Ubiquitin‐like domains can target to the proteasome but proteolysis requires a disordered region
2016; Springer Nature; Volume: 35; Issue: 14 Linguagem: Inglês
10.15252/embj.201593147
ISSN1460-2075
AutoresHouqing Yu, Grace Kago, Christopher M. Yellman, Andreas Matouschek,
Tópico(s)Protein Degradation and Inhibitors
ResumoArticle27 May 2016free access Source DataTransparent process Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region Houqing Yu Houqing Yu Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA Search for more papers by this author Grace Kago Grace Kago Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Christopher M Yellman Christopher M Yellman Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Andreas Matouschek Corresponding Author Andreas Matouschek orcid.org/0000-0001-6016-2341 Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA Search for more papers by this author Houqing Yu Houqing Yu Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA Search for more papers by this author Grace Kago Grace Kago Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Christopher M Yellman Christopher M Yellman Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Andreas Matouschek Corresponding Author Andreas Matouschek orcid.org/0000-0001-6016-2341 Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA Search for more papers by this author Author Information Houqing Yu1,2, Grace Kago1, Christopher M Yellman1 and Andreas Matouschek 1,2 1Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA 2Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA *Corresponding author. Tel: +1 512 2324045; E-mail: [email protected] The EMBO Journal (2016)35:1522-1536https://doi.org/10.15252/embj.201593147 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin-like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin-like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitin-like domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin-like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation. Synopsis The lack of proteolysis-initiating sequences stabilizes yeast ubiquitin-like domain-containing proteins despite their targeting to the proteasome, showing that protein stability is not solely controlled by proteasome affinity as determined by the UbL domains but also by the presence or absence of sites at which the proteasome can engage substrates to initiate the degradation. 10 out of 11 ubiquitin-like (UbL) domains tested can bind to proteasome and target model proteins to degradation in vitro and in yeast. Protein stability does not correlate directly with proteasome affinity but is modulated by accessibility of disordered region at which proteasomes can initiate the degradation. Burying of a disordered region in the UbL domain protein Mdy2 by its binding partner Get4 protects Mdy2 from proteasomal degradation. UbL domain protein Ubp6 is a stable proteasome-associated subunit but becomes degraded when a disordered region is provided. Introduction Ubiquitin is a small single-domain protein consisting of 76 amino acids that is conserved in all eukaryotes. It becomes attached to proteins post-translationally, often in long polyubiquitin chains in which the first ubiquitin is modified with additional ubiquitin moieties. The first role discovered for polyubiquitin chains was to target proteins to the proteasome for degradation, but it is now clear that ubiquitin chains are also involved in the regulation of cellular processes via non-proteolytic mechanisms ranging from membrane trafficking to DNA repair (Komander & Rape, 2012). It is not fully understood how cells use ubiquitin chains to specify distinct cellular processes. Biochemically, ubiquitin chains function as transferable protein–protein interaction tags and are recognized by ubiquitin binding domains, sometimes within the same protein and sometimes in interacting proteins (Husnjak & Dikic, 2012). Individual ubiquitin moieties in polyubiquitin chains are connected to each other through different lysine residues, and chains with different linkage patterns are recognized with different affinities by ubiquitin receptors (Husnjak & Dikic, 2012; Komander & Rape, 2012). However, other properties of the modified proteins also affect their fate and provide additional information to the ubiquitin code (Prakash et al, 2004; Takeuchi et al, 2007; Zhao et al, 2010; Fishbain et al, 2011, 2015; Heinen et al, 2011; Inobe et al, 2011; van der Lee et al, 2014). Cells also encode a number of ubiquitin homologues and in budding yeast (S. cerevisiae), at least eleven proteins show homology with ubiquitin. The sequence identity within these ubiquitin-like (UbL) domains ranges between 7 and 53%, and they all share ubiquitin's β-grasp fold (Grabbe & Dikic, 2009) (Fig 1). Although some UbL domains bind to the proteasome, it is thought that none of the proteins that carry UbL domains are degraded by the proteasome. Figure 1. UbL domains Phylogenetic relationship of UbL domains in S. cerevisiae and schematic representations of the corresponding UbL proteins. Sequences were clustered and mapped onto a N-J tree by ClustalX2 (Larkin et al, 2007) and the PHYLIP package (Felsenstein, 1989). ASP, aspartyl protease domain; UBA, ubiquitin-associated domain; USP, ubiquitin-specific protease domain; DD, dimerization domain; RBD, Rad4-binding domain; ST2, Sti1/Sti1 domain pair. Structural models of UbL domains generated by PyMOL (The PyMOL Molecular Graphics System, Version 0.99 Schrödinger, LLC). PDB IDs: Atg8 2ZPN, Atg12 3W1S, Dsk2 2BWF, Hub1 1M94, Mdy2 4GOC, Rad23 3M62, hHR23B 1P1A, Rub1 1BT0, Smt3 1EUV, Urm1 2PK0, ubiquitin 1UBQ. Download figure Download PowerPoint Ubiquitin-like proteins fall into two classes, ubiquitin-like modifiers (ULMs) and ubiquitin-like domain proteins (UDPs). Ubiquitin-like modifiers are attached to proteins reversibly and post-translationally, like ubiquitin (van der Veen & Ploegh, 2012). In yeast, the ULMs are Atg8, Hub1, Rub1 (Nedd8), Smt3 (SUMO), and Urm1, and these proteins are not thought to bind to the proteasome (Table 1). Rub1 may be an exception in that it can be incorporated into polyubiquitin chains, which in turn can be recognized by the proteasome (Singh et al, 2012). Ubiquitin-like domain proteins are larger than ubiquitin and typically contain multiple domains, with only one homologous to ubiquitin. Four of the six UDPs in yeast are associated with the ubiquitin proteasome system (UPS) and interact directly with the proteasome through their UbL domains. Rad23, Dsk2, and Ddi1 contain N-terminal UbLs, which are recognized by the proteasome subunits Rpn1, Rpn10, and Rpn13 (Elsasser et al, 2002; Funakoshi et al, 2002; Kaplun et al, 2005; Husnjak et al, 2008; Zhang et al, 2009; Gomez et al, 2011). Rad23, Dsk2, and Ddi1 also contain one or more ubiquitin-associated (UBA) domains, which bind to polyubiquitin chains and allow the proteins to serve as diffusible proteasome substrate receptors (Saeki et al, 2002a; Elsasser et al, 2004; Kim et al, 2004; Verma et al, 2004; Elsasser & Finley, 2005; Kaplun et al, 2005; Zhang et al, 2009). The Ubp6 protein binds to the proteasome subunit Rpn1 through a UbL domain at its N-terminus and through interactions of its catalytic domain (Leggett et al, 2002; Rosenzweig et al, 2012; Aufderheide et al, 2015; Bashore et al, 2015; Shi et al, 2016). Ubp6 modulates proteasome activity by trimming ubiquitin chains on proteasome substrates and by modulating proteasome activity allosterically (Crosas et al, 2006; Hanna et al, 2006; Koulich et al, 2008; Peth et al, 2009; Lee et al, 2010; Bashore et al, 2015). Neither the UbL-UBA proteins nor Ubp6 are degraded by the proteasome. The remaining two UDPs are not associated with the UPS. Atg12 contains a C-terminal UbL domain and participates in the autophagy pathway (Mizushima et al, 1998), whereas Mdy2 contains a central UbL domain and is involved in the biogenesis of tail-anchored proteins in the endoplasmic reticulum (Wang et al, 2010; Chartron et al, 2012a). Table 1. Physical and sequence properties of UbL domains analyzed in this study as well as the function of the relevant proteins Protein Class Full length (aa) UbL domain (aa) Identity to Ub (%) Identity to UbLRad23 (%) Secondary structure identity to Ub (%SSE) Function Ki relative to UbLRad23 (μM) Rad23 UDP 398 1–77 22 – 83 DNA excision repair; UPS 0.45 ± 0.04 hHR23B UDP 409 1–82 30 27 100 DNA excision repair; UPS 2.0 ± 0.2 Dsk2 UDP 373 1–76 29 28 67 Spindle pole body duplication; UPS 3.3 ± 0.3 Atg8 ULM 117 1–117 20 14 83 Autophagy 3.5 ± 0.2 Hub1 ULM 73 1–73 22 18 83 Pre-mRNA splicing; morphogenesis 9.8 ± 0.8 ubiquitin – 76 1–76 – 22 – Ubiquitination 16 ± 1 Ddi1 UDP 428 1–80 20 19 N/A Mating type switching; UPS 17 ± 2 Ubp6 UDP 499 6–80 17 23 N/A Deubiquitination 24 ± 3 Urm1 ULM 99 1–99 28 6 83 Sulfur carrier in tRNA modification 30 ± 3 Mdy2 UDP 212 74–152 22 34 83 Biogenesis of TA proteins 33 ± 3 Rub1 ULM 77 1–77 53 22 100 Cullin proteins neddylation 34 ± 2 Smt3 ULM 101 22–98 16 17 50 Sumoylation 65 ± 4 Atg12 UDP 186 101–186 7 9 83 Autophagy N/A Ub, ubiquitin; UDP, ubiquitin-like domain protein; ULM, ubiquitin-like modifier; UPS, ubiquitin proteasome system; N/A, not available. Here, we ask how the UbL domains specify different functions. The simplest model would be that domains in proteins not involved in the UPS do not bind to the proteasome. To test this model, we selected twelve ubiquitin-like domains, eleven encoded by S. cerevisiae and one by H. sapiens, and examined whether they could target proteins to the yeast proteasome. We found that the yeast proteasome can bind all UbL domains tested, albeit with different affinities. The in vivo functions of the domains do not correlate with their apparent proteasome affinities; some UbL domains not involved in the UPS can bind the proteasome more tightly than bona fide interactors. We then asked why the proteasome does not degrade UbL proteins. We propose that proteins escape proteasomal degradation when they lack unstructured regions that allow the proteasome to initiate degradation. We tested this hypothesis on two ubiquitin domain proteins, Ubp6 and Mdy2. Ubp6 naturally lacks sites at which the proteasome can initiate degradation, but was depleted rapidly when a disordered region was introduced. The N-terminal domain of Mdy2 is disordered but is buried by its binding partner Get4. Deleting Get4 exposed the disordered region and led to the degradation of Mdy2. We conclude that protein stability is determined not only by proteasome affinity, but also by the presence of sites at which the proteasome can engage its substrates and initiate degradation. Results UbL domains bind the proteasome To investigate whether UbL domains bind to the proteasome, we tested whether the purified domains could displace a proteasome substrate and prevent its degradation. We constructed a model substrate with the UbL domain of S. cerevisiae Rad23 at its N-terminus followed by a superfolder green fluorescent protein (GFP) domain (Pédelacq et al, 2006) and finally a previously characterized C-terminal disordered region of 95 amino acids derived from S. cerevisiae cytochrome b2 to allow the proteasome to initiate the degradation (Inobe et al, 2011) (UbLRad23-GFP-95; Fig 2A). We then expressed the protein in E. coli, isolated it, and presented it to purified yeast proteasome. The protein was degraded efficiently and at a rate comparable to that observed for model substrates targeted to the proteasome by ubiquitin tags under equivalent conditions (Fishbain et al, 2011; Kraut & Matouschek, 2012). The degradation followed Michaelis–Menten behavior and the measured KM value was independent of proteasome concentration, whereas Vmax values scaled linearly with it (Fig 2C). Thus, it was possible to characterize the degradation process using standard kinetic approaches. Figure 2. UbL domains bind with proteasome Sketch of UbLRad23-GFP-95 consisting of the UbL domain of S. cerevisiae Rad23, followed by superfolder GFP and a 95-amino acid-long tail derived from S. cerevisiae cytochrome b2. In vitro degradation of UbLRad23-GFP-95 by purified S. cerevisiae proteasome. The graph plots the amount of substrate over time, estimated by fluorescence intensity monitored by plate reader (green) or electronic autoradiography of SDS–PAGE gel bands (red), normalized to the initial substrate amount as described in Materials and Methods. Michaelis–Menten analysis of UbLRad23-GFP-95 degradation by different concentrations of purified S. cerevisiae proteasome (green, 10 nM; red, 40 nM). UbLRad23 inhibits UbLRad23-GFP-95 degradation. The initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition. The UbLDsk2 domain is a competitive inhibitor of UbLRad23-GFP-95 degradation. Lineweaver–Burk plot of UbLRad23-GFP-95 degradation with different concentrations of purified UbLDsk2 domain. Data information: (B–E) Proteasomal degradation of UbLRad23-GFP-95 monitored by fluorescence intensity in a Tecan plate reader as described in Materials and Methods. Download figure Download PowerPoint To investigate the binding of different UbL domains to the proteasome, we cloned and expressed the domains in E. coli and purified the corresponding proteins. We first characterized the UbL domains from Rad23 and Dsk2 because these two domains are known to bind to the proteasome (Schauber et al, 1998; Elsasser et al, 2002; Funakoshi et al, 2002; Saeki et al, 2002b). Adding increasing amounts of the purified UbL domains inhibited the degradation of UbLRad23-GFP-95 (Figs 2D and E, and EV1). Conversely, increasing amount of substrate overcame the inhibition by the UbL domains (Fig 2E). Thus, substrate and UbL domains competed for binding to the proteasome. The apparent equilibrium constants (Kis) with which the competing UbL domains inhibited substrate degradation reflect the affinity of the domains to the proteasome (Table 1, Fig EV1). The measured Ki values report only on binding to the sites at which the Rad23 UbL domain interacts with the proteasome. It is possible that the other UbL domains also bind to other sites on the proteasome (Gomez et al, 2011; Shi et al, 2016) but these interactions would not be detected by our assay, causing us to underestimate affinities in these cases. Click here to expand this figure. Figure EV1. UbL domains inhibit UbLRad23-GFP-95 degradation A–L. Initial degradation rates of UbLRad23-GFP-95 in the presence of different concentrations of purified UbL domains plotted against the competitor concentration and fitted to the equation describing competitive inhibition. The UbL domains were from yeast Rad23 (A), Dsk2 (C), Atg8 (D), Hub1 (E), ubiquitin (F), Ddi1 (G), Ubp6 (H), Urm1 (I), Mdy2 (J), Rub1 (K), Smt3 (L), and hHR23B (B). Graphs show one representative dataset of at least three independent experiments. Download figure Download PowerPoint We then measured the ability of nine additional UbL domains, as well as monoubiquitin, to compete with Rad23's UbL domain for the proteasome (Table 1). The apparent Kis fell into a range from 0.5 to 65 μM. UbL domains that are known to associate with the proteasome were distributed over the entire range of affinities, with monoubiquitin roughly in the middle. UbL domains can target artificial substrates for proteasomal degradation in vitro Next we examined whether all UbL domains that bound to the proteasome could also target proteins for degradation. We first addressed this question in vitro with model proteasome substrates consisting of a central E. coli dihydrofolate reductase (DHFR) domain with the relevant UbL domain fused to its N-terminus and the 95-amino acid tail described above fused to its C-terminus, creating UbL-DHFR-95 (Fig 3A). We expressed these proteins by coupled in vitro transcription and translation and presented them to purified yeast proteasome. The UbL domain of Rad23 mediated efficient degradation. However, DHFR-95 without the UbL domain and UbLRad23-DHFR without the tail were stable (Fig 3A). Treatment with the proteasome inhibitor MG132 stabilized the UbLRad23-DHFR-95 protein, indicating that degradation was proteasome dependent (Fig 3A). Strikingly, all of the other UbL domains tested also mediated degradation (Fig 3B). As before, degradation depended on the presence of a disordered tail and was repressed by the proteasome inhibitor MG132. The rates and extent of degradation in the in vitro assays varied somewhat for the different UbL domains, but the variation did not seem to depend on whether the domains were naturally involved in proteasome-dependent processes, nor did they correlate with the apparent inhibition constants (Kis). For example, the yeast SUMO homolog Smt3 targeted DHFR for degradation as effectively as the Rad23 UbL domain but bound to the proteasome more weakly (Fig 3A and B, Table 1). Figure 3. UbL domains target substrate to proteasome degradation in vitro A, B. In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95-amino acid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino acid tail (no tail, blue diamonds), or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS–PAGE gel bands over time as normalized to the initial substrate amount as described in Materials and Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m. Source data are available online for this figure. Source Data for Figure 3 [embj201593147-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint UbL domains can target proteins for proteasome degradation in vivo We next investigated whether UbL domains could mediate degradation by the proteasome in yeast. We created model substrates based on imidazoleglycerol-phosphate dehydratase (His3) and yellow fluorescent protein (YFP). The abundance of His3 protein was estimated by its ability to support growth, and the abundance of yellow fluorescent protein (YFP) by measuring cellular fluorescence. His3 catalyzes an essential step in histidine production in yeast, so that his3 mutant strains cannot grow in medium lacking histidine unless the mutation is complemented by a plasmid-borne wild-type HIS3 gene (Alifano et al, 1996). However, if the His3 protein expressed from the plasmid is degraded, then the complementation will fail. Proteasomal degradation of His3 protein can therefore modulate the yeast growth rate. We tested whether UbL domains could target His3 protein for degradation in yeast by attaching them to the N-terminus of His3. The fusion proteins were expressed from a GAL1 promoter on a 2-micron (multicopy) plasmid in a his3 mutant yeast strain (Fleming et al, 2002) (Appendix Table S1). We added 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of the His3 enzyme, to make the growth assay more sensitive (Hawkes et al, 1995). To allow the proteasome to initiate the degradation of any bound His3 fusion protein, we also fused a 51-amino acid disordered tail (derived from subunit 9 of the Fo component of the N. crassa ATPase, abbreviation Su9) to the C-terminus of His3 (Fig 4A). Expression of His3 with an N-terminal DHFR domain and the Su9 tail complemented his3 mutant cells, indicating that N- and C-terminal modifications alone did not impede His3 activity (Fig 4B). However, fusing the Rad23 UbL domain to the N-terminus of His3 with a Su9 tail prevented the complementation and inhibited cell growth (Fig 4B). His3 fusion protein could not be detected by Western blotting in cell lysate, unless the proteasome was inhibited by the proteasome inhibitor bortezomib (Fig EV2A), suggesting that the growth defect was caused by the depletion of UbL-His3-tail protein by the proteasome. Indeed, most of the yeast UbL domains we investigated prevented His3 from complementing the his3 mutation to some extent. The exceptions were the UbL domains of Ddi1 and Urm1, which affected growth similarly under selective and nonselective conditions, indicating that UbLDdi1 and UbLUrm1 failed to target His3 fusion proteins to degradation effectively. None of the UbL domains themselves disrupted His3 function as UbL-His3 fusion proteins lacking the C-terminal proteasome initiation tails were stable (Fig EV2B) and supported growth (Fig 2B). Expressing either UbL-His3-tail or UbL-His3 constructs when histidine was present in the medium did not affect yeast growth. The simplest interpretation of our observations is that the different UbL domains target His3 to the proteasome. Furthermore, degradation by the proteasome required a disordered region at which the proteasome could initiate the degradation. Figure 4. UbL domains target His3 substrates to proteasome degradation in yeast Schematic representation of yeast growth assay. The model proteins consisted of an UbL domain and S. cerevisiae imidazoleglycerol-phosphate dehydratase (His3), followed by stop codon (no tail) or a 51-amino acid tail derived from subunit 9 of the Fo component of the Neurospora crassa ATPase (Su9) at the C-terminus. In his3 mutant cells, the absence of His3 protein caused a growth defect when grown on selective media. Stable His3 proteins escaped proteasome degradation and rescued the his3 mutant cells from the growth defect. UbL domains mediated the degradation of His3 fusion proteins with a Su9 tail and affected yeast growth under selective condition (+3-AT, −his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. The plates were incubated at 30°C for 3 days for imaging. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. UbL domain-mediated His3 protein degradation depends on proteasome, but not ubiquitination In the E1 temperature-sensitive strain uba1-204, the cellular abundance of UbL-His3-tail proteins was similar at the permissive (30°C) and restrictive (37°C) temperatures. In contrast, a His3 fusion protein containing a classic N-end rule degron was stabilized at the restrictive temperature (37°C), showing that its degradation depended on the ubiquitination machinery. Bortezomib stabilized both the UbL-His3-tail and N-degron-His3-tail proteins. UbL-His3 proteins without initiation region were stable and not affected by the shift to the restrictive temperature or treatment with bortezomib. Data information: Protein levels were determined by Western blotting for the HA-tag and for Scs2 protein as a loading control in SDS–PAGE gels of S. cerevisiae protein extracts. Proteasome degradation was tested by the addition of the proteasome inhibitor bortezomib. Source data are available online for this figure. Download figure Download PowerPoint The his3 complementation experiments report on cellular protein abundance indirectly through the His3 protein's enzymatic activity and its effect on cell metabolism and growth. The abundance of fluorescent proteins can be estimated directly from the total cell fluorescence measured by flow cytometry (Yen et al, 2008; Sharon et al, 2012). Therefore, we repeated the degradation experiments with yellow fluorescent protein (YFP) as the reporter protein. We fused either one of the eleven UbL domains or a DHFR domain to the N-terminus of YFP and a Su9 tail to its C-terminus, and then expressed the fusion proteins (UbL-YFP-tail) from the constitutive promoter pACT1 on a CEN plasmid derived from pYCplac33 (Gietz & Sugino, 1988). The same plasmid also expressed the red fluorescent protein dsRed Express2 (Strack et al, 2008) from a second pACT1 promoter as a normalization control for variation in plasmid copy number, global protein expression levels, and cell size. We again first investigated the yeast Rad23 UbL domain, measuring the fluorescence of yeast cells expressing the fusion protein UbLRad23-YFP-tail together with dsRed (Fig 5A). Replacing the UbL domain with DHFR increased the yellow fluorescence roughly 9-fold, while the red fluorescence remained unchanged (Fig 5A). Inhibiting the proteasome with bortezomib increased yellow fluorescence without changing red fluorescence (Figs 5A and EV3B). Degradation required a C-terminal tail; removing the tail increased YFP fluorescence 9-fold and bortezomib did not affect the fluorescence of cells expressing UbLRad23-YPF protein without the tail (Fig EV3B and C). Figure 5. UbL domains target fluorescence substrates to proteasome degradation in yeast Fluorescence-based degradation assay in S. cerevisiae. The substrate proteins consisted of a UbL domain, followed by a yellow fluorescent protein (YFP) domain and a disordered tail (51 amino acids derived from subunit 9 of the Fo component of the Neurospora crassa ATPase, Su9). Control substrates lacked the disordered tail. Substrates and a red fluorescent protein were expressed from consecutive constitutive promoters (pACT1) on the same CEN plasmid (YCplac33). Fluorescence profiles of cells expressing different substrates monitored by flow cytometry. UbLRad23 targeted YFP-Su9 protein for degradation. Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilized YFP protein in cells. UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots the abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence was also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Materials and Methods. Data points represent the mean values determined from at least three repeat experiments; error bars indicate s.e.m. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. UbL domain-mediated YFP protein degradation depends on proteasome, but not ubiquitination Schematic representation of UbL-YFP constructs. The fluorescent substrates consisted of a UbL domain, followed by a yellow fluorescent protein (YFP) domain and a tail (51 amino acids derived from subunit 9 of the Fo component of the Neurospora crassa ATPase, Su9) or a stop codon at the C-terminus. dsRed Express2 (expression control) and UbL-YFP-Su9 (substrate) were expressed from the consecutive constitutive ACT1 promoters on the same CEN plasmid (YCplac33). Stabilization of UbL-YFP substrates with tails (Su9, gray) or without tails (no tail, red) by proteasome inhibitor (bortezomib) treatment. The extent of recovery was calculated as the fraction of the YFP/RFP ratios of cells expressing each construct after bortezomib treatment divided by the YFP/RFP ratios for cells with the same construct after DMSO treatment. Levels of UbL-YFP proteins with tails (Su9, gray) or without tails (no tail, red) in the E1 temperature-sensitive strain uba1-204 at the permissive temperature (30°C; UbL-YFP-Su9, dark gray; UbL-YFP, dark red) and the restrictive temperature (37°C; UbL-YFP-Su9, light
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