCA (Calbiochem, CA), were carried out as described previously [23]. 2.5. Promoter shut-off system analysis Promoter shut-off system analysis was performed as described previously [24]. Samples were analyzed at the indicated time points after promoter shut-off (0 min time point). Whole cell lysate was subjected to SDS–PAGE and Western blotting with monoclonal anti-myc (9E10) (Santa Cruz Biotechnology, CA) and anti-Pgk1p (Molecular Probes, OR) as an internal control. Gcn4p protein bands were quantified using the TINA image analysis software. 2.6. Immunoprecipitation and accumulation of polyubiquitinated Gcn4p pGAL1GCN4-myc-transformed cells were induced by 2% galactose for 12 h at 30 C. Cell extracts were prepared in the same volume of buffer A containing 1 mM DTT, 5 mM N-ethylmaleimide, 0.1 mM MG132, and protease inhibitors, and were disrupted with glass beads. The extracts were precleared with 20 ll of the protein A-agarose for 1 h at 4 C. Two micrograms of monoclonal anti-myc antibody were then added to the precleared lysate and incubated for 4 h at 4 C. Gcn4p recognized by anti-myc antibody was recovered with 30 ll of protein A-agarose for 2 h at 4 C. The beads were washed extensively with buffer A and bead-bound materials were eluted with SDS-loading buffer and subjected to SDS–PAGE, followed by Western blotting. Prior to elution, immunoprecipitated Gcn4p was also used for receptor-binding and proteasome-binding assays. 2.7. Bacterial expression and purification of His6-tagged proteins E. coli strain BL21-CodonPlus (DE3)-RIL cells (Stratagene, CA) were used for the expression of His6-tagged proteins. Bacterially-expressed proteins were purified with Ni-NTA agarose (Quiagene, CA) following the protocol of the manufacturer, and were dialyzed against phosphate-buffered saline with 125 mM HEPES (pH 7.5) for in vitro binding assays with the polyubiquitinated Gcn4p.
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573 2.8. Polyubiquitinated Gcn4p-binding assay with proteasome and recombinant proteins Twenty micrograms of purified recombinant protein were added to bead-bound Gcn4p in buffer A. After extensive washing, SDS-loading dye was added and eluted by boiling. Elutes were subjected to SDS– PAGE and Western blotting with anti-His probe (Santa Cruz Biotechnology, CA). For the proteasome-binding assay, 20 lg of purified 26S proteasome from each strain were added to the polyubiquitinated Gcn4p.
3. Results 3.1. The isolation of the 26S proteasome in the Drpn13 and Drpn14 strains To determine whether the deletion of the RPN13 and RPN14 genes affects the native composition and proteolytic function of the 26S proteasome complex, we isolated the 26S proteasome from deleted mutant strains using RPN10-TAP as bait. Purified 26S proteasome was resolved on a 10% polyacrylamide gel, followed by silver staining and immunoblotting with antibodies for Rpt5p in 19S RP and a/b subunits in 20S CP. The 26S proteasome purified from the Drpn13 and Drpn14 strains showed normal assembly of the 26S proteasome, similar to that of the wild-type strain (Fig. 1A). Each subunit was identified by liquid chromatography and mass spectrometry (Fig. 1B, Supplementary Table S3). To assess the proteolytic function of the purified 26S proteasome, we performed in-gel peptidase assays using Suc-LLVY-MCA (Fig. 1C). The patterns of signals resulting from the peptidase activities of the proteasomes from deleted strains were also identical to those of the wild-type strain. The deletion of RPN13 and RPN14 had no effects on the assembly or activity of the 26S proteasome complex. Rpn14p/Ygl004cp has been previously identified as a protein that interacts with 19S RP in large-scale protein–protein interaction studies [20]. To investigate whether Rpn14p is an intrinsic component of the 19S RP, we attempted to purify the 26S proteasome with RPN14-TAP. Although we repeated the purification of the 26S proteasome, whole subunits of 26S proteasome were not isolated completely using RPN14-TAP apart from Rpn10-TAP (data not shown). Nevertheless, Rpt5p, one of six ATPase subunits in the base of the 19S RP was likely to more strongly interact with Rpn14p than other subunit proteins (Fig. 1D). This result was also confirmed by washing the purified fraction with a high concentration of salt (Fig. 1E). 3.2. The stability of Gcn4p is increased in the non-ATPase subunit deletion strains Gcn4p is a good physiological substrate for 26S proteasome apart from the synthetic substrates of UPS such as Ub-Pro-bgal, UbV76-b-gal, and UbV76-V-DHFR [25–27]. Therefore, we examined whether the degradation of Gcn4p was affected by deletion of each gene. A galactose-inducible GCN4 expression system with C-terminus myc7-tag was introduced due to its rapid degradation. Interestingly, the breakdown of Gcn4p was delayed significantly in each Drpn13 and Drpn14 strain (Fig. 2A,B). The half-life (s1/2) of Gcn4p in the deleted mutant strains was about two times longer than that of the wild-type strain. Because deletion of RPN10 was known to increase the accumulation of ubiquitinated Gcn4p [28], we also compared the ubiquitinated Gcn4p of each deleted strain with those of the Drpn10 and wild-type strains. Remarkably, the
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
2569
Fig. 1. Affinity purification of the 26S proteasome from TAP-tagged cells of the Drpn13 and Drpn14 strains. (A) The purification of the 26S proteasome from YJK 401 (wild type), YJK411 (Drpn13), and YJK421 (Drpn14) strains was performed as described in Section 2. (B) Proteins from isolated 26S proteasomes were resolved on a 10–20% polyacrylamide gradient gel and subsequently stained with silver. Some of the identified protein bands were indicated at the right of the gel. (C) The same proteasome samples were subjected to in-gel peptidase assay. The isolated complex contained both asymmetric singly-capped (RP1CP) and symmetric doubly-capped (RP2CP) proteasomes. (D) The purification of the 26S proteasome was performed using the Rpn14p-TAP cells. (E) The 26S proteasome was purified by the high salt concentration washes. The bead-bound 26S proteasomes were washed three times with the binding buffer containing 0.1 M, 0.3 M, and 0.5 M NaCl, respectively, and were then washed once with the binding buffer.
ubiquitinated Gcn4p accumulated to a much higher degree in all three deletion strains than in the wild-type strain (Fig. 2C). To check whether this difference results from defects of global UPS in cells, we evaluated the ubiquitination of total lysate (Fig. 2D) and the resistance of cells to canavanine, an arginine analog, which induces protein misfolding, thereby increasing the demands on proteasome to clear them (Fig. 2E). RPN10deleted cells only showed large-scale UPS defects, but Drpn13 and Drpn14 cells did not. Taken together, we found that the deletions of RPN13 and RPN14 did not affect the large-scale UPS in cells but increased the stability in particular substrates. 3.3. Rpn13p and Rpn14p are required for the substrate recognition by 26S proteasome Biochemical studies have shown that Rpn10p serves as a true ubiquitin receptor for the proteasome, recognizing targets via its UIM (ubiquitin-interacting motif) [5,29]. In addition, the polyubiquitinated Gcn4p in Drpn13 and Drpn14 accumulated considerably, but to a somewhat lower level than that observed in Drpn10 (Fig. 2C). Therefore, we could regard the Rpn13 and Rpn14p as factors related to the recognition of ubiquitinated Gcn4p. First, we tested whether both proteins
work as receptors for the ubiquitinated Gcn4p using an in vitro binding assay. Gcn4p-myc7 was immunoprecipitated with the RPN10-deleted cell extracts using lysis buffer containing the inhibitors for proteolytic activity such as MG132 and N-ethylmalemide. These immunoprecipitated Gcn4 proteins were incubated with the C-terminus His6-tagged proteins purified from E. coli. Rpn10p interacted with ubiquitinated Gcn4p as expected, but Rpn13p and Rpn14p were not detected with anti-His probe (Fig. 3A). These data showed that Rpn13p and Rpn14p did not directly bind to the ubiquitinated Gcn4p. We then attempted to determine their specific roles in the recognition step of the 26S proteasome to the substrates because the effects of RPN13 and RPN14 deletions were similar to that of the deletion of RPN10 in the accumulation of ubiquitinated Gcn4p. To probe these possibilities, we carried out a proteasome-binding assay with immunoprecipitated Gcn4p from Drpn10 and Dubi4 cell extracts. UBI4 was the only gene that was not fused with a ribosomal gene and encoded the ubiquitin for polyubiquitination [30]. Because Gcn4p was only slightly polyubiquitinated in Dubi4 cells as shown in Fig. 3B, we used it as a negative control. Purified 26S proteasomes from the Drpn13 and Drpn14 strains were added to the
2570
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
Fig. 2. Deletions of RPN13 and RPN14 lead to increased Gcn4p stability. (A) Gcn4p stability was analyzed with pGAL1GCN4-myc-transformed cells. Protein levels of Gcn4p-myc7 and Pgk1p as loading control were determined in each of the deleted mutant cells at 0, 5, 10, 20, and 30 min after the GAL1 promoter shut-off. (B) Quantitation of experimental data shown in (A). Each bar indicated remaining Gcn4p percentages that were normalized using Pgk1p. The values for average and S.D. in this graph were obtained from five independent experiments. (C) Accumulation of polyubiquitinated Gcn4p was detected with the immunoprecipitated Gcn4p from each of the deletion strains using the total lysate extracted 5 min after the promoter shut-off with glucose. Equal amounts of immunoprecipitated Gcn4p are shown in the upper panel, and accumulation of ubiquitinated Gcn4p is shown in the lower panel. Ig (H), heavy chain of immunoglobulin. (D) Each deleted strain was grown in YPD media until mid-log phase, and total lysate was extracted. An equal amount of lysate was determined by Bradford protein assay and subjected to SDS–PAGE, followed by Western blotting with anti-Ub and anti-Pgk1p. (E) To assess the defects of the deletion of each gene in the global ubiquitin-proteasome pathway, 0.5 and 1.0 lg/ml canavanine plates were used. Serially-diluted cells were spotted and incubated at 30 C for 4 days.
polyubiquitinated Gcn4p immobilized with Protein A-agarose and anti-myc antibodies. The interaction between the Gcn4p and each subcomplex was determined by immunoblotting (Fig. 3B). We found that the 26S proteasome of each deleted strain showed significantly decreased binding activities to the ubiquitinated Gcn4p. We estimated that the 26S proteasome purified from the deleted strains was defective in the recognition of the ubiquitinated Gcn4p because these decreased binding activities were found in all of the subunits. 3.4. Rpn13p and Rpn14p work for the stable assembly of 26S proteasome Previous studies suggested that the interaction between the 19S RP subunits was an important factor in the complex formation and the regulation of 26S proteasome activity [31–33]. To characterize the structural function of Rpn13p and Rpn14p in the 26S proteasome, we tested the stability of the 26S proteasome purified from the deleted mutant cells by washing them with high salt concentration buffer. Each salt-washed 26S pro-
teasome was analyzed by Western blotting with antibodies as indicated in Fig. 4. In all strains, each subcomplex in the 26S proteasome was detected in equal amounts by washing with 0.1 M NaCl buffer (Fig. 4, lanes 4–6). The 26S proteasome treated with 0.3 M and 0.5 M NaCl washing showed different patterns in terms of composition. The 20S CP of the proteasome in deleted mutant cells dissociated more rapidly than that of the proteasome in wild-type cells during the 0.3 M NaCl washing (Fig. 4, lanes 7–9), whereas the 20S CP of wild-type 26S proteasome was found to be associated in the complex after the 0.5 M NaCl washing (Fig. 4, lanes 10–12). In addition, 20S CP was more easily dissociated from the 26S proteasome compared to other subcomplexes. The Rpt5p of the base complex and the Rpn11p of the lid complex remained throughout the 0.3 M NaCl washing, but both of the deletion mutant cells were dissociated by the 0.5 M NaCl washing (Fig. 4, lanes 10–12). Data gathered in this experiment imply that the 26S proteosomes from the Drpn13 and Drpn14 strains were less stable to NaCl washing than the 26S proteasome of the wild-type strain.
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
2571
Fig. 3. Efficient recognition of Rpn10p for ubiquitinated Gcn4p requires Rpn13p and Rpn14p. (A) For the preparation of the polyubiquitinated Gcn4p, pGAL1GCN4-myc was transformed into the Drpn10 strain. Immunoprecipitated Gcn4p was analyzed by Western blotting with 9E10 and FK2 antibodies; (S), short exposure time to film; (L), long exposure time to film. Immunoprecipitated Gcn4p was verified in equal amounts as shown in the short exposure time panel. Modification of Gcn4p was determined in the long exposure time panel. Bacterially-expressed and purified recombinant proteins were subjected to SDS–PAGE and subsequently analyzed by Coomassie blue staining. This showed that the amounts of purified proteins used in the in vitro binding assay were equal. (B) The purified 26S proteasome from YJK411 (Drpn13) and YJK421 (Drpn14) were also added to the immobilized ubiquitinated Gcn4 proteins.
4. Discussion The 26S proteasome is a complex 2-megadalton protease that is composed of at least 32 different subunits. Each subunit has unique functions that enhance the turnover of ubiquitinated substrates in the proteasome complex. Although a number of subunits have been characterized in the structural and functional roles of proteasome complex, uncharacterized subunits still remain. In addition, while most subunits of the proteasome complex are essential components in budding yeast, some of these subunits are non-essential. This allowed us to consider the idea that non-essential subunits of 19S RP play a special role in the proteasomal function. It is very interesting to find that the deletion of RPN13 and RPN14 increased the stability of Gcn4p and the accumulation of ubiquitinated Gcn4p, as seen in the deletion strain of RPN10 (Fig. 2). This phenomenon might have been caused by the inefficient recognition of the substrates by the 26S proteasome when RPN13 orRPN14 was deleted (Fig. 3). Similar to our data, in human
Rpn13-knockdown cells with siRNA, the degradation of short-lived protein was also reduced without alteration in the expression of other proteasomal subunits and accumulation of polyubiquitination in total extracts [18,19]. Taken together, we propose that Rpn13p and Rpn14p are required for the substrate recognition by the 26S proteasome, and the accumulation of ubiquitinated Gcn4p and the increase of Gcn4p stability result from the defect in the recognition of the substrates in proteasomal degradation. Because the purified 26S proteasomes from Drpn13 and Drpn14 mutant strains were not defective in structural assembly and proteolytic function (Fig. 1), we considered that RPN13 and RPN14 might not be essential genes for proteasomal activity, but act as auxiliary genes for specific functions. We were not able to isolate the intact 26S proteasome using RPN14-TAP, but found that only Rpt5p strongly interacted with Rpn14p (Fig. 1D,E). PAAF-1, a mammalian ortholog of RPN14, directly interacted with the proteasomal ATPases of 19S RP but not with the 20S core particle [34]. Thus,
2572
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
binding and the functional links between each receptor and other substrates should be conducted in the future. Acknowledgments: This work was supported in part by FPR05C2-390 Proteomics Grant and R01-2005-000-10798-0 Grant from Korean Ministry of Science & Technology.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2007. 04.064. Fig. 4. 26S proteasomes lacking Rpn13p and Rpn14p are unstable in high salt buffer. The 26S proteasome was pulled down from YJK411 (Drpn13) and YJK421 (Drpn14) strains using calmodulin-binding beads, and was then washed with binding buffer containing the indicated concentration of NaCl. Samples were resolved on 10% SDS– polyacrylamide gels, and each subunit was analyzed by immunoblotting with antibodies for each subcomplex as shown in the figure. Total lysate used in the purification was equal to the amounts appearing in input panels.
Rpn14p is not a genuine component of the 26S proteasome, but is, in fact, an auxiliary factor that interacts with the proteasomal ATPase of 19S RP. Whereas our data showed that deletion of RPN14 compromised the recognition and degradation of ubiquitinated Gcn4p, recent studies suggested that PAAF-1 negatively regulates proteasomal activities by promoting the dissociation of proteasome into 19S and 20S particles [34,35]. This discrepancy might reflect a fundamental functional difference between yeast and mammalian proteasome. We revealed that Rpn13p and Rpn14p play structural roles in the stability of the 26S proteasome in high salt concentration washing conditions, as shown in Fig. 4. Additionally, the assembly was not influenced by the deletion of both genes in washing conditions at concentrations lower than 0.1 M NaCl. Thus, we suggest that both subunits may also contribute to the stability of the 26S proteasome in some stress conditions. We could not discriminate the differences in the functions of Rpn13p and Rpn14p. However, we believed that the functions of the two proteins were not identical because Rpn13p is known to be a stoichiometric subunit of the 26S proteasome [15], while Rpn14p was found as an associated factor that interacted with the specific subcomplex in the proteasome rather than the intrinsic proteasome subunit in our experiment. These proteins related to the recognition of proteasome might function in different steps and/or with different pairs of the unknown substrates. Rpn10p can bind the substrates into the 26S proteasome in both its free form and its incorporated form [5,6]. Therefore, we cannot exclude the possibility that Rpn13p and Rpn14p were able to guide the ubiquitinated Gcn4p recognized by some receptors to the proteasome stably. We also suggest that Rpn13p and Rpn14p could contribute to the substrate specificity of the 26S proteasome. When the recognition of ubiquitinated substrates is carried out by some gate subunits in the 26S proteasome, these polyubiquitin chain receptors can specifically recognize their proper substrates, and can also share the substrate layers without specificity [36]. To further elucidate the specificity between polyubiquitin chain receptors and ubiquitinated substrates with respect to Rpn13p and Rpn14p, more thorough investigations on the
References [1] Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67, 425–479. [2] Schwartz, A.L. and Ciechanover, A. (1999) The ubiquitinproteasome pathway and pathogenesis of human diseases. Annu. Rev. Med. 50, 57–74. [3] Voges, D., Zwickl, P. and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068. [4] Glickman, M.H. et al. (1998) A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615– 623. [5] Elsasser, S. et al. (2002) Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat. Cell Biol. 4, 725–730. [6] Kominami, K. et al. (1997) Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1. Mol. Biol. Cell 8, 171–187. [7] van Nocker, S. et al. (1996) The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol. Cell Biol. 16, 6020–6028. [8] Verma, R., Aravind, L., Oania, R., McDonald, W.H., Yates 3rd, J.R., Koonin, E.V. and Deshaies, R.J. (2002) Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615. [9] Yao, T. and Cohen, R.E. (2002) A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407. [10] Kornitzer, D., Raboy, B., Kulka, R.G. and Fink, G.R. (1994) Regulated degradation of the transcription factor Gcn4. EMBO J. 13, 6021–6030. [11] Irniger, S. and Braus, G.H. (2003) Controlling transcription by destruction: the regulation of yeast Gcn4p stability. Curr. Genet. 44, 8–18. [12] Chi, Y., Huddleston, M.J., Zhang, X., Young, R.A., Annan, R.S., Carr, S.A. and Deshaies, R.J. (2001) Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase. Genes Dev. 15, 1078–1092. [13] Meimoun, A., Holtzman, T., Weissman, Z., McBride, H.J., Stillman, D.J., Fink, G.R. and Kornitzer, D. (2000) Degradation of the transcription factor Gcn4 requires the kinase Pho85 and the SCF(CDC4) ubiquitin-ligase complex. Mol. Biol. Cell 11, 915– 927. [14] Lipford, J.R., Smith, G.T., Chi, Y. and Deshaies, R.J. (2005) A putative stimulatory role for activator turnover in gene expression. Nature 438, 113–116. [15] Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Dohmen, J. and Deshaies, R.J. (2000) Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439. [16] Jorgensen, J.P., Lauridsen, A.M., Kristensen, P., Dissing, K., Johnsen, A.H., Hendil, K.B. and Hartmann-Petersen, R. (2006) Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor. J. Mol. Biol. 360, 1043–1052.
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573 [17] Yao, T. et al. (2006) Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat. Cell Biol. 8, 994–1002. [18] Hamazaki, J., Iemura, S., Natsume, T., Yashiroda, H., Tanaka, K. and Murata, S. (2006) A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 25, 4524–4536. [19] Qiu, X.B., Ouyang, S.Y., Li, C.J., Miao, S., Wang, L. and Goldberg, A.L. (2006) hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J. 25, 5742–5753. [20] Ho, Y. et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183. [21] Sherman, F. (1991) Getting started with yeast. Methods Enzymol. 194, 3–21. [22] Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229. [23] Elsasser, S., Schmidt, M. and Finley, D. (2005) Characterization of the proteasome using native gel electrophoresis. Methods Enzymol. 398, 353–363. [24] Pries, R., Bomeke, K., Irniger, S., Grundmann, O. and Braus, G.H. (2002) Amino acid-dependent Gcn4p stability regulation occurs exclusively in the yeast nucleus. Eukaryot. Cell 1, 663– 672. [25] Penney, M. et al. (1998) The Pad1+ gene encodes a subunit of the 26 S proteasome in fission yeast. J. Biol. Chem. 273, 23938– 23945. [26] 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.
2573 [27] Xie, Y. and Varshavsky, A. (2002) UFD4 lacking the proteasomebinding region catalyses ubiquitination but is impaired in proteolysis. Nat. Cell Biol. 4, 1003–1007. [28] Mayor, T., Lipford, J.R., Graumann, J., Smith, G.T. and Deshaies, R.J. (2005) Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol. Cell Proteomics 4, 741–751. [29] Fisher, R.D., Wang, B., Alam, S.L., Higginson, D.S., Robinson, H., Sundquist, W.I. and Hill, C.P. (2003) Structure and ubiquitin binding of the ubiquitin-interacting motif. J. Biol. Chem. 278, 28976–28984. [30] Finley, D., Sadis, S., Monia, B.P., Boucher, P., Ecker, D.J., Crooke, S.T. and Chau, V. (1994) Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol. Cell Biol. 14, 5501–5509. [31] Ferrell, K., Wilkinson, C.R., Dubiel, W. and Gordon, C. (2000) Regulatory subunit interactions of the 26S proteasome, a complex problem. Trends Biochem. Sci. 25, 83–88. [32] Takeuchi, J., Fujimuro, M., Yokosawa, H., Tanaka, K. and Tohe, A. (1999) Rpn9 is required for efficient assembly of the yeast 26S proteasome. Mol. Cell Biol. 19, 6575–6584. [33] Sone, T., Saeki, Y., Toh-e, A. and Yokosawa, H. (2004) Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae. J. Biol. Chem. 279, 28807–28816. [34] Park, Y., Hwang, Y.P., Lee, J.S., Seo, S.H., Yoon, S.K. and Yoon, J.B. (2005) Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases. Mol. Cell Biol. 25, 3842–3853. [35] Lassot, I. et al. (2007) The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms. Mol. Cell 25, 369–383. [36] Verma, R., Oania, R., Graumann, J. and Deshaies, R.J. (2004) Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118, 99–110.
Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome Ki Moon Seonga,1, Je-Hyun Baeka,b,1, Myeong-Hee Yub, Joon Kima,* b
a Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea Functional Proteomics Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Received 12 March 2007; revised 6 April 2007; accepted 20 April 2007 Available online 30 April 2007 Edited by Horst Feldmann
Abstract The 26S proteasome, composed of the 20S core and 19S regulatory complexes, is important for the turnover of polyubiquitinated proteins. Each subunit of the complex plays a special role in proteolytic function, including substrate recruitment, deubiquitination, and structural contribution. To assess the function of some non-essential subunits in the 26S proteasome, we isolated the 26S proteasome from deletion strains of RPN13 and RPN14 using TAP affinity purification. The stability of Gcn4p and the accumulation of ubiquitinated Gcn4p were significantly increased, but the affinity in the recognition of proteasome was decreased. In addition, the subcomplexes of the isolated 26S proteasomes from deletion mutants were less stable than that of the wild type. Taken together, our findings indicate that Rpn13p and Rpn14p are involved in the efficient recognition of 26S proteasome for the proteolysis of ubiquitinated Gcn4p. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: 26S proteasome; 19S regulatory particle; Ubiquitinated Gcn4p; Rpn13p; Rpn14p
1. Introduction In eukaryotic cells, the breakdown of proteins by the ubiquitin-proteasome system (UPS) is a central regulatory mechanism for various cellular processes including the cell cycle, apoptosis, signal transduction, and gene expression [1,2]. In this pathway, substrate proteins are polyubiquitinated by ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. The 26S proteasome then recognizes and degrades the ubiquitin-conjugated proteins in an ATP-dependent manner [3]. The 26S proteasome is a multicatalytic protein complex that is composed of two subcomplexes, the 670 kDa 20S core particle (CP), which contains proteolytic active sites, and the 900 kDa 19S regulatory particle (RP), which binds to either or both ends of the 20S CP [3]. The 19S complex can be divided into lid and base subcomplexes. The lid consists of at least eight non-ATPase subunits (Rpn3p, Rpn5-9p, Rpn11p, and Rpn12p), while the base is * Corresponding author. Fax: +82 2 927 9028. E-mail address: [email protected] (J. Kim). 1
These authors contributed equally to this work.
made up of six AAA-ATPase subunits (Rpt1-6p) and two non-ATPase subunits (Rpn1-2p) [4]. Several components of the 19S RP play unique roles in the 26S proteasome. For example, Rpn1p acts as a receptor for ubiquitin-like proteins such as Rad23p [5]. Rpn10p is responsible for the binding of polyubiquitinated substrates with both the free form and the incorporated form in the 26S proteasome, and strengthens the interaction between the lid and base [6,7]. Rpn11p functions as a metallo-isopeptidase during proteasomal degradation [8,9]. Ubiquitin-mediated degradation ensures that key cellular factors are regulated at the appropriate time and location. Gcn4p is a typical eukaryotic transcriptional activator that is degraded by the 26S proteasome [10,11]. Under normal growth conditions, Gcn4p is rapidly degraded with a very short half-life of only 2–3 min [10]. This efficient degradation is mediated through the UPS pathway depending on the phosphorylation of specific residues, which leads to the ubiquitination and breakdown of Gcn4p [12,13]. The degradation of Gcn4p by UPS is also required for the recruitment of RNA polymerase II in the promoter of the target gene [14]. Therefore, Gcn4p seems to be a remarkable candidate molecule for use in investigating the function of the 26S proteasome in proteolysis. Considering the unique function of each subunit in the 19S complex, it is likely that each individual subunit of the 19S RP will be responsible for a specific function in the regulation of the Gcn4p level. In this study, we focused on the two non-ATPase subunits (Rpn13p and Rpn14p) of the 19S RP in the stability of Gcn4p. These are non-essential genes related to cell viability. However, non-essential subunits of 19S RP might also play a role in the fine regulation of proteasome. Rpn13p was shown to be a stoichiometric subunit of the proteasome and affected the proteasomal degradation of ubiquitinated substrates [15]. ADRM1, a human ortholog of Rpn13p, was recently reported to recruit and activate the deubiquitinating enzyme Uch37 [16– 19]. However, it seems likely that Rpn13p plays a different role considering the absence of ortholog of Uch37 in budding yeast. Rpn14p, which is reported only through high-throughput mass spectrometric protein complex identification (HMSPCI), has not yet been characterized in terms of its function [20]. We verified the normal proteolytic function of the 26S proteasome that was purified from these two deleted strains, and showed that their deletion is related to the degradation of Gcn4p. The data presented here suggested that the Rpn13p and Rpn14p were involved in the stable formation of 26S proteasome and the recognition of the ubiquitinated Gcn4p in proteasomal degradation.
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.04.064
2568
2. Materials and methods 2.1. Strains, plasmids, genetic manipulation, and growth media Yeast strains, plasmids and primers used in this study are described in Supplementary Table S1, S2. The YJK411 and YJK421 strains were generated by introducing the PCR product containing the kanMX module that was recovered from the genomic DNA of YJK302 (Drpn13) and YJK303 (Drpn14) strains with RPN13KO and RPN14KO primers, respectively. Standard genetic manipulation and transformation were used and standard yeast media such as YPD and synthetic complete media were prepared as previously described [21]. 2.2. TAP affinity purification of 26S proteasome The 26S proteasome was isolated by a modification of the previously described method [22]. The TAP-tagged and untagged strains of Saccharomyces cerevisiae were grown to an absorbance of 3.0 at 600 nm in YPD medium at 30 C. Cells were harvested and suspended in 5 ml of buffer A (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol, 5 mM ATP, 10 mM MgCl2, and 10· ATP regeneration system), and lysed with glass beads using a FastPrep-24 (MP Biomedicals Products, CA). The purified 26S proteasome was dialyzed with an excess of appropriate buffer for other experiments. Each subcomplex was verified by Western blotting with antibodies for TAP epitope (Open Biosystems, AL), Rpn11p (Santa Cruz Biotechnology, CA), Rpt5p (BIOMOL, UK), and 20S CP (BIOMOL, UK). 2.3. Identification of purified proteasome subunits by mass spectrometry The isolated 26S proteasome subunits were separated by SDS–polyacrylamide gel with a gradient of 10–20%, visualized by silver staining, digested with trypsin, and subjected to liquid chromatography and tandem mass spectrometry. Tandem mass spectra were assigned using SEQUEST searching against the Stanford yeast database (6948 entries) and validated with Trans-Proteomic Pipeline provided from the Institute for Systems Biology. 2.4. In-gel peptidase assay Non-denaturing PAGE on 4% polyacrylamide gel and the subsequent in-gel peptidase assay for the proteasome using a fluorogenic substrate, Suc-LLVY-MCA (Calbiochem, CA), were carried out as described previously [23]. 2.5. Promoter shut-off system analysis Promoter shut-off system analysis was performed as described previously [24]. Samples were analyzed at the indicated time points after promoter shut-off (0 min time point). Whole cell lysate was subjected to SDS–PAGE and Western blotting with monoclonal anti-myc (9E10) (Santa Cruz Biotechnology, CA) and anti-Pgk1p (Molecular Probes, OR) as an internal control. Gcn4p protein bands were quantified using the TINA image analysis software. 2.6. Immunoprecipitation and accumulation of polyubiquitinated Gcn4p pGAL1GCN4-myc-transformed cells were induced by 2% galactose for 12 h at 30 C. Cell extracts were prepared in the same volume of buffer A containing 1 mM DTT, 5 mM N-ethylmaleimide, 0.1 mM MG132, and protease inhibitors, and were disrupted with glass beads. The extracts were precleared with 20 ll of the protein A-agarose for 1 h at 4 C. Two micrograms of monoclonal anti-myc antibody were then added to the precleared lysate and incubated for 4 h at 4 C. Gcn4p recognized by anti-myc antibody was recovered with 30 ll of protein A-agarose for 2 h at 4 C. The beads were washed extensively with buffer A and bead-bound materials were eluted with SDS-loading buffer and subjected to SDS–PAGE, followed by Western blotting. Prior to elution, immunoprecipitated Gcn4p was also used for receptor-binding and proteasome-binding assays. 2.7. Bacterial expression and purification of His6-tagged proteins E. coli strain BL21-CodonPlus (DE3)-RIL cells (Stratagene, CA) were used for the expression of His6-tagged proteins. Bacterially-expressed proteins were purified with Ni-NTA agarose (Quiagene, CA) following the protocol of the manufacturer, and were dialyzed against phosphate-buffered saline with 125 mM HEPES (pH 7.5) for in vitro binding assays with the polyubiquitinated Gcn4p.
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573 2.8. Polyubiquitinated Gcn4p-binding assay with proteasome and recombinant proteins Twenty micrograms of purified recombinant protein were added to bead-bound Gcn4p in buffer A. After extensive washing, SDS-loading dye was added and eluted by boiling. Elutes were subjected to SDS– PAGE and Western blotting with anti-His probe (Santa Cruz Biotechnology, CA). For the proteasome-binding assay, 20 lg of purified 26S proteasome from each strain were added to the polyubiquitinated Gcn4p.
3. Results 3.1. The isolation of the 26S proteasome in the Drpn13 and Drpn14 strains To determine whether the deletion of the RPN13 and RPN14 genes affects the native composition and proteolytic function of the 26S proteasome complex, we isolated the 26S proteasome from deleted mutant strains using RPN10-TAP as bait. Purified 26S proteasome was resolved on a 10% polyacrylamide gel, followed by silver staining and immunoblotting with antibodies for Rpt5p in 19S RP and a/b subunits in 20S CP. The 26S proteasome purified from the Drpn13 and Drpn14 strains showed normal assembly of the 26S proteasome, similar to that of the wild-type strain (Fig. 1A). Each subunit was identified by liquid chromatography and mass spectrometry (Fig. 1B, Supplementary Table S3). To assess the proteolytic function of the purified 26S proteasome, we performed in-gel peptidase assays using Suc-LLVY-MCA (Fig. 1C). The patterns of signals resulting from the peptidase activities of the proteasomes from deleted strains were also identical to those of the wild-type strain. The deletion of RPN13 and RPN14 had no effects on the assembly or activity of the 26S proteasome complex. Rpn14p/Ygl004cp has been previously identified as a protein that interacts with 19S RP in large-scale protein–protein interaction studies [20]. To investigate whether Rpn14p is an intrinsic component of the 19S RP, we attempted to purify the 26S proteasome with RPN14-TAP. Although we repeated the purification of the 26S proteasome, whole subunits of 26S proteasome were not isolated completely using RPN14-TAP apart from Rpn10-TAP (data not shown). Nevertheless, Rpt5p, one of six ATPase subunits in the base of the 19S RP was likely to more strongly interact with Rpn14p than other subunit proteins (Fig. 1D). This result was also confirmed by washing the purified fraction with a high concentration of salt (Fig. 1E). 3.2. The stability of Gcn4p is increased in the non-ATPase subunit deletion strains Gcn4p is a good physiological substrate for 26S proteasome apart from the synthetic substrates of UPS such as Ub-Pro-bgal, UbV76-b-gal, and UbV76-V-DHFR [25–27]. Therefore, we examined whether the degradation of Gcn4p was affected by deletion of each gene. A galactose-inducible GCN4 expression system with C-terminus myc7-tag was introduced due to its rapid degradation. Interestingly, the breakdown of Gcn4p was delayed significantly in each Drpn13 and Drpn14 strain (Fig. 2A,B). The half-life (s1/2) of Gcn4p in the deleted mutant strains was about two times longer than that of the wild-type strain. Because deletion of RPN10 was known to increase the accumulation of ubiquitinated Gcn4p [28], we also compared the ubiquitinated Gcn4p of each deleted strain with those of the Drpn10 and wild-type strains. Remarkably, the
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
2569
Fig. 1. Affinity purification of the 26S proteasome from TAP-tagged cells of the Drpn13 and Drpn14 strains. (A) The purification of the 26S proteasome from YJK 401 (wild type), YJK411 (Drpn13), and YJK421 (Drpn14) strains was performed as described in Section 2. (B) Proteins from isolated 26S proteasomes were resolved on a 10–20% polyacrylamide gradient gel and subsequently stained with silver. Some of the identified protein bands were indicated at the right of the gel. (C) The same proteasome samples were subjected to in-gel peptidase assay. The isolated complex contained both asymmetric singly-capped (RP1CP) and symmetric doubly-capped (RP2CP) proteasomes. (D) The purification of the 26S proteasome was performed using the Rpn14p-TAP cells. (E) The 26S proteasome was purified by the high salt concentration washes. The bead-bound 26S proteasomes were washed three times with the binding buffer containing 0.1 M, 0.3 M, and 0.5 M NaCl, respectively, and were then washed once with the binding buffer.
ubiquitinated Gcn4p accumulated to a much higher degree in all three deletion strains than in the wild-type strain (Fig. 2C). To check whether this difference results from defects of global UPS in cells, we evaluated the ubiquitination of total lysate (Fig. 2D) and the resistance of cells to canavanine, an arginine analog, which induces protein misfolding, thereby increasing the demands on proteasome to clear them (Fig. 2E). RPN10deleted cells only showed large-scale UPS defects, but Drpn13 and Drpn14 cells did not. Taken together, we found that the deletions of RPN13 and RPN14 did not affect the large-scale UPS in cells but increased the stability in particular substrates. 3.3. Rpn13p and Rpn14p are required for the substrate recognition by 26S proteasome Biochemical studies have shown that Rpn10p serves as a true ubiquitin receptor for the proteasome, recognizing targets via its UIM (ubiquitin-interacting motif) [5,29]. In addition, the polyubiquitinated Gcn4p in Drpn13 and Drpn14 accumulated considerably, but to a somewhat lower level than that observed in Drpn10 (Fig. 2C). Therefore, we could regard the Rpn13 and Rpn14p as factors related to the recognition of ubiquitinated Gcn4p. First, we tested whether both proteins
work as receptors for the ubiquitinated Gcn4p using an in vitro binding assay. Gcn4p-myc7 was immunoprecipitated with the RPN10-deleted cell extracts using lysis buffer containing the inhibitors for proteolytic activity such as MG132 and N-ethylmalemide. These immunoprecipitated Gcn4 proteins were incubated with the C-terminus His6-tagged proteins purified from E. coli. Rpn10p interacted with ubiquitinated Gcn4p as expected, but Rpn13p and Rpn14p were not detected with anti-His probe (Fig. 3A). These data showed that Rpn13p and Rpn14p did not directly bind to the ubiquitinated Gcn4p. We then attempted to determine their specific roles in the recognition step of the 26S proteasome to the substrates because the effects of RPN13 and RPN14 deletions were similar to that of the deletion of RPN10 in the accumulation of ubiquitinated Gcn4p. To probe these possibilities, we carried out a proteasome-binding assay with immunoprecipitated Gcn4p from Drpn10 and Dubi4 cell extracts. UBI4 was the only gene that was not fused with a ribosomal gene and encoded the ubiquitin for polyubiquitination [30]. Because Gcn4p was only slightly polyubiquitinated in Dubi4 cells as shown in Fig. 3B, we used it as a negative control. Purified 26S proteasomes from the Drpn13 and Drpn14 strains were added to the
2570
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
Fig. 2. Deletions of RPN13 and RPN14 lead to increased Gcn4p stability. (A) Gcn4p stability was analyzed with pGAL1GCN4-myc-transformed cells. Protein levels of Gcn4p-myc7 and Pgk1p as loading control were determined in each of the deleted mutant cells at 0, 5, 10, 20, and 30 min after the GAL1 promoter shut-off. (B) Quantitation of experimental data shown in (A). Each bar indicated remaining Gcn4p percentages that were normalized using Pgk1p. The values for average and S.D. in this graph were obtained from five independent experiments. (C) Accumulation of polyubiquitinated Gcn4p was detected with the immunoprecipitated Gcn4p from each of the deletion strains using the total lysate extracted 5 min after the promoter shut-off with glucose. Equal amounts of immunoprecipitated Gcn4p are shown in the upper panel, and accumulation of ubiquitinated Gcn4p is shown in the lower panel. Ig (H), heavy chain of immunoglobulin. (D) Each deleted strain was grown in YPD media until mid-log phase, and total lysate was extracted. An equal amount of lysate was determined by Bradford protein assay and subjected to SDS–PAGE, followed by Western blotting with anti-Ub and anti-Pgk1p. (E) To assess the defects of the deletion of each gene in the global ubiquitin-proteasome pathway, 0.5 and 1.0 lg/ml canavanine plates were used. Serially-diluted cells were spotted and incubated at 30 C for 4 days.
polyubiquitinated Gcn4p immobilized with Protein A-agarose and anti-myc antibodies. The interaction between the Gcn4p and each subcomplex was determined by immunoblotting (Fig. 3B). We found that the 26S proteasome of each deleted strain showed significantly decreased binding activities to the ubiquitinated Gcn4p. We estimated that the 26S proteasome purified from the deleted strains was defective in the recognition of the ubiquitinated Gcn4p because these decreased binding activities were found in all of the subunits. 3.4. Rpn13p and Rpn14p work for the stable assembly of 26S proteasome Previous studies suggested that the interaction between the 19S RP subunits was an important factor in the complex formation and the regulation of 26S proteasome activity [31–33]. To characterize the structural function of Rpn13p and Rpn14p in the 26S proteasome, we tested the stability of the 26S proteasome purified from the deleted mutant cells by washing them with high salt concentration buffer. Each salt-washed 26S pro-
teasome was analyzed by Western blotting with antibodies as indicated in Fig. 4. In all strains, each subcomplex in the 26S proteasome was detected in equal amounts by washing with 0.1 M NaCl buffer (Fig. 4, lanes 4–6). The 26S proteasome treated with 0.3 M and 0.5 M NaCl washing showed different patterns in terms of composition. The 20S CP of the proteasome in deleted mutant cells dissociated more rapidly than that of the proteasome in wild-type cells during the 0.3 M NaCl washing (Fig. 4, lanes 7–9), whereas the 20S CP of wild-type 26S proteasome was found to be associated in the complex after the 0.5 M NaCl washing (Fig. 4, lanes 10–12). In addition, 20S CP was more easily dissociated from the 26S proteasome compared to other subcomplexes. The Rpt5p of the base complex and the Rpn11p of the lid complex remained throughout the 0.3 M NaCl washing, but both of the deletion mutant cells were dissociated by the 0.5 M NaCl washing (Fig. 4, lanes 10–12). Data gathered in this experiment imply that the 26S proteosomes from the Drpn13 and Drpn14 strains were less stable to NaCl washing than the 26S proteasome of the wild-type strain.
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
2571
Fig. 3. Efficient recognition of Rpn10p for ubiquitinated Gcn4p requires Rpn13p and Rpn14p. (A) For the preparation of the polyubiquitinated Gcn4p, pGAL1GCN4-myc was transformed into the Drpn10 strain. Immunoprecipitated Gcn4p was analyzed by Western blotting with 9E10 and FK2 antibodies; (S), short exposure time to film; (L), long exposure time to film. Immunoprecipitated Gcn4p was verified in equal amounts as shown in the short exposure time panel. Modification of Gcn4p was determined in the long exposure time panel. Bacterially-expressed and purified recombinant proteins were subjected to SDS–PAGE and subsequently analyzed by Coomassie blue staining. This showed that the amounts of purified proteins used in the in vitro binding assay were equal. (B) The purified 26S proteasome from YJK411 (Drpn13) and YJK421 (Drpn14) were also added to the immobilized ubiquitinated Gcn4 proteins.
4. Discussion The 26S proteasome is a complex 2-megadalton protease that is composed of at least 32 different subunits. Each subunit has unique functions that enhance the turnover of ubiquitinated substrates in the proteasome complex. Although a number of subunits have been characterized in the structural and functional roles of proteasome complex, uncharacterized subunits still remain. In addition, while most subunits of the proteasome complex are essential components in budding yeast, some of these subunits are non-essential. This allowed us to consider the idea that non-essential subunits of 19S RP play a special role in the proteasomal function. It is very interesting to find that the deletion of RPN13 and RPN14 increased the stability of Gcn4p and the accumulation of ubiquitinated Gcn4p, as seen in the deletion strain of RPN10 (Fig. 2). This phenomenon might have been caused by the inefficient recognition of the substrates by the 26S proteasome when RPN13 orRPN14 was deleted (Fig. 3). Similar to our data, in human
Rpn13-knockdown cells with siRNA, the degradation of short-lived protein was also reduced without alteration in the expression of other proteasomal subunits and accumulation of polyubiquitination in total extracts [18,19]. Taken together, we propose that Rpn13p and Rpn14p are required for the substrate recognition by the 26S proteasome, and the accumulation of ubiquitinated Gcn4p and the increase of Gcn4p stability result from the defect in the recognition of the substrates in proteasomal degradation. Because the purified 26S proteasomes from Drpn13 and Drpn14 mutant strains were not defective in structural assembly and proteolytic function (Fig. 1), we considered that RPN13 and RPN14 might not be essential genes for proteasomal activity, but act as auxiliary genes for specific functions. We were not able to isolate the intact 26S proteasome using RPN14-TAP, but found that only Rpt5p strongly interacted with Rpn14p (Fig. 1D,E). PAAF-1, a mammalian ortholog of RPN14, directly interacted with the proteasomal ATPases of 19S RP but not with the 20S core particle [34]. Thus,
2572
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573
binding and the functional links between each receptor and other substrates should be conducted in the future. Acknowledgments: This work was supported in part by FPR05C2-390 Proteomics Grant and R01-2005-000-10798-0 Grant from Korean Ministry of Science & Technology.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2007. 04.064. Fig. 4. 26S proteasomes lacking Rpn13p and Rpn14p are unstable in high salt buffer. The 26S proteasome was pulled down from YJK411 (Drpn13) and YJK421 (Drpn14) strains using calmodulin-binding beads, and was then washed with binding buffer containing the indicated concentration of NaCl. Samples were resolved on 10% SDS– polyacrylamide gels, and each subunit was analyzed by immunoblotting with antibodies for each subcomplex as shown in the figure. Total lysate used in the purification was equal to the amounts appearing in input panels.
Rpn14p is not a genuine component of the 26S proteasome, but is, in fact, an auxiliary factor that interacts with the proteasomal ATPase of 19S RP. Whereas our data showed that deletion of RPN14 compromised the recognition and degradation of ubiquitinated Gcn4p, recent studies suggested that PAAF-1 negatively regulates proteasomal activities by promoting the dissociation of proteasome into 19S and 20S particles [34,35]. This discrepancy might reflect a fundamental functional difference between yeast and mammalian proteasome. We revealed that Rpn13p and Rpn14p play structural roles in the stability of the 26S proteasome in high salt concentration washing conditions, as shown in Fig. 4. Additionally, the assembly was not influenced by the deletion of both genes in washing conditions at concentrations lower than 0.1 M NaCl. Thus, we suggest that both subunits may also contribute to the stability of the 26S proteasome in some stress conditions. We could not discriminate the differences in the functions of Rpn13p and Rpn14p. However, we believed that the functions of the two proteins were not identical because Rpn13p is known to be a stoichiometric subunit of the 26S proteasome [15], while Rpn14p was found as an associated factor that interacted with the specific subcomplex in the proteasome rather than the intrinsic proteasome subunit in our experiment. These proteins related to the recognition of proteasome might function in different steps and/or with different pairs of the unknown substrates. Rpn10p can bind the substrates into the 26S proteasome in both its free form and its incorporated form [5,6]. Therefore, we cannot exclude the possibility that Rpn13p and Rpn14p were able to guide the ubiquitinated Gcn4p recognized by some receptors to the proteasome stably. We also suggest that Rpn13p and Rpn14p could contribute to the substrate specificity of the 26S proteasome. When the recognition of ubiquitinated substrates is carried out by some gate subunits in the 26S proteasome, these polyubiquitin chain receptors can specifically recognize their proper substrates, and can also share the substrate layers without specificity [36]. To further elucidate the specificity between polyubiquitin chain receptors and ubiquitinated substrates with respect to Rpn13p and Rpn14p, more thorough investigations on the
References [1] Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67, 425–479. [2] Schwartz, A.L. and Ciechanover, A. (1999) The ubiquitinproteasome pathway and pathogenesis of human diseases. Annu. Rev. Med. 50, 57–74. [3] Voges, D., Zwickl, P. and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068. [4] Glickman, M.H. et al. (1998) A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615– 623. [5] Elsasser, S. et al. (2002) Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat. Cell Biol. 4, 725–730. [6] Kominami, K. et al. (1997) Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1. Mol. Biol. Cell 8, 171–187. [7] van Nocker, S. et al. (1996) The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol. Cell Biol. 16, 6020–6028. [8] Verma, R., Aravind, L., Oania, R., McDonald, W.H., Yates 3rd, J.R., Koonin, E.V. and Deshaies, R.J. (2002) Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615. [9] Yao, T. and Cohen, R.E. (2002) A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407. [10] Kornitzer, D., Raboy, B., Kulka, R.G. and Fink, G.R. (1994) Regulated degradation of the transcription factor Gcn4. EMBO J. 13, 6021–6030. [11] Irniger, S. and Braus, G.H. (2003) Controlling transcription by destruction: the regulation of yeast Gcn4p stability. Curr. Genet. 44, 8–18. [12] Chi, Y., Huddleston, M.J., Zhang, X., Young, R.A., Annan, R.S., Carr, S.A. and Deshaies, R.J. (2001) Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase. Genes Dev. 15, 1078–1092. [13] Meimoun, A., Holtzman, T., Weissman, Z., McBride, H.J., Stillman, D.J., Fink, G.R. and Kornitzer, D. (2000) Degradation of the transcription factor Gcn4 requires the kinase Pho85 and the SCF(CDC4) ubiquitin-ligase complex. Mol. Biol. Cell 11, 915– 927. [14] Lipford, J.R., Smith, G.T., Chi, Y. and Deshaies, R.J. (2005) A putative stimulatory role for activator turnover in gene expression. Nature 438, 113–116. [15] Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Dohmen, J. and Deshaies, R.J. (2000) Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439. [16] Jorgensen, J.P., Lauridsen, A.M., Kristensen, P., Dissing, K., Johnsen, A.H., Hendil, K.B. and Hartmann-Petersen, R. (2006) Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor. J. Mol. Biol. 360, 1043–1052.
K.M. Seong et al. / FEBS Letters 581 (2007) 2567–2573 [17] Yao, T. et al. (2006) Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat. Cell Biol. 8, 994–1002. [18] Hamazaki, J., Iemura, S., Natsume, T., Yashiroda, H., Tanaka, K. and Murata, S. (2006) A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 25, 4524–4536. [19] Qiu, X.B., Ouyang, S.Y., Li, C.J., Miao, S., Wang, L. and Goldberg, A.L. (2006) hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J. 25, 5742–5753. [20] Ho, Y. et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183. [21] Sherman, F. (1991) Getting started with yeast. Methods Enzymol. 194, 3–21. [22] Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229. [23] Elsasser, S., Schmidt, M. and Finley, D. (2005) Characterization of the proteasome using native gel electrophoresis. Methods Enzymol. 398, 353–363. [24] Pries, R., Bomeke, K., Irniger, S., Grundmann, O. and Braus, G.H. (2002) Amino acid-dependent Gcn4p stability regulation occurs exclusively in the yeast nucleus. Eukaryot. Cell 1, 663– 672. [25] Penney, M. et al. (1998) The Pad1+ gene encodes a subunit of the 26 S proteasome in fission yeast. J. Biol. Chem. 273, 23938– 23945. [26] 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.
2573 [27] Xie, Y. and Varshavsky, A. (2002) UFD4 lacking the proteasomebinding region catalyses ubiquitination but is impaired in proteolysis. Nat. Cell Biol. 4, 1003–1007. [28] Mayor, T., Lipford, J.R., Graumann, J., Smith, G.T. and Deshaies, R.J. (2005) Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol. Cell Proteomics 4, 741–751. [29] Fisher, R.D., Wang, B., Alam, S.L., Higginson, D.S., Robinson, H., Sundquist, W.I. and Hill, C.P. (2003) Structure and ubiquitin binding of the ubiquitin-interacting motif. J. Biol. Chem. 278, 28976–28984. [30] Finley, D., Sadis, S., Monia, B.P., Boucher, P., Ecker, D.J., Crooke, S.T. and Chau, V. (1994) Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol. Cell Biol. 14, 5501–5509. [31] Ferrell, K., Wilkinson, C.R., Dubiel, W. and Gordon, C. (2000) Regulatory subunit interactions of the 26S proteasome, a complex problem. Trends Biochem. Sci. 25, 83–88. [32] Takeuchi, J., Fujimuro, M., Yokosawa, H., Tanaka, K. and Tohe, A. (1999) Rpn9 is required for efficient assembly of the yeast 26S proteasome. Mol. Cell Biol. 19, 6575–6584. [33] Sone, T., Saeki, Y., Toh-e, A. and Yokosawa, H. (2004) Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae. J. Biol. Chem. 279, 28807–28816. [34] Park, Y., Hwang, Y.P., Lee, J.S., Seo, S.H., Yoon, S.K. and Yoon, J.B. (2005) Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases. Mol. Cell Biol. 25, 3842–3853. [35] Lassot, I. et al. (2007) The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms. Mol. Cell 25, 369–383. [36] Verma, R., Oania, R., Graumann, J. and Deshaies, R.J. (2004) Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118, 99–110.