the ubiquitin - proteasome system ( ups ) is the major pathway for protein degradation in eukaryotic cells , regulating most cellular processes , including cell division , signal transduction , and development ( finley , 2009 ) . before degradation , proteins are conjugated to ubiquitin chains that act as recognition signals for the 26s proteasome , a large proteolytic complex that degrades substrate proteins ( finley , 2009 ) . although proteasome function has been extensively studied , our knowledge of how this particle recognizes ubiquitylated substrates remains incomplete . since the identification of the first intrinsic proteasomal ubiquitin receptor , rpn10 , studies have identified a group of so - called ubl - uba domain proteins that act as transient , extrinsic proteasome substrate receptors ( deveraux et  al . , 1994 ; seeger et  al . , 2003 ; su and lau , 2009 ; wilkinson et  al . , more recently , an additional novel intrinsic receptor , rpn13 , was identified ( husnjak et  al . , 2008 ; however , budding yeast cells , deleted for the ubl - uba domain proteins and mutated in both the rpn10 and rpn13 ubiquitin - interacting regions , are still viable ( husnjak et  al . , 2008 ) . moreover , ubiquitin conjugates still bind to 26s proteasomes lacking the ubiquitin - interacting regions of rpn10 and rpn13 ( peth et  al . , 2010 ) . as proteasome function is essential , at least one additional ubiquitin receptor remains to be discovered ( saeki and tanaka , 2008 ) . here , we present structural , biochemical , and genetic data that the disordered and multifunctional protein dss1 ( known as sem1 in budding yeast ) , is another ubiquitin - binding subunit of the 26s proteasome . in fission yeast , substrate recognition by the 26s proteasome is accomplished by two intrinsic proteasome subunits , rpn10 and rpn13 , and two extrinsic ubl - uba domain proteasome cofactors , rhp23 and dph1 ( finley , 2009 ; hartmann - petersen et  al . , 2003 ; sakata et  al . , 2012 ; wilkinson et  al . , 2001 ) studies have shown these receptors to be functionally redundant ( husnjak et  al . , 2008 ; peth et  al . , 2010 it was previously demonstrated , both in budding and fission yeast , that the gene for the ubl - uba domain protein rad23 ( rhp23 in fission yeast ) functionally overlapped with the  gene encoding the 26s proteasome ubiquitin receptor subunit rpn10 . specifically , only a double deletion mutant ( rpn10rhp23 ) displayed severe growth defects ( wilkinson et  al . in addition , rhp23 variants unable to bind ubiquitin or the proteasome could not rescue the growth defects of the double mutant , implying that substrate recognition was at least partly responsible for the observed phenotypes ( wilkinson et  al . , 2001 ) . therefore , we asked whether lack of the ubiquitin- or proteasome - binding functions of rpn10 contribute to the severe phenotype of the rpn10rhp23 double mutant . to this end , we cloned constructs of rpn10 that lacked the ubiquitin interaction motif ( uim ) , rpn10uim , or the n - terminal proteasome - binding region , rpn1082 ( figure  1b ) ( seeger et  al . , 2003 ) . these strains were then crossed , and the ability of the rpn10 constructs to rescue the growth defects of the rpn10rhp23 double mutant were assayed by plating and selecting for the relevant spores . surprisingly , this revealed that the rpn10uim construct rescued the growth defects as efficiently as the full - length construct ( figure  1c ; figure  s1a available online ) , while the rpn1082 proteasome - binding mutant did not ( figure  1c ) . this implies that loss of rpn10 ubiquitin binding does not contribute to the severe phenotype of the rpn10rhp23 double mutant . the fact that the rhp23rpn10uim mutant is viable is consistent with previous work , suggesting that the vwa domain has some unknown facilitator function in the ups ( mayor et  al . , 2007 ; peth et  al . , 2010 ; verma et  al . , 2004 ) and shows that other proteasomal substrate receptors functionally overlap with rpn10 and rhp23 . currently , the remaining known receptors and shuttle proteins are the ubl - uba protein dsk2 ( dph1 in fission yeast ) and rpn13 ( rpn13a and rpn13b in fission yeast ) that associate with both ubiquitin and the proteasome . to test these candidates genetically , null mutants were constructed for each and subsequently crossed to create the appropriate genetic backgrounds . we postulated that , if either of these receptors functionally overlapped with rpn10 and rad23 , then  deletion of its gene in the rpn10rhp23 background should prevent rescue of the rpn10rhp23 phenotype by the rpn10uim construct . surprisingly , the rpn10uim construct once again rescued the dph1rpn10rhp23 triple ( figure  s1b ) and rpn13arpn13brpn10rhp23 quadruple deletion mutants ( figure  s1c ) . this implies that neither dph1 nor rpn13 were responsible for the rescue of the rpn10rhp23 growth defects by rpn10uim . such candidates should either be proteasome subunits or proteasome - associated proteins and would be expected to display synthetic phenotypes with mutants in rpn10 or rhp23 . when searching the saccharomyces genome database , we found that the proteasome subunit , called sem1 in budding yeast ( funakoshi et  al . 2004 ) and dss1 in humans and fission yeast ( joss et  al . , 2006 ) , fulfills these criteria . to assess if dss1 functions as a proteasomal ubiquitin receptor , we first tested its ability to interact directly with ubiquitin chains . we performed an in  vitro ubiquitin - binding assay using glutathione s - transferase ( gst)-dss1 and k48- and k63-linked ubiquitin chains . indeed , under these conditions , gst - dss1 efficiently interacted with both k48 and k63 ubiquitin chains , while gst alone did not ( figure  2a ) . in general , ubiquitin receptors recognize ubiquitin via a conserved hydrophobic patch around ile44 ( husnjak et  al . , 2008 ) . to test if dss1 also binds ubiquitin via this hydrophobic area , we assayed the ability of dss1 to interact with the i44a ubiquitin mutant . compared to wild - type ubiquitin that clearly interacted with dss1 , i44a ubiquitin did not efficiently associate with dss1 or rhp23 ( figure  2b ) . this suggests that the ubiquitin ile44 patch is important for efficient dss1 and rhp23 binding . scrutinizing the dss1 sequence left us unable to identify any resemblance to known ubiquitin - binding sites ( ubss ) or domains ( husnjak and dikic , 2012 ) . structural prediction analyses of dss1 suggested it to belong to the intrinsically disordered proteins ( idps ) ( figure  2c ) ( uversky , 2011 ) . pondr ( obradovic et  al . , 2003 ) , but not iupred ( dosztnyi et  al . , 2005 ) , predicted that a short stretch in the dss1 c terminus is structured ( figure  2c ) . to probe this further , we analyzed dss1 by heteronuclear nuclear magnetic resonance ( nmr ) spectroscopy . assigned c chemical shifts relative to random coil shifts ( figure  2d ) ( kjaergaard et  al . , 2011 ) , combined with a low - dispersion n , h - heteronuclear single quantum correlation ( hsqc ) spectrum ( figure  2e ; figure  s2a ) , conclusively identified dss1 as intrinsically disordered with a single , transiently populated  helix from f55 through k66 . successive addition of excess ubiquitin and analysis by nmr uncovered two distinct ubss , identified from chemical shift perturbation analyses . titration analyses with increasing amounts of ubiquitin disclosed the strongest binding to ubiquitin by binding site i ( ubs - i ) , which is located at d38d49 ( dissociation constant , kd ,  = 50   30  m ) and disclosed the second and weakest site , ubs - ii , located at d16n25 ( apparent kd > 1  mm ) ( figure  2f ; figures s2b and s2c ) . these ubss are conserved and located in the disordered region of dss1 ( figure  s3 ) . notably , both sites have a similar sequence , characterized by a series of hydrophobic residues flanked by acidic residues ( figure  s3 ) . we subsequently mapped the corresponding interaction surface on ubiquitin by nmr , using c , n - labeled ubiquitin ( figure  3 ) . the perturbations of peak intensities of ubiquitin , imposed by addition of dss1 ( figure  3a ) , mapped consistently to the surface - exposed common hydrophobic binding surface of ubiquitin involving the  sheet and the hydrophobic residues i13 , l69 , and i44 ( figures 3b3d ) but is also extended to the c terminus , resembling the binding site exploited by the e2 ubiquitin - conjugating enzyme cdc34 ( arrigoni et  al . , 2012 ; choi et  al . , 2010 ; spratt and shaw , 2011 ) . several positively charged residues located on the same surface were also significantly perturbed , whereas no perturbations were seen on the opposite face of ubiquitin ( figure  3c ) . a representation of the electrostatic surface of ubiquitin revealed a tripartite binding site of a hydrophobic patch flanked by two positively charged regions ( figures 3e and 3f ) . this directly mirrors the architecture of the ubss identified in dss1 ( figure  s3 ) . moreover , the size of the interaction surface and the length of each ubs in dss1 strongly suggest that the two ubss bind independently to  each their ubiquitin moiety . of note , we observe that , depending on the linkages , there are unequal distances from the dss1 binding site on ubiquitin to a second dss1 binding site on a linked ubiquitin , suggesting that dss1 may express a preference in the selection of different lysine - linked ubiquitin chains . as expected from the nmr data , mutation of either ubs - i ( l40a , w41a , w45a ) or ubs - ii ( f18a , f21a , w26a ) clearly reduced binding to ubiquitin , and no ubiquitin binding was observed for dss1 mutated at both sites ( figure  4a ) . consistent with ubs - i being the stronger of the two binding sites , mutation of this site also had a greater effect on ubiquitin binding ( figure  4a ) . for better understanding of the functional relevance of dss1 and the importance of its ubiquitin - binding activity , a range of yeast mutants was created and tested in growth assays under various conditions . expression of dss1 or any of the dss1 variants did not affect cell growth of wild - type cells ( figure  s4a ) , whereas deletion of the dss1 gene resulted in a growth defect that was especially pronounced at higher temperatures ( figure  4b ) . when introducing the dss1 variants into the dss1 strain , we observed that cells expressing dss1 , mutated at both ubs - i and ubs - ii , displayed a significant growth defect ( figure  4b ) , while each of the single ubs mutants or wild - type human dss1 only partially restored growth ( figure  4b ) . similar effects were observed on media containing canavanine ( figure  s4b ) , a drug that inhibits protein folding and induces cell stress . notably , these genetic effects correlated with the cellular accumulation of ubiquitin - protein conjugates . thus , ubiquitin - protein conjugates accumulated in the dss1 strain , and this accumulation was not affected by ectopic expression of dss1 mutated in both ubs - i and ubs - ii ( figure  4c ) . expression of either dss1  ubs - i or dss1  ubs - ii mutants partially reduced the level of ubiquitin conjugates in the dss1 strain , while expression of wild - type s.  pombe dss1 or human dss1 fully reduced ubiquitin - protein conjugates to wild - type levels ( figure  4c ) . we next analyzed if any of the dss1 mutants were also compromised in proteasome binding . we found that wild - type dss1 , as well as individual dss1  ubs - i and dss1  ubs - ii mutants , all efficiently coprecipitated 26s proteasomes ( figure  4d ) . however , dss1 mutated in both ubs - i and ubs - ii failed to interact with 26s proteasomes ( figure  4d ) . hence , the strong phenotype of dss1 mutated in both ubs - i and ubs - ii is likely caused by both loss of ubiquitin binding and loss of proteasome binding . in contrast , the intermediate phenotypes of dss1 with single mutations in ubs - ii or , in particular , in ubs - i can likely be attributed to a reduced ubiquitin binding since they still bind to the proteasome . recently , dss1 was shown to function in proteasome assembly ( tomko and hochstrasser , 2014 ) . to assess the importance of dss1 on overall proteasome integrity , we isolated 26s proteasomes from a dss1 strain and analyzed them biochemically . we found that proteasomes lacking dss1 still efficiently interacted with polyubiquitylated proteins ( figure  s4c ) and were proteolytically active ( figure  s4d ) . this suggests that , structurally , 26s proteasomes are not strongly affected by loss of dss1 and that the contribution of dss1 to the proteasomal substrate binding capacity in  vitro is lower compared to the already known substrate receptors . this agrees with previous in  vitro activity studies of purified proteasomes , lacking all known ubss , which suggest the existence of an additional low - affinity substrate binding site ( peth et  al . , 2010 ) . to further rule out that the observed phenotype of the dss1 null mutant was not caused by a general loss of 26s proteasome integrity , we performed label - free quantitative mass spectroscopy , comparing 26s proteasomes purified from wild - type , rpn10 , rpn10uim , and dss1 cells ( figures s4e and s4f ) . in agreement with data from budding yeast ( bohn et  al . , 2013 ; tomko and hochstrasser , 2014 ) , loss of dss1 caused a modest reduction in 26s proteasome integrity ( figures s4e mutation of the dss1  ubs - i only slightly reduced the amount of rpn10 in the 26s proteasome ( figure  s4h ) . loss of rpn10 was more disruptive , with the amounts of 26s proteasomes being reduced to around 10% of that found in wild - type cells ( figures s4e collectively , these data imply that ubiquitin binding is important for the function of dss1 in the 26s proteasome in  vivo and that dss1 could be responsible for the viability of the  rhp23rpn10uim strain ( figure  1c ) . this being the case ,  then loss of dss1 should impart growth defects in the  rhp23rpn10uim strain . indeed , spore viability of the dss1rhp23rpn10uim strain was reduced compared to cells expressing the full - length rpn10 protein ( figures 4e and 4f ) . when introducing wild - type dss1 and the dss1  ubs - i and ubs - ii mutants in the dss1rhp23rpn10uim strain , we found that neither the dss1  ubs - i mutant nor the dss1  ubs - ii mutant was able to fully restore growth of the dss1rhp23rpn10uim strain ( figure  4f ) , suggesting that the ubiquitin - binding function of dss1 , described here , is important for proteasomal function and cell viability . in this article , we demonstrate that dss1 has a previously uncharactized function as a ubiquitin - binding protein of the 26s proteasome : unlike other receptors , dss1 interacts with ubiquitin via an unstructured ubs . given the highly conserved nature of the ups and the dss1 gene itself ( 47% identity between fission yeast and human dss1 ) , and given that human dss1 complements the phenotype of a fission yeast dss1 mutant , we propose that dss1 acts as a ubiquitin receptor in all eukaryotes . most ubiquitin - binding proteins have well - defined and structured ubiquitin - binding domains or small motifs ( husnjak and dikic , 2012 ) . this is in sharp contrast to proteins interacting with the ubiquitin - like modifier sumo that , in general , associate via short motifs located in intrinsically disordered regions ( vogt and hofmann , 2012 ) . we suspect that other ubiquitin - binding proteins may interact by a similar mechanism . in general , disordered proteins are not well conserved in sequence ( uversky , 2011 ) , and by homology searches , we have not been able to identify other proteins containing any dss1-like ubss . however , we did note some similarity between the sites in dss1 and the ubss found in the e2 - 3r family of e2 ubiquitin - conjugating enzymes ( arrigoni et  al . , 2012 ) such as cdc34 ( choi et  al . , 2010 ) . intriguingly , a recently described disordered region of cdc34 binds an area on ubiquitin similar to the area we identified for dss1 ( arrigoni et  al . , 2012 ; , 2010 ; spratt and shaw , 2011 ) , suggesting that these binding regions are required to be unstructured . previous studies in budding yeast have shown that cells lacking all known proteasomal ubss still remain viable ( husnjak et  al . , 2008 ) . the data presented here reveal that the same is true for fission yeast , but this viability , at least in part , depends on dss1 . what happens to ubiquitylated substrates after reaching the 26s proteasome , but prior to or during degradation , is still an open question . for instance , we know little about the events taking place during the initial substrate capture by rpn10 and rpn13 , localized at the tip of the regulatory particle , and the translocation to the central atpase ring . it is possible that substrates are handed over from the outer receptors to an inner receptor more proximal to the atpase ring . the localization of dss1 near the atpase pore and the deubiquitylating subunit rpn11 ( bohn et  al . , 2013 ) the disordered and flexible nature of dss1 could then allow for interaction with substrates presented in various orientations . however , like most disordered proteins ( uversky , 2011 ) , dss1 is multifunctional , even within the 26s proteasome , where it appears to act both structurally and functionally . recently , budding yeast sem1 was shown to play an important role in proteasome assembly ( tomko and hochstrasser , 2014 ) . specifically , sem1 catalyzes incorporation of subunits rpn3 and rpn7 into the 19s regulatory complex through sites that overlap with ubs - i and ubs - ii in fission yeast dss1 . although our proteomic analyses of dss1 26s proteasomes do not indicate that the level of rpn3 or rpn7 is reduced compared to that of other subunits of the lid complex , we also noted that dss1 , mutated in both ubs - i and ubs - ii , is not incorporated into 26s proteasomes . notably , the dss1 mutant in ubs - i alone was still incorporated into 26s proteasomes but continued to display the temperature - dependent growth defect and ubiquitin - conjugate stabilization . this suggests that the phenotypes connected with the dss1 ubiquitin - binding activity is limited to that of the dss1  ubs - i , which has a much greater affinity for ubiquitin compared to ubs - ii . however , dss1 also has proteasome - independent functions , including associating with dna repair proteins ( yang et  al . , 2002 ) and the transcription - export complex ( ellisdon et  al . , 2012 ; faza et  al . , we speculate that the ubiquitin - binding activity of dss1 may also play a functional role for these cellular processes . in conclusion , our studies suggest the intrinsically disordered protein dss1 as a ubiquitin receptor for the 26s proteasome in fission yeast . since dss1 is phylogenetically conserved , we propose that dss1 acts as a ubiquitin receptor in all eukaryotes . the strains were all derived from the s.  pombe wild - type heterothallic 972h and 975h . standard genetic methods and media were used ( moreno et  al . , 1991 ) . the plasmids used for expression of rpn10 and dss1 in fission yeast were prep41 carrying the budding yeast leu2 gene for selection and the nmt41 promoter or the pdual vector carrying ura4 for selection and the nmt1 promoter ( matsuyama et  al . , 2004 ) . antibodies to mts4/rpn1 have been described elsewhere ( wilkinson et  al . , 2001 ) . other antibodies were commercially available : flag ( sigma ) , green fluorescent protein ( gfp ; sigma ) , tubulin ( abcam ) , 20s proteasome mcp231 ( enzo ) , t7 ( bethyl ) , and ubiquitin ( dako ) . the 26s proteasomes , flag - tagged on mts4 ( rpn1 ) , were purified as described elsewhere ( verma et  al . , 2002 ) . the proteolytic activity of affinity - purified 26s proteasomes with or without dss1 was measured in the presence or absence of 5  m of the proteasome inhibitor bortezomib ( lc laboratories ) using the suc - llvy - amc substrate ( enzo ) as described elsewhere ( groll et  al . , 2006 ) . all dss1 proteins were expressed in escherichia coli bl21 ( de3 ) from the pgex6p1 or pdest15 vectors by standard methods . harvested cells were lysed by sonication in a buffer containing 12.5  mm tris - hcl , ph 7.5 , 37.5  mm nacl , 1  mm phenylmethylsulfonyl fluoride and complete mini protease inhibitor tablets ( roche ) . following centrifugation at 13,000   g , the cleared lysates were tumbled with glutathione - sepharose beads ( ge healthcare ) for 1  hr at 4c and extensively washed with the lysis buffer . coprecipitation assays were performed as described elsewhere ( wilkinson et  al . , 2001 ) . for the ubiquitin precipitation studies , 3  g of k48- and k63-linked ubiquitin chains ( boston biochemicals ) were used per precipitation in 100  l buffer a , containing 12.5  mm tris - hcl , ph 7.5 , 37.5  mm nacl . the protein / bead ratio was adjusted to about 1  mg / ml , and 10  l of beads were used per assay . after 2  hr of tumbling at 4c , the beads were washed twice with 1  ml of buffer a with 0.5% triton x-100 and once with buffer a. bound protein was eluted by boiling with sds sample buffer . some ubiquitin blots were boiled for 30  min after transfer to enhance reactivity and blocked with 5% bsa in pbs . the t7-tagged sic1-py was purified and in  vitro ubiquitylated as described elsewhere ( kriegenburg et  al . , 2008 ) . k.p . performed the protein purification experiments in figures 2a , 2b , and s4 .
summarythe ubiquitin - proteasome system is the major pathway for protein degradation in eukaryotic cells . 
 proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26s proteasome . 
 the proteasome subunits rpn10 and rpn13 are known to bind ubiquitin , but genetic and biochemical data suggest the existence of at least one other substrate receptor . here 
 , we show that the phylogenetically conserved proteasome subunit dss1 ( sem1 ) binds ubiquitin chains linked by k63 and k48 . 
 atomic resolution data show that dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues . 
 the complementary binding region in ubiquitin is composed of a hydrophobic patch formed by i13 ,  i44 , and l69 flanked by two basic regions . 
 mutations in the ubiquitin - binding site of dss1 cause growth defects and accumulation of ubiquitylated proteins .