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• W2052302154 abstract "Article11 January 2013free access Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes? James A Nathan James A Nathan Department of Cell Biology, Harvard Medical School, Boston, MA, USA Department of Medicine, NIHR Cambridge Biomedical Research Centre, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Hyoung Tae Kim Hyoung Tae Kim Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Lily Ting Lily Ting Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Steven P Gygi Steven P Gygi Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alfred L Goldberg Corresponding Author Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author James A Nathan James A Nathan Department of Cell Biology, Harvard Medical School, Boston, MA, USA Department of Medicine, NIHR Cambridge Biomedical Research Centre, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Hyoung Tae Kim Hyoung Tae Kim Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Lily Ting Lily Ting Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Steven P Gygi Steven P Gygi Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alfred L Goldberg Corresponding Author Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information James A Nathan1,2, Hyoung Tae Kim1, Lily Ting1, Steven P Gygi1 and Alfred L Goldberg 1 1Department of Cell Biology, Harvard Medical School, Boston, MA, USA 2Department of Medicine, NIHR Cambridge Biomedical Research Centre, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK *Corresponding author. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Tel.:+1 617 432 1855; Fax:+1 617 232 0173; E-mail: [email protected] The EMBO Journal (2013)32:552-565https://doi.org/10.1038/emboj.2012.354 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 Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway. Introduction Degradation of intracellular proteins by the 26S proteasome is mediated primarily by the conjugation of polyubiquitin (polyUb) chains to substrates (Finley, 2009). The formation of these chains requires three types of cellular enzymes (E1, E2, and E3), which catalyse the covalent attachment of Ub molecules to lysine residues in the target protein (Hershko and Ciechanover, 1998; Pickart, 2001). As a result, at least seven different types of Ub linkages can be formed, depending on whether the Ubs are attached to K6, K11, K27, K29, K33, K48, or K63 on the proximal Ub (Ikeda and Dikic, 2008; Kim et al, 2009). In addition to homogeneous chains composed of a single type of specific linkage, chains composed of mixed linkages are also formed (Kim et al, 2007). K48-Ub chains appear to be the primary signal for proteasomal degradation, and attachment of four or more Ub molecules to the protein is sufficient to target proteins to the proteasome (Thrower et al, 2000). In vivo, all types of Ub chains, except K63-chains, accumulate when proteasome function is blocked (Jacobson et al, 2009; Xu et al, 2009). However, formation of K63 chains on cell proteins directs them to other fates, especially endosomal trafficking to the lysosome, intracellular signalling, and DNA repair (Ikeda and Dikic, 2008). Nevertheless, isolated K48- and K63-ubiquitinated proteins bind to purified 26S proteasomes with similar affinities (Kim et al, 2007; Peth et al, 2010) and support substrate degradation at comparable rates (Hofmann and Pickart, 2001; Kim et al, 2007; Saeki et al, 2009). This surprising lack of chain preference in vitro is clearly opposite to observations in vivo, where treatment with proteasome inhibitors does not affect the stability of proteins conjugated to K63 chains (Xu et al, 2009). Furthermore, only K48 but not K63-polyubiquitinated proteins have been observed to co-localize with proteasomes in cells (Newton et al, 2008). The eukaryotic 26S proteasome is a 2.5 megadalton, ATP-dependent complex composed of the hollow cylindrical 20S core particle, which contains the proteolytic sites, and the 19S regulatory particle, which binds ubiquitinated substrates. This particle contains two subunits that function as high affinity receptors for ubiquitinated proteins Rpn10 (S5a) and Rpn13 (Husnjak et al, 2008; Schreiner et al, 2008; Peth et al, 2010), which do not distinguish K48 or K63 chains in vitro (Peth et al, 2010). The present studies were undertaken to learn why proteins linked to K63 chains in cells do not become bound to proteasomes, as they do with purified 26S particles. Three types of mechanisms can explain why K63-ubiquitinated proteins do not undergo proteasomal degradation in vivo. (1) Factors may exist in cells that either prevent their binding to the 19S complex or (2) promote their deubiquitination by one or more of the 26S-associated deubiquitinating enzymes (DUBs), Usp14, Uch37, or Rpn11 (Finley, 2009), and release from the 19S without proteolysis. Alternatively, (3) cytosolic factors may exist that selectively enhance the binding of K48-ubiquitinated substrates to the 26S. To determine how K63-ubiquitinated proteins are protected from proteasomal degradation, we examined in mammalian cell extracts the binding of K48- and K63-ubiquitinated proteins to the 26S complex. Using more physiological approaches than in prior studies of Ub-binding proteins (UBPs), we have identified cell proteins that bind preferentially to K63-ubiquitinated proteins and block their binding to proteasomes. We show here that components of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway (Williams and Urbe, 2007) associate strongly with K63 chains, provided they contain more than four Ubs, and can prevent binding of purified as well as endogenous K63-ubiquitinated proteins to the 26S, thus targeting them to the endosomal-lysosomal pathway. In addition, the Rad23 proteins (human homologues of yeast Rad23 (hHR23)), which have been proposed to ‘shuttle’ proteins to the proteasome (Elsasser et al, 2004), associate specifically with K48 conjugates and promote their binding to the 26S complex. Together, these chain-specific UBPs seem to determine whether Ub conjugates are degraded by lysosomes or proteasomes in vivo. Results In cell extracts proteasomes bind preferentially to K48-polyUb chains To determine whether cells contain factors that influence the binding of K63-polyUb chains to the 26S, we first compared the binding of purified 26S to resin-bound K63- and K48-polyubiquitinated proteins, using the assay described by Peth et al (2010). Ub conjugate-affinity columns were formed by incubating the ligases, E6AP or Nedd4 (bound to a GST resin), with E1, E2, Ub and ATP. E6AP forms homogenous K48-polyUb chains, and Nedd4 homogenous K63-polyUb chains (Supplementary Figure S2A; Kim et al, 2007). Kim et al showed that both these HECT E3 ligases form Ub chains on a single lysine and not through multiple short monoUb chains. The washed resin-bound ubiquitinated proteins were incubated with pure 26S proteasomes at 4°C, and the amounts of bound proteasomes were measured by assaying the cleavage of LLVY-AMC at 37°C (Peth et al, 2010). This assay of activity was shown to accurately reflect the amount of 26S proteasomes bound to the Ub conjugates (Peth et al, 2010), as measured by immunoblot (see below), but the activity assay was faster and easier to quantitate. Using this method, we confirmed that purified 26S bind both types of chains with similar high affinities (Figure 1A, right panel), as reported previously (Peth et al, 2010). To learn whether mammalian cell extracts contain factors that might inhibit the binding of the Nedd4-K63 conjugates to proteasomes, rat muscle extracts were incubated with the resin-bound conjugates at 4°C (Figure 1A; Supplementary Figure S1A). To ensure that only proteasome activity was being measured with this assay, the control lysate was treated with the proteasome inhibitor, Bortezomib/Velcade, and the very low amounts of Bortezomib-insensitive activity were subtracted. After washing the resin, we found that in these tissue lysates, proteasomes bound efficiently to the K48 conjugates, but not to the K63 chains (Figure 1A, left panel). Thus, in the presence of cell extracts, proteasomes and Ub chains behave as they do in vivo. Figure 1.Proteins in lysate prevent proteasome binding to K63-polyUb chains. (A) Although pure proteasomes bind to K48 and K63 chains, proteasomes in the muscle lysate bind efficiently only to K48 conjugates. Ubiquitinated E6AP and Nedd4 were incubated with purified 26S particles and the bound proteasomes were measured by LLVY-AMC cleavage (right panel). These same ubiquitinated conjugates were incubated with a rat muscle extract (120 μg) (left panel), with or without the addition of Bortezomib (1 μM) and the proteasomes from the lysate that bound to the conjugates were measured by LLVY-AMC cleavage (Bortezomib residual activity was subtracted). (B) The cell lysate contains factors that reduce binding of K63, but not K48-polyUb conjugates to the proteasome. The proteasome depleted of rabbit muscle extract (200 μg) was incubated with the ubiquitinated substrates and proteasomes as shown in Supplementary Figure S1B. (C) DUB inhibitors do not influence the inhibition of K63-conjugate binding to proteasomes by cell extracts. Proteasome binding to the Ub conjugates was measured as in (B), with or without 4 mM N-ethylmaleimide (NEM) and 1 mM 1,10-o-phenanthroline (oPT). (D) Factors within the cell extract that inhibit the binding of K63 conjugates to the proteasomes, bind tightly to K63 chains and can be depleted from cell extracts. The rat muscle extract, ubiquitinated Nedd4 and 26S proteasomes were incubated as in (B). The unbound fraction of the lysate (flow-through) was then assayed for its capacity to block proteasome binding. The flow-through was incubated with a fresh column of ubiquitinated Nedd4 and proteasomes, and the conjugate bound proteasome fraction was then measured. (E) Multiple cellular proteins or complexes can prevent K63 conjugates binding to the proteasome. Proteins in the rat muscle lysate (4 mg/ml) were separated according to their molecular weight using a Sephacryl S300HR column. After a void volume of 7 ml, 0.5 ml lysate fractions were collected and incubated with the ubiquitinated Nedd4 and proteasomes, to assay their ability to block K63-conjugate binding. All values are the means±s.e.m. See also Supplementary Figure S2. E6, E6AP; N4, Nedd4. Download figure Download PowerPoint To ensure that this failure of the ubiquitinated Nedd4 to bind to the proteasome was due to the K63 chain and not the Nedd4 protein, we forced Nedd4 to attach to itself shorter K63 or K48 chains by incubating resin-bound Nedd4 with E1, E2, ATP, and K48 or K63-tetraUb (Ub4) in place of monoUb. With time, each type of tetraUb chain became covalently bound to Nedd4 (Supplementary Figure S2B). These conjugates were then incubated with the muscle extract. As expected, many more proteasomes became bound to the Nedd4-K48 chains than to the Nedd4-K63 conjugates (Supplementary Figure S2C). Thus, factors within the lysate must block 26S binding to the K63 conjugates. Lysate proteins bind to K63 conjugates and prevent proteasomal binding We therefore examined whether proteins in the cell lysate could prevent the binding of K63 chains to purified 26S particles. The muscle extract was first ultra-centrifuged for 6 h to remove the endogenous proteasomes (Supplementary Figure S2F and G; Gaczynska et al, 1993). This lysate and a set amount of purified 26S proteasomes were incubated with the resin-bound conjugates at 4°C, and the amount of bound proteasomes measured (Figure 1B; Supplementary Figure S1B). In contrast to the untreated K48- and K63-polyUb chains, which bound the proteasomes similarly, incubation with the lysate prevented K63 conjugates from binding to the purified 26S, but did not reduce K48-chain binding (Figure 1B). A similar selective inhibition of K63-chain binding was observed with lysates of rabbit and rat muscle, as well as HEK293 cells (Supplementary Figure S2E). Thus, soluble factors present in many (presumably all) mammalian cells inhibit K63-conjugate binding to 26S proteasomes. One possible explanation of these results could be that enzymes in the cell lysate caused the K63 conjugates to be deubiquitinated much more rapidly than the K48 conjugates. Although there should be little or no deubiquitination occurring since binding was assayed at 4°C, we examined whether there might be some deubiquitination during incubation with the extract. Immunoblots of the polyubiquitinated E6AP and Nedd4 did not show any significant difference in the levels of ubiquitination before or after incubation (Supplementary Figure S2H). To confirm that destruction of the K63 chains was not responsible for their failure to bind proteasomes, the resin-bound substrates and lysate were treated with N-ethylmaleimide (NEM) and 1,10-o-phenanthroline (oPT) to inactivate the two classes of DUBs, cysteine and metalloproteinases. Despite this treatment, the cell extracts still blocked only K63-chain binding to the proteasomes (Figure 1C; Supplementary Figure S2I). Although polyubiquitinated Nedd4 and E6AP bind strongly to purified proteasomes, they are not rapidly degraded in vitro (Peth et al, 2010). We therefore examined whether the cell extracts also prevent binding and degradation of K63-polyubiquitinated Sic1, a short-lived protein in vitro and in vivo (Saeki et al, 2009). This substrate was preincubated with pure 26S proteasomes, the proteasome-depleted cell extract, or both together for 15 min at 4°C, before incubation at 37°C, during which we assayed the degradation of polyubiquitinated Sic1 by immunoblot. As expected, the polyubiquitinated Sic1 was rapidly degraded by the 26S proteasomes (Supplementary Figure S3A, left). Although the Sic1-Ub conjugates were not deubiquitinated in the cell extract at 4°C, at 37°C the Ub chains were completely removed within 20 min. This rapid disassembly of K63 chains in mammalian extracts is consistent with prior reports (Cooper et al, 2009). Treatment of these samples with NEM and oPT prevented this deubiquitination, and also prevented the efficient degradation of the polyubiquitinated Sic1 by the proteasome (Supplementary Figure S3A, right). To differentiate the initial binding of the Ub conjugates to the proteasome from the subsequent deubiquitination, we measured the binding of the polyubiquitinated Sic1 to the 26S at 4°C. While K63-polyubiquitinated Sic1 bound to the pure proteasomes (Supplementary Figure S3B), incubation of the cell extract with the polyubiquitinated Sic1 prevented conjugate binding to the 26S (Supplementary Figure S3B). Thus, the capacity of the extract to block K63-chain binding to the proteasome was observed with multiple K63-polyubiquitinated substrates (Sic1 and Nedd4) and caused an inhibition of degradation. To determine whether the cell factors that prevent conjugate binding to the 26S did so by binding with higher affinity to the K63 chains, the cell extract was first depleted of these K63-binding proteins by incubation with the ubiquitinated Nedd4 column. To ensure depletion of K63-binding proteins, the resin-bound ubiquitinated Nedd4 was first incubated with different concentrations of the extract. The unbound (flow-through) fraction was removed and incubated with the fresh resin-bound ubiquitinated Nedd4. The conjugates were then incubated with pure 26S proteasomes, and the 26S bound fraction measured. Pretreatment with increasing amounts of cell extract caused greater inhibition of K63-conjugate binding to proteasomes (Figure 1D). The extract proteins reduced K63 chain binding up to 80% below the levels found with no extract. By contrast, after exposure to the K63 chain, the unbound fraction (flow-through) lost most of its capacity to prevent K63 conjugates binding to proteasomes. By contrast, pretreatment of the cell extract with the K48-Ub conjugates (which should deplete it of K48-binding proteins) had no effect on the ability of the extract to prevent K63-Ub chains binding to the 26S (Supplementary Figure S2J). Therefore, the cell proteins that bound specifically to K63 chains were also the ones that blocked proteasomal binding to K63 chains. To define the size distribution of the cell proteins that can block K63 conjugates from binding to proteasomes, we used gel filtration on a Sephacryl S300HR column to fractionate lysate proteins according to size. These fractions were then incubated with the resin-bound K63 chains to determine which fractions prevented K63 binding to the 26S (Figure 1E). Inhibition of Ub-conjugate binding was observed over a wide range of molecular weights, with maximal inhibition observed from 150 to 670 kDa. This wide range suggests that multiple proteins, and/or multimeric complexes inhibit K63 chains from binding to the 26S (see below). To define the nature of this association between these cellular proteins and the K63 conjugates, we examined whether increasingly stringent washes could release the K63-associated proteins and restore the ability of the conjugates to bind the 26S. After incubating the ubiquitinated Nedd4 with cell extracts, the resin-bound conjugates were washed with increasing sodium chloride concentrations (up to 900 mM) or detergents (1% Triton X-100 or 0.1% SDS), and then they were incubated with purified proteasomes and the bound 26S species measured (Supplementary Table S1). Neither the high salt concentrations nor the detergent reduced the inhibition of conjugate binding to proteasomes. Thus, a number of cellular proteins can block proteasome binding by associating tightly with K63 chains through non-ionic, presumably hydrophobic, interactions. Identification by mass spectrometry of K63- and K48-specific binding proteins These findings suggested that the proteins responsible for blocking proteasome binding contain one or more Ub binding domains (UBDs). If so, a large excess of a UBD should elute the UBD-containing cell proteins from the resin-bound conjugates and allow us to identify them by mass spectrometry (MS). After the resin-bound K48- and K63-polyUb conjugates were incubated with the HEK293 lysate and extensively washed, the proteins remaining bound to the Ub chains were eluted with a large excess of the Ub interacting motif (UIM) of S5a/Rpn10. The His-tagged UIM was then removed using NiNTA, and the eluted proteins separated by SDS–PAGE (Figure 2A; Supplementary Figure S1C). A few dominant protein bands were observed only in the Ub-conjugate samples (Figure 2A). The major protein migrating at 90 kDa that bound to both types of Ub chains was identified as USP5 by immunoblot (Supplementary Figure S4A). (This association of USP5 with conjugates was also used to measure the efficiency of the elution of UBPs from the resin; Supplementary Figure S4A, right.) MS analysis of the dominant bands in K48-polyUb sample identified them as hHR23B, hHR23A, and S5a/Rpn10 (a UIM-containing subunit of the 26S proteasome that is also found free in the cytosol; Kim et al, 2009). The Rad23 proteins contain a Ub-like (Ubl) domain that can bind to the proteasome as well as a UBA (Ub-associated) domain, which binds directly to Ub chains (Chen and Madura, 2002). Such UBA-Ubl proteins appear to help shuttle ubiquitinated substrates to the 26S (Elsasser et al, 2004; Verma et al, 2004). Figure 2.Identification of K63- and K48-specific Ub-binding proteins. (A) Representative silver-stained gel of proteins eluted from K48 or K63 conjugates. After washing columns, proteins bound to the control or ubiquitinated substrates (E6AP and Nedd4) were eluted with His10-UIM, and 10% of eluted volume was separated by SDS–PAGE. (B) Identified UBD-containing proteins demonstrate specificity for K48 or K63 chains. Control or ubiquitinated substrates (E6AP and Nedd4) were incubated with an HEK293 lysate. Following washing, proteins were eluted from the resin in SDS-loading buffer and immunoblotted for the Rad23 proteins, TOM1, Hrs, and STAM. The size range of ubiquitinated E6AP and Nedd4 is shown with the anti-Ub antibody. *Non-specific band with anti-hHR23B antibody. (C) The ESCRT0 components, Hrs and STAM, bind specifically to K63 chains, while hHR23A and hHR23B are selective for K48 conjugates. Purified recombinant hHR23A, hHR23B, Hrs, and STAM at concentrations of 10, 50, and 100 nM were incubated with the ubiquitinated E6AP or Nedd4 resins. (D) HHR23A and B bind specifically to K48 chains. Resin bound GST-hHR23B (100 nM) was incubated with increasing concentrations (10, 50, and 100 nM) of K48- or K63-Ub tetramers in TBSG with 0.1% Triton X-100. Following washing, proteins were eluted from the resin and immunoblotted for hHR23B or Ub. (E) ESCRT0 preferentially binds to longer K63 chains. GST–Hrs and His–STAM were incubated with increasing concentrations of K63-Ub tetramers or K63 chains of mixed lengths (3–9mers), and immunoblotted for Ub (left panel). A Coomassie stained gel of the Ub-chain inputs is also shown (right panel). (F) K48 or K63 tetramers were forced onto GST–Nedd4 as described in Supplementary Figure S2. These conjugates were then incubated with hHR23A, hHR23B, and STAM (10, 50, and 100 nM) and the bound protein fraction visualized by immunoblotting. Download figure Download PowerPoint In contrast surprisingly, few discrete gel bands were observed for proteins bound to the K63 conjugates (Figure 2A, right panel). Therefore, we concentrated the K63-eluted fractions by precipitation with TCA and analysed the precipitate by LS-MS/MS. In total, 80 proteins were identified in the K63-chain analysis and 106 in the K48-chain MS analysis (Table I; Supplementary MS data file). Among the K63-specific proteins were several components of the endosomal trafficking system, namely, Hrs, STAM, Vps37, and Epsin (Table I), none of which were found in the K48-bound fraction. Interestingly, other components of the endosomal pathway were found to bind to both K48 and K63 chains, including TOM(target of Myb)1, an endosomal trafficking protein (Yamakami et al, 2003; Seet et al, 2004), although more TOM1 peptides were identified in the K63 sample. Similarly, TOM1L2, a protein closely related to TOM1, and Tollip, a TOM1 interacting protein, were detected in both K48- and K63-bound fractions. Table 1. Proteins identified by mass spectrometry that bound to K48- or K63-polyubiquitin conjugates K48 specific K63 specific Both K48 and K63 Deubiquitinating enzymes Ataxin 3a USP13a USP25a USP5a Ubiquitin interacting proteins hHR23B HRS TOM1 hHR23A STAM1 TOM1L2 S5a Epsin1 Tollip FAF1 P62 (SQSTM1) WRNIP1 UFD1L Ubiquitin ligases HUWE1a Endosomal proteins VPS37C a aThese proteins also contain known UBDs. In addition to hHR23A and B, the other predominant proteins identified as binding to K48 conjugates are also known to be involved in proteasomal degradation. FAF1 and UFD1 form a complex with p97/VCP (homologue of yeast cdc48) (Meyer et al, 2000; Song et al, 2005), an AAA ATPase involved in targeting of ubiquitinated proteins by the proteasome for degradation. Also, HUWE1 is a Ub ligase that can associate with the 26S (Besche et al, 2009). All the proteins that were identified with confidence by MS (>1 peptide or manually verified), which encode UBDs and bound to the Ub conjugates, are listed in Table I. Two DUBs were identified as binding selectively to the K48 chains, USP25 and Ataxin 3, and two that bound to both types of conjugates, USP5 and USP13. Characterization of K48- and K63-binding proteins To learn which proteins influence proteasome binding to Ub conjugates, we focused on those proteins that demonstrated clear specificity for Ub linkages: the Rad23 proteins for K48 chains and the ESCRT0 complex (composed of Hrs and STAM) for K63 conjugates, as well as TOM1, which associated with both types of chains. Hrs, STAM, and TOM1 were all found by western blot in the gel filtration fractions of the lysates that showed the greatest inhibition of 26S binding (Supplementary Figure S4B). We also confirmed by immunoblotting the association of these UBD-containing proteins with the Ub conjugates identified by MS. When the resin-bound conjugates and non-ubiquitinated E3s were incubated with the cell extract, and the bound cellular proteins separated by SDS–PAGE, Hrs and STAM were bound only to the K63-ubiquitinated substrate, whereas hHR23B became bound only to K48 chains (Figure 2B). Again, TOM1 was found to associate with both K48 and K63 conjugates, but it showed greater binding to K63 chains. In addition, these UBD proteins did not bind to the non-ubiquitinated proteins; in particular, we did not detect any hHR23B binding to E6AP, as had been reported previously (Kumar et al, 1999). To confirm that these UBD-containing proteins interacted directly with the Ub conjugates and not through an association with another UBP in the extract, we expressed and purified recombinant forms of these proteins (Supplementary Figure S4C) and examined their ability to associate with the ubiquitinated substrates. The resin-bound polyubiquitinated E6AP and Nedd4 were incubated with increasing concentrations of the purified UBD-containing proteins, hHR23A, hHR23B, Hrs and STAM, and the fraction of each that bound to the resin was determined by immunoblotting. HHR23B, at concentrations up to 100 nM, bound only to the K48 conjugates (Figure 2C). Furthermore, we also found that GST-hHR23B bound selectively K48-linked tetraUb chains and showed no binding of K63 tetramers (Figure 2D). HHR23A also showed a clear preference for K48 conjugates, but some binding to K63 chains was observed at concentrations of 50 and 100 nM hHR23A (Figure 2C). Conversely, at all concentrations tested, the ESCRT0 proteins Hrs and STAM bound only to K63 conjugates (Figure 2C). Interestingly, using resin-bound GST–Hrs and His–STAM, we could barely detect any interaction between these ESCRT0 proteins with pure K63 tetramers (Figure 2E), suggesting a preference for longer chains. Accordingly, when we used a mixture of chains containing 3–9 Ub monomers, we observed selective binding of Hrs and STAM to chains composed of 6–9 Ub molecules, with minimal binding to K63 tetramers and no binding to Ub trimers (Figure 2E) or a K48-linked mixture of chains (Supplementary Figure S4D). Thus, the ESCRT0 proteins preferentially bind to K63 conjugates longer than 4 Ub molecules. It is noteworthy that nearly all prior in vitro studies of UBPs have utilized much shorter constructs (monoUb or di-Ub chains), whose behaviour clearly differs from that of these long Ub chains. Finally, to confirm that the UBD-containing proteins bound to these specific Ub chains and not to the E6AP or Nedd4 molecules, we incubated Nedd4 with E1, E2, ATP and either K63- or K48-tetraUb to force the attachment of K63 or K48 chains to Nedd4 (Figure 2F). HHR23B bound only to these Nedd4-K48 conjugates, whereas STAM predominantly associated with the Nedd4-K63 chains (Figure 2F). Thus, the Rad23 proteins show clear specificity for K48 conjugates, and the ESCRT0 proteins for the K63 chains. ESCRT0 prevents K63 conjugates from binding to the 26S proteasome To determine if the K63-specific proteins could in fact prevent the binding of proteasomes to the K63-conjugated E3, we incubated the resin-bound ubiquitinated Nedd4 or E6AP with increasing concentrations of pure Hrs or STAM and purified proteasomes. After 30 min, the resins were washed and the binding of 26S measured. Both Hrs and STAM inhibited the binding of K63-polyUb conjugates to the 26S in a concentration-dependent manner. Similarly, low concentrations (80 nM) of" @default.
• W2052302154 created "2016-06-24" @default.
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• W2052302154 date "2013-01-11" @default.
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• W2052302154 title "Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?" @default.
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