The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells

FEBS Letters xxx (2014) xxx–xxx

journal homepage: www.FEBSLetters.org

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells A.V. Morozov ⇑, D.S. Spasskaya, D.S. Karpov, V.L. Karpov W.A. Engelhardt Institute of Molecular Biology RAS, Moscow, Russia

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Article history: Received 9 May 2014 Revised 8 August 2014 Accepted 13 August 2014 Available online xxxx Edited by Noboru Mizushima Keywords: Rpn4 Proteasome Protein degradation signals

a b s t r a c t Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction The turnover of intracellular proteins is a tightly regulated and constantly adjusted process, with the 26S proteasome complex as one of the key players. In most cases, protein substrates are tagged with ubiquitin (Ub) chains that are recognized by the 19S regulatory particle of the 26S proteasome. On the other hand, increasing numbers of proteins are shown to be degraded by the proteasome without prior ubiquitination [4]. Among these: ornithine decarboxylase (ODC), thymidylate synthase, yeast Rpn4, c-jun, p-21 and others [15,10,8,18]. Furthermore, it is estimated that up to 22% of all cellular proteins undergo proteasome-dependent proteolysis when omitting ubiquitination [3]. Special motifs denoted as degradation signals (degrons) are responsible for ubiquitin-dependent and ubiquitin-independent degradation [28]. Ubiquitindependent degrons consist of a preferential ubiquitination site (mostly lysine) and a distinct sequence. Conversely, ubiquitinindependent degradation signals frequently share structural, rather than sequence, similarities. They contain an intrinsically disordered region and an alpha helix, which serve two major functions: the initiation of degradation and tethering to the proteasome, respectively [17,21]. Little is known about interspecies

Abbreviations: ODC, ornithine decarboxylase; GFP, green fluorescent protein; AFP, Alpha-fetoprotein; Ub, ubiquitin; CMV, cytomegalovirus ⇑ Corresponding author. Address: 119991 Vavilova Str. 32, Moscow, Russia. Fax: +7 (499) 135 14 05. E-mail address: [email protected] (A.V. Morozov).

degrons and the evolution of the degradation machinery from lower to higher eukaryotes. The rare example, except classical N-degrons [25], is the mammalian ODC degron, which promotes the degradation of chimeric proteins in mammalian and yeast cells [5,23,11,19]. However, no yeast-derived, degradation signals, capable to work in mammalian cells have been discovered. Yeast protein Rpn4 (yRpn4) is a transcription factor with extremely short half-life (1–2 min). It controls proteasome abundance through a negative feedback mechanism [16,27]. Turnover of yRpn4 plays an important role in the resistance to various stresses [26,12] and is tightly regulated. It was shown that degradation of yRpn4 can be both Ub-dependent and -independent [10]. The N-terminal portion of yRpn4 (a.a. 1–80) contains a portable Ub-independent degron with an intrinsically disordered domain and a folded segment [7] (Fig. 1A and Suppl. Fig. 1A). This degron mediates the cotranslational degradation of yRpn4 [6]. Portable ubiquitindependent degron of yRpn4 was mapped to 172–229 a.a. [9]. It contains the preferable polyubiquitination site K187 and proximal acidic domain that mediates the interaction with the cognate ubiquitin ligase Ubr2 [9] (Fig. 1A). Given that yRpn4 degradation is mediated by the evolutionarily conservative components of the ubiquitin–proteasome system, we suggested that yRpn4 could be highly unstable in higher eukaryotic cells and contain degrons capable of promoting chimeric protein degradation in mammalian cells. In this study, we demonstrated that human proteasomes effectively degrade yRpn4 as well as two different stable proteins fused to the central domain of yRpn4.

http://dx.doi.org/10.1016/j.febslet.2014.08.017 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017

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A a.a.

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pDAFP by cloning RPN4 gene fragment upstream of the DAFP gene. The constructs were verified by restriction analysis and sequencing. Plasmids were amplified in DH5a cells and isolated using the Qiagen Plasmid Mini kit (Qiagen, Germany). All proteins encoded by these vectors bore a stabilizing amino acid at their N-termini next to methionine to avoid degradation by the N-terminal rule. 2.3. Transfection and preparation of cellular lysates

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HEK 293T cells (3  105) were transfected with 1 lg of plasmids using Mirus TransIT-293 reagent (Mirus Bio, USA) according to the manufacturer’s instructions. 48 h post transfection cells were first examined under a DMI 4000B fluorescent microscope (Leica, Germany) and then washed, scraped and analyzed by flow cytometry or lysed in NP-40 buffer (Tris–HCl, pH 8.0 50 mM, NaCl 150 mM, NP-40 1%, EDTA 5 mM, containing protease inhibitor cocktail (Roche, Germany)). Lysates were collected and stored at 80 °C before use.

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42 Anti-beta-actin Fig. 1. Recombinant yRpn4 accumulates in transfected HEK 293T cells after treatment with proteasome inhibitor. Schematic representation of yRpn4 degrons (A). Lysine 187 is marked. (B) Proteasome inhibition assay. Western blot analysis of lysates of transfected 293T cells. Cells were maintained in growth media containing no proteasome inhibitor (track 1), 10 lM MG132 or 10 lM lactacystin (tracks 2 and 3 respectively). yRpn4 was detected by Western blotting with anti-yRpn4 antibodies. Equal protein loading was confirmed by staining the membrane with anti-b-actin antibodies. yRpn4 and b-actin are marked by arrow heads.

2. Materials and methods 2.1. Cell lines The human embryonic kidney cell line HEK 293T (ATCC CRL-3216) was used for transfection. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, L-glutamine and antibiotics (all from PanEco, Russia) and kept at 37 °C, 5% CO2 and 95% humidity. 2.2. Vectors Using the pEGFP-N1 (Clontech, USA) backbone, two eukaryotic expression vectors encoding the yRpn4 and GFP-ODC degradation signal chimera were designed. The RPN4 gene and mouse ODC degradation signal were cloned from previously obtained plasmids [13,19], and the constructs were denoted pyRpn4 and pGFP-ODCsignal. The design of additional control vectors pDAFP (encoding murine Alpha-fetoprotein (AFP) without leader sequence) and pDAFPODCsignal (encoding murine Alpha-fetoprotein without leader sequence and carrying ODC degron) is described elsewhere [20,19]. The leader sequence was removed in order to ensure cytoplasmic localization of the recombinant proteins. Plasmids pyRpn4(1–82)-N-GFP, pyRpn4(1–110)-N-GFP, pyRpn4(1–176)-N-GFP, pyRpn4(172–229)-N-GFP, pyRpn4(177–327)N-GFP and pyRpn4(230–327)-N-GFP were obtained by cloning in-frame RPN4 gene fragments encoding yRpn4 portions (the corresponding amino acids are indicated in brackets) upstream of the GFP gene in pEGFP-N1. Plasmids pGFP-C-yRpn4(1–82), pGFPC-yRpn4(1–110), and pGFP-C-yRpn4(172–229) were obtained by cloning the RPN4 fragments downstream of the GFP gene in pEGFP-N1. Plasmid pyRpn4(177–327)-N-DAFP was derived from

Proteins were separated in 10% or 13% Tris–Glycine polyacrylamide gels followed by transfer onto nitrocellulose membranes (BioRad, USA). The GFP and GFP-yRpn4 chimeras were detected using polyclonal rabbit anti-GFP antibodies (Abcam, USA) taken 1:1000 or mouse monoclonal anti-GFP antibodies taken 1:1000 (ProteinSynthesis, Russia) and goat anti-mouse, or anti-rabbit HRP conjugated antibodies, both were used 1:20,000 (Enzo, USA and Abcam, USA respectively). AFP was detected using polyclonal rabbit anti-AFP antibodies diluted 1:1000 and goat anti-rabbit HRP conjugates (both from Abcam, USA). To stain yRpn4, we used rabbit anti-yRpn4 antibodies [14]. Ubiquitin was revealed using rabbit anti-Ub antibodies taken 1:1000 (Abcam, USA). The blots were detected using ECL kit (GE Healthcare, UK). To ensure equal loading of protein in each lane, the membranes were stripped and stained with anti-b-actin antibodies diluted from 1:1000 to 1:2000 (Sigma–Aldrich, Germany). 2.5. Proteasome inhibition assay with MG132/lactacystin and cycloheximide/puromycin chase Thirty hours post transfection, HEK 293T cells were treated with 10 lM MG132/lactacystin (both from Sigma–Aldrich, Germany) and incubated for 18 h at 37 °C. Treated cells were then examined using a DMI 4000B fluorescent microscope (Leica, Germany), after that, samples were investigated by flow cytometry or lysed and analyzed by Western blotting. The translation inhibitors cycloheximide (CHX, Sigma–Aldrich, Germany) and puromycin (Sigma–Aldrich, Germany) were used to evaluate the kinetics of recombinant protein degradation; 48 h post transfection, HEK 293T cells were treated with 100 lg/ml (final concentration) cycloheximide or 10 lg/ml puromycin. Immediately after treatment and after 2, 4 and 6 h of incubation with the translation inhibitors, the cells were scraped and lysed, and the cellular lysates were analyzed by Western blotting. Experiments were performed twice. 2.6. Accumulation chase Forty-eight hours post transfection, the cell culture media was supplemented with MG132 (10 lM); and after incubation for 2, 4 and 6 h, the cells were lysed and analyzed for recombinant protein accumulation by Western blotting. The signals were normalized by staining the membrane with anti-b-actin antibodies (Sigma–Aldrich, Germany). X-ray films with the detected bands

Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017

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were analyzed using ImageJ software (for reference http:// www.di.uq.edu.au/sparqimagejblots can be used). In brief, rectangular sections were placed over the detected protein bands; histograms indicating the intensity of each of the bands were obtained; the peaks were separated from the background and the peak area value indicating the intensity of the band was calculated. To normalize signal intensities the same was performed with the b-actin bands. Relative protein band intensity values were multiplied on the coefficients derived from b-actin band measurements. Finally, the normalized signal intensity values were used to build accumulation curves using Microsoft Excel. The method is further described in the results.

and prepared for Western-blotting by boiling protein-G-agarose with protein loading buffer. 3. Results and discussion 3.1. yRpn4 is degraded by proteasomes of higher eukaryotes To assess the stability of yRpn4 in human cells, HEK 293T cells were transfected with the plasmid expressing yRpn4 under control of the cytomegalovirus (CMV) promoter, and the yRpn4 level was measured by Western blotting. Under normal conditions, we were unable to detect yRpn4. However, the protein band corresponding to yRpn4 was clearly observed when cells were treated with MG132 or lactacystin. Our results highlight the involvement of human proteasomes in the fast turnover of yRpn4 (Fig. 1B). To evaluate the rate of yRpn4 hydrolysis in HEK 293T cells, we performed a cycloheximide chase. However, we failed to assess the yRpn4 degradation kinetics due to the extremely low level of yRpn4 in the cells incubated without proteasome inhibitors (data not shown). To solve this problem, we designed an experiment to estimate yRpn4 degradation by monitoring its accumulation and called it an accumulation chase. To build reference accumulation curves,

2.7. Immunoprecipitation Protein-G-agarose slurry (Millipore, USA) was inactivated with bovine serum albumin in PBS at RT with shaking. Mouse monoclonal anti-GFP antibodies (ProteinSynthesis, Russia) (1:200) were incubated with cell lysates at 10 °C during 2 h. 20 ll of 50% BSA inactivated Protein-G-agarose slurry was added to each mixture and incubation was prolonged for 1.5 h at 10 °C. Slurries were washed once with 20 volumes of ice-cold PBS and twice with 20 volumes of PBS with 0.1% Tween. Immunocomplexes were eluted

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Fig. 2. Analysis of recombinant protein stability by accumulation chase. (A) Proteasome inhibition assay (left panels), cycloheximide chase (middle panels) and accumulation chase (right panels) of GFP, DAFP, GFP-ODCsignal and DAFPODCsignal. (B) Dynamics of recombinant protein accretion in HEK 293T cells. Accumulation curves for GFP, DAFP, GFP-ODCsignal, DAFPODCsignal and yRpn4 were calculated using the ImageJ software. Tests were performed in triplicates. The average values and standard deviation are shown. (C) Accumulation chase of yRpn4. b-Actin signal intensity values were used for normalization. Recombinant proteins and b-actin are indicated by arrow heads.

Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017

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A.V. Morozov et al. / FEBS Letters xxx (2014) xxx–xxx

we used plasmids expressing two stable proteins, Alpha-fetoprotein lacking export signal (DAFP) and green fluorescent protein (GFP), and unstable chimeric proteins, GFP and DAFP fused to the ODC degron (GFP-ODCsignal and DAFPODCsignal respectively). It should be noted that, in the presence of a proteasome inhibitor, CMV-driven transcription could be stimulated and contribute to protein accumulation [1]. To account for this effect, all proteins were expressed from vectors where genes of interest were under control of identical CMV promoters. Using a proteasome inhibition assay and cycloheximide chase, we confirmed the slow degradation kinetics of GFP and DAFP and fast proteasomal hydrolysis of GFP-ODCsignal and DAFPODCsignal (both half-lives approximately 3 h) in transfected cells (Fig. 2A). Interestingly, in the accumulation chase, we observed accumulation with the constant rate of stable GFP and DAFP for 6 h (Fig. 2A and B). In contrast, unstable GFPODCsignal DAFPODCsignal accumulated rapidly within 2 h after the addition of MG132 followed by a much slower rate of accumulation. The initial brisk accumulation of fast-degrading proteins may be explained as an effect of proteasome inhibition. The subsequent slow rate of accumulation likely represents partial reversibility of inhibition and enhancement of proteasome activity due to a negative feedback mechanism of proteasome regulation [27]. We have shown that the accumulation kinetics of yRpn4 is closer to that of the fast-degrading GFP-ODCsignal and DAFPODCsignal (Fig. 2B and C). Therefore, we can conclude that yRpn4 is an unstable protein and a proteasome substrate in human cells.

3.2. N-terminal degron of yRpn4 does not induce proteasomal degradation of the yRpn4-GFP chimera in HEK 293T cells It was shown that the first 80 N-terminal amino acids of yRpn4 comprise a portable ubiquitin-independent degron in yeast [7] (Fig. 1A). To estimate its activity in HEK 293T cells, we analyzed the stability of chimeras containing N-terminal fragments of yRpn4 fused to a reporter protein (GFP) using a proteasome inhibition assay, cycloheximide chase, fluorescent imaging and flow cytometry. First, we again confirmed the stability of wild type GFP (Fig. 3A). Next, we found that administration of MG132 into the culture growth media of transfected cells only slightly influenced the yRpn4(1–82)-N-GFP and GFP-C-yRpn4(1–82) levels (Fig. 3B and C). These chimeras were also stable in the cycloheximide chase (Fig. 3B and C). These data suggest that the N-degron is likely inactive in HEK 293T cells. Bioinformatic analysis of yRpn4 identified a potential PEST signal downstream of the N-terminal degron at a.a 88–110 (Suppl. Fig. 1A and B). To check its activity, we analyzed the stability of yRpn4(1–110)-N-GFP and GFP-C-yRpn4(1–110). We observed a stronger effect of MG132 on the accumulation of GFP-C-yRpn4(1–110) in transfected cells; however, both chimeras were again stable in the cycloheximide chase (Fig. 3B and C). It has been shown that the addition of cycloheximide into cell culture media prevented heterologously expressed IgM heavy chains from ER-associated degradation, while another translation inhibitor, puromycin, did not [2]. In this regard, we investigated

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Fig. 3. No evidence of the yRpn4N-terminal part engagement in recombinant protein hydrolysis in HEK 293T cells. Data obtained from cells expressing GFP (A), data from cells expressing yRpn4 fragments as N-terminal or C-terminal fusions of GFP (B) and (C), respectively. Proteasome inhibition assay of cells transfected with the GFP-reporter constructs (left and middle panels). Cells were analyzed by fluorescent microscopy. GFP was detected in cellular lysates by Western blotting with anti-GFP antibodies. Presence (+) or absence ( ) of proteasome inhibitor MG132 in the transfected cell culture media is indicated. The stability of the recombinant proteins was evaluated using cycloheximide chase (right panels). Recombinant proteins and b-actin are indicated by arrow heads.

Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017

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A.V. Morozov et al. / FEBS Letters xxx (2014) xxx–xxx

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Fig. 4. The central domain of yRpn4 contains efficient degron(s) active in HEK 293T cells. Data obtained from cells expressing yRpn4 fragments as N-terminal or C-terminal fusions of GFP (A) and (B), respectively. Data from cells expressing GFP-ODCsignal (C). Proteasome inhibition assay of cells transfected with the GFP-reporter constructs (left and middle panels). Cells were analyzed by fluorescent microscopy. GFP was detected in cellular lysates by Western blotting with anti-GFP antibodies. Presence (+) or absence ( ) of proteasome inhibitor MG132 in the transfected cell culture media is indicated. The stability of recombinant proteins was evaluated by cycloheximide chase (right panels). Recombinant proteins and b-actin are indicated by arrow heads.

the degradation kinetics of GFP-C-yRpn4(1–110) in the presence of puromycin and confirmed the stability of the chimeric protein (Suppl. Fig. 2). The observed limited accumulation of yRpn4-GFP fusion proteins after the addition of MG132 may be explained by enhanced expression from the CMV promoter [1]. Indeed, RT-PCR showed that the mRNA levels of wild type yRpn4 and all of the four chimeras were slightly increased (Suppl. Fig. 3A and B). Therefore, we suggest only a minor influence of the N-degron and PEST-motif on GFP degradation in HEK 293T cells. To identify other potential degrons in the N-terminal domain of yRpn4, we analyzed the stability of yRpn4(1–176)-N-GFP. Using a proteasome inhibition assay, fluorescent imaging and flow cytometry, we demonstrated that the chimeric protein was nearly undetectable in transfected cells (Fig. 3B, Suppl. Figs. 4 and 5). In inhibitor-free conditions, only 12% of cells were confirmed positive by flow cytometry, compared to 81% for wild type GFP. After the addition of MG132, 27% of cells transfected with yRpn4(1–176)N-GFP were slightly above the threshold, in contrast to 85% for GFP (Suppl. Fig. 4). Moreover, the amount of yRpn4(1–176)-NGFP in transfected cell lysates was marginally increased after the administration of proteasome inhibitors (Fig. 3B, Suppl. Fig. 5). Interestingly, the levels of yRpn4(1–176)-N-GFP mRNA were extremely low (Suppl. Fig. 3B). Thus, we suggest that the N-terminal domain of yRpn4 does not contain degradation signals capable of stimulating hydrolysis of chimeric proteins in human cells.

3.3. The central domain of yRpn4 stimulates fast degradation of GFP and AFP Next, we examined the central portion of yRpn4. It was shown that the yRpn4 fragment a.a. 172–229 is a portable, ubiquitindependent degron in yeast [9]. To evaluate its activity, we fused it to GFP making the N-terminal and C-terminal fusions. The HEK 293T cells transfected with the corresponding constructs were subjected to a proteasome inhibition assay. After treatment with MG132, we observed insignificant increase in GFP fluorescence in transfected cells (Fig. 4A and B). Moreover, the chimeras were also stable during the 6 h cycloheximide chase (Fig. 4A and B). These data indicate that the ubiquitin-dependent degron of yRpn4 is ineffective in cells of higher eukaryotes. To search for other potential degrons in the central part of yRpn4, we analyzed the stability of yRpn4(177–327)-N-GFP. Transfected HEK 293T cells displayed significantly increased GFP fluorescence after MG132 treatment (Fig. 4A, Suppl Fig. 4), and analysis of cellular lysates by Western blotting suggested proteasome-dependent degradation of yRpn4(177–327)-N-GFP (Fig. 4A). These results were further confirmed using another proteasome inhibitor, lactacystin (Suppl. Fig. 5). Moreover, cycloheximide chase experiments showed that the half-life of yRpn4(177–327)-N-GFP was 1–1.5 h, which was in average 2 times lower than that of GFP-ODCsignal (half-life 2–2.5 h) (the experiments were performed twice) (Fig. 4A and C). Therefore, we suggest that the central domain of yRpn4 contains efficient degradation signal(s). Using previously

Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017

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Fig. 5. Immunoprecipitated yRpn4(177–327)-N-GFP is not tagged by Ub in lysates of transfected cells and central domain of yRpn4 induce rapid proteasomal hydrolysis of DAFP. (A) Immunoprecipitation with anti-GFP antibodies and Western blot of lysates of HEK 293T cells transfected with pEGFP-N1 and pyRpn4(177–327)-N-GFP and cultured in presence or absence of MG132. Membranes were stained with anti-UB (left panel) and anti-GFP antibodies (right panel). Samples in order: 1 – mock, 2 – mock +MG132, 3 – GFP, 4 – GFP +MG132, 5 – yRpn4(177–327)-N-GFP, 6 – yRpn4(177–327)-N-GFP +MG132, 7 – not immunoprecipitated mock lysate. Positions of marker-proteins of protein ladder are indicated. (B) Examination of proteasome-dependent degradation and evaluation of yRpn4(177–327)-N-DAFP stability in transfected HEK 293T cells by proteasome inhibition assay and cycloheximide chase. Presence (+) or absence ( ) of proteasome inhibitor MG132 in the transfected cell culture media is indicated. Recombinant proteins and b-actin are indicated by arrow heads.

described protocol [24,22] we utilized Escherichia coli Dam methylase as a reporter protein and confirmed that the yRpn4 fragment a.a. 177–327 represents a transferable degron in yeast (Suppl. Fig. 6). In addition we performed immunoprecipitation of recombinant GFP, yRpn4(177–327)-N-GFP from transfected cellular lysates with anti-GFP immunoglobulins. No precipitated proteins were detected as shown by Western blot with anti-Ub antibodies (Fig. 5A). Further on we studied the degradation of a truncated fusion protein bearing (a.a 230–327) fragment of the central part of yRpn4 fused with GFP. By fluorescent microscopy, proteasome inhibition assay and cycloheximide chase it was shown that the chimera is stable and undergoes slow degradation in HEK 293T cells (Fig. 4A). Interestingly, besides expected 37 kDa protein we observed two larger proteins of 42 and 50 kDa. These forms may represent proteins with different modifications such as ubiquitination or sumoylation, however it was not further investigated. Furthermore, Dam methylase activity test confirmed that the a.a 230–327 is a weak degron in yeast (Suppl. Fig. 6). Obtained data are in favor of the fact that the central domain of yRpn4 may represent a conformational degron. However, one can not exclude that central domain of yRpn4 contains two degrons: previously identified Ub-dependent that works in yeast, but doesn’t work in human cells and a conformational degron which is capable to induce degradation at least in mammalian cells and likely in yeast. Finally, to test if the elucidated part (a.a. 177–327) of the yRpn4 can serve as an effective interspecies degron, capable to destabilize different proteins we fused it with DAFP. The proteasome inhibition assay and the cycloheximide chase were performed on transfected HEK 293T cells. In the presence of MG132 the drastic increase of recombinant yRpn4(177–327)-N-DAFP in the cellular

lysate was demonstrated by Western blotting (Fig. 5B). Using cycloheximide chase half-life of the recombinant protein was estimated 2–3 h, suggesting efficient degradation. 4. Conclusions Degradation of the yeast transcription factor yRpn4 in human cells was investigated. It was found that yRpn4 expressed in transfected HEK 293T cells undergoes fast proteasomal degradation. Interestingly, N-terminal ubiquitin-independent degron of yRpn4 that was shown to function in yeast cells was incapable to promote the degradation of chimeric proteins in human cells. However, the central domain of yRpn4, which contains an ubiquitin-dependent degron effectively induced the degradation of GFP. Notably, fusion of the sole yRpn4 ubiquitin-dependent degron to the C- or N-termini was insufficient to destabilize the substrate. Accordingly, using immunoprecipitation with anti-GFP antibodies we detected no ubiquitinated proteins in yRpn4(177–327)-GFP transfected cellular lysates even though cells were treated with MG132. Thus, indicating that other structures in the central part of yRpn4 are important for proteosomal degradation in HEK 293T cells. Moreover, using Dam methylase assay we obtained evidence that yRpn4(177–327) can serve as a degron in yeast. Finally, we demonstrated that another recombinant protein carrying this degron yRpn4(177–327)-DAFP is rapidly hydrolyzed by the human proteasomes. Therefore, the central domain of yRpn4 might be considered as an efficient interspecies degradation signal. Key elements inside this part of yRpn4 responsible for degradation have yet to be elucidated. In general, the identification of generic structures that are essential for protein degradation can be beneficial for a variety of applications, including in therapies when artificial proteasome targeting of a specific protein is necessary. In this regard,

Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017

A.V. Morozov et al. / FEBS Letters xxx (2014) xxx–xxx

yeast degradation signals could be more favorable to mammalian degrons, such as that of ODC, due to a reduced risk of autoimmune reactions. Finally, future investigations of degradation signals could deepen our understanding of common principles of protein degradation that are shared by lower and higher eukaryotes. Acknowledgments We would like to thank Dr. Vladimir Morozov for important remarks, suggestions and editing the text. We also would like to acknowledge Dr. Olga Preobrazhenskaja for editing the manuscript and contribution to the discussions. The study was supported by the Grants from the Russian Foundation for Basic Research #1204-31863 and #14-04-00834, the ‘‘Molecular and Cellular Biology’’ and ‘‘Fundamental research for the development of biomedical technologies’’ programs of the Russian Academy of Sciences to V.K. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014.08. 017. References [1] Alvarez-Castelao, B., Martín-Guerrero, I., García-Orad, A. and Castaño, J.G. (2009) CMV promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors. J. Biol. Chem. 284 (41), 28253–28262. [2] Amshoff, C., Jäck, H.M. and Haas, I.G. (1999) Cycloheximide, a new tool to dissect specific steps in ER-associated degradation of different substrates. Biol. Chem. 380, 669–677. [3] Baugh, J.M., Viktorova, E.G. and Pilipenko, E.V. (2009) Proteasomes can degrade a significant proportion of cellular proteins independent of ubiquitination. J. Mol. Biol. 386 (3), 814–827. [4] Erales, J. and Coffino, P. (2014) Ubiquitin-independent proteasomal degradation. Biochim. Biophys. Acta 1843 (1), 216–221. [5] Ghoda, L., Van Daalen Wetters, T., Macrae, M., et al. (1989) Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 243 (4897), 1493–1495. [6] Ha, S.W., Ju, D. and Xie, Y. (2014) Nuclear import factor Srp1 and its associated protein Sts1 couple ribosome-bound nascent polypeptides to proteasomes for cotranslational degradation. J. Biol. Chem. 289 (5), 2701–2710. [7] Ha, S.W., Ju, D. and Xie, Y. (2012) The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits. Biochem. Biophys. Res. Commun. 419 (2), 226–231. [8] Jariel-Encontre, I., Bossis, G. and Piechaczyk, M. (2008) Ubiquitin-independent degradation of proteins by the proteasome. Biochim. Biophys. Acta 1786 (2), 153–177. [9] Ju, D. and Xie, Y. (2006) Identification of the preferential ubiquitination site and ubiquitin-dependent degradation signal of Rpn4. J. Biol. Chem. 281 (16), 10657–10662.

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Please cite this article in press as: Morozov, A.V., et al. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells. FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2014.08.017