Uch2/Uch37 is the Major Deubiquitinating Enzyme Associated with the 26 S Proteasome in Fission Yeast

doi:10.1016/j.jmb.2004.09.057 J. Mol. Biol. (2004) 344, 697–706

Uch2/Uch37 is the Major Deubiquitinating EnzymeAssociated with the 26 S Proteasome in Fission Yeast

Miranda Stone1†, Rasmus Hartmann-Petersen1†, Michael Seeger2

Dawadschargal Bech-Otschir1, Mairi Wallace1 and Colin Gordon1*

1MRC Human Genetics UnitWestern General HospitalCrewe Road, Edinburgh, EH42XU Scotland, UK

2Medical Faculty ChariteHumboldt UniversityMonbijoustrasse 2, D-10117Berlin, Germany

0022-2836/$ - see front matter q 2004 E

† M.S. & R.H.-P. contributed equaAbbreviations used: GST, glutath

DAPI, 4 0,6-diamidino-2-phenylindoE-mail address of the correspond

[email protected]

Conjugation of proteins to ubiquitin plays a central role for a number ofcellular processes including endocytosis, DNA repair and degradation bythe 26 S proteasome. However, ubiquitination is reversible as a number ofdeubiquitinating enzymes mediate the disassembly of ubiquitin–proteinconjugates. Some deubiquitinating enzymes are associated with the 26 Sproteasome contributing to and regulating the particle’s activity.

Here, we characterise fission yeast Uch2 and Ubp6, two proteasomeassociated deubiquitinating enzymes. The human orthologues of theseenzymes are known as Uch37 and Usp14, respectively. We report that thesubunit Uch2/Uch37 is the major deubiquitinating enzyme associated withthe fission yeast 26 S proteasome. In contrast, the activity of Ubp6 appears toplay a more regulatory and/or structural role involving the proteasomesubunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6 becomesessential when activity of these subunits is compromised by conditionalmutations.

Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted,the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes mayremedy for the redundancy of these enzymes.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: hydrolase; proteasome; ubiquitin; UBP/UCH
*Corresponding author
Introduction

Intracellular protein degradation in eukaryoticcells plays an important role in a series of cellularand molecular functions, including the turnover ofbulk proteins, cell cycle control, DNA repair,antigen presentation, vesicle transport and in theregulation of signal transduction.1

To become degraded, proteins are normallyconjugated to a chain of ubiquitin moieties. Thisreaction is catalysed by three consecutively actingenzymes, E1, E2 and E3. First ubiquitin is activatedand bound to the E1 enzyme in an ATP-consumingprocess. Then ubiquitin is transferred to an E2enzyme and finally to the target protein associatedwith the E3 enzyme. Several rounds of thisconjugation reaction yields proteins carrying chainsof ubiquitin moieties.1,2 The ubiquitin chains lend

lsevier Ltd. All rights reserve

lly to this work.ione-S-transferase;le dihydrochloride.ing author:

the proteins affinity for the 26 S proteasome,3 amultisubunit protease responsible for most intra-cellular proteolysis. The 26 S proteasome is com-posed of two stable sub-complexes, the 20 Scatalytic core, a hollow cylindrical structure, and a19 S regulatory complex, which binds to one or bothends of the cylindrical 20 S particle.4

The 20 S proteasome on its own is a broad spectrumprotease, composed of four stacked heptameric ringsenclosing two antechambers and a central catalyticchamber.5 In yeast and higher eukaryotes the ringsare formed by two sets of subunits designated as a orb-type. Seven different a-subunits compose the twoidentical outer rings of the cylinder and sevendifferent b-subunits form each of the two identicalinner rings of the 20 S proteasome. Some of theb-subunits are threonine-type proteases and accountfor the proteolytic activity of the proteasome.5

The 19 S particle is an asymmetrical w700 kDaprotein complex which possesses a variety offunctions associated with proteasome-dependentproteolysis. These include the recognition ofubiquitinated substrates, substrate unfolding andthe translocation of substrates into the 20 S particle.4

d.

698 Proteasome-associated Deubiquitination

The process of ubiquitination is reversible.6,7 Atleast 18 different deubiquitinating enzymes, whichall potentially are able to remove or trim theubiquitin chains conjugated to the substrateproteins, are encoded in the Schizosaccharomycespombe genome (our unpublished results). When theproteasome degrades the ubiquitin-conjugatedsubstrate, the ubiquitin chains become detached.Several deubiquitinating isopeptidases orubiquitin-specific proteases are associated with the19 S particle of the 26 S proteasome, and thusprotein degradation and ubiquitin release seem tobe tightly coupled.8–12 Indeed deubiquitination canin certain cases be rate-limiting for proteasome-mediated protein degradation.11

Of the deubiquitinating enzymes associatedwith the 26 S proteasome, most notable arePad1/Rpn11,10–12 Uch2/Uch37,13,14 Doa48 andUbp6/Usp14.12,15–18 Whereas Pad1/Rpn11 andUch2/Uch37 are integral proteasome subunits,Doa4 and Ubp6/Usp14 associate more loosely withthe complex. The Doa4 enzyme has been shown toplay important roles in regulating the deubiquitina-tion pathways involved in vacuolar sorting19 anddegradation of membrane-bound proteins.20

Though most proteasome subunits andproteasome-associated proteins are remarkablywell conserved throughout eukaryotes, no obviousorthologue of the budding yeast Doa4 enzyme ispresent in either fission yeast or humans. However,these organisms contain the Uch2/Uch37 subunit,which is not encoded in the budding yeast genome.

Recently, a study showed that Ubp6/Usp14 isresponsible for most deubiquitinating activityassociated with the 26 S proteasome in buddingyeast.17 However, as this organism does not contain

an orthologue of Uch2/Uch37, we set out to analysethe proteasome-associated deubiquitinationactivity using the fission yeast S. pombe as a modelorganism.

Here, we show, contrary to the results frombudding yeast, that of the deubiquitinatingenzymes at the fission yeast 26 S proteasome,Uch2/Uch37 is responsible for the majority of theactivity. By extrapolation this means thatUch2/Uch37 is likely to be of similar importancein higher eukaryotes. Contrarily Ubp6/Usp14appears to play an elusive structural and/orregulatory role in cooperation with variousproteasome subunits, such as Mts1/Rpn9,Mts2/Rpt2 and Mts3/Rpn12, but curiously notwith Mts4/Rpn1, Pus1/Rpn10, Mts8/b1 and thedeubiquitinating subunit Pad1/Rpn11.

Results

The 26 S proteasomes from fission yeastcontain Uch2 and Ubp6

In order to study proteasome-mediateddeubiquitination activity in S. pombe, we purified26 S proteasomes from a strain carrying a proteinA-tagged Pus1/Rpn10 subunit. As shown, the 26 Sproteasomes from this strain were readily purifiedon IgG Sepharose beads (Figure 1a). Next, wewished to verify the presence of Uch2 and Ubp6 inour proteasome preparations. The purified proteinA-tagged proteasomes were separated on SDS/polyacrylamide gels and subjected to Western blotanalyses, probing with antibodies against Uch2/Uch37 and Ubp6/Usp14. As shown, both Uch2/

Figure 1. Purification of 26 Sproteasomes from S. pombe.a, Pus1/Rpn10 protein A-tagged26 S proteasomes were precipi-tated using IgG Sepharose beads,eluted and separated on SDS/polyacrylamide gels. The presenceof both the 20 S catalytic particleand the 19 S regulatory complexare indicated with brackets.b, Western blot analyses revealedthat the proteasome preparationscontained the deubiquitinatingenzymes Uch2/Uch37 and Ubp6/Usp14 as well as the 26 S protea-some subunit Mts4/Rpn1. Thoughthe calculated M of Ubp6 is only52.1 kDa, we consistently foundUbp6 at about 60 kDa on SDS-PAGE.

Proteasome-associated Deubiquitination 699

Uch37 and Ubp6/Usp14 were present in ourproteasome preparations (Figure 1b).

Direct interactions between proteasomesubunits and deubiquitinating enzymes

Ubp6/Usp14 is a UBL domain-containingprotein, whereas Uch2/Uch37 does not contain aUBL domain. Previously, we have shown that UBLdomain proteins like Rhp23/Dph1 and Udp7interact with the 26 S proteasome Mts4/Rpn1.21 Totest whether this was also the case for Ubp6 weperformed in vitro co-precipitation experimentsusing glutathione-S-transferase (GST)-taggedUbp6 and a preparation of Mts4/Rpn1. As expectedwe found that Ubp6 interacts with Mts4/Rpn1(Figure 2a), data which are in agreement withstudies from budding yeast.22 During the course ofthese experiments we found that Uch2 interactedweakly with another 26 S proteasome subunitcalled Pus1/Rpn10 (Figure 2a). However, Rpn10cannot be the sole 26 S proteasome subunit to whichUch2 can bind, as Pad1-protein A-tagged 26 Sproteasomes from wild-type and pus1D strainsboth contain Uch2 (Figure 2b).

Association of Uch2 and Ubp6 with the 26 Sproteasome in vivo

In order to determine whether these protein–protein interactions also occurred in vivo, weutilised our antibodies to carry out immuno-localisation studies on fixed S. pombe cells. Asshown, the deubiquitinating enzymes in question,Uch2/Uch37 and Ubp6/Usp14, showed a specificsubcellular localisation in a punctate patternaround the nuclear rim (Figure 3), a signal thatco-localises with other 26 S proteasome subunits,Mts4/Rpn1 and Pad1/Rpn11 (Figure 3). This resultis in accordance with previous cytology studies ofthe S. pombe 26 S proteasome, which showed thatthe 26 S proteasome localises to the interior of thenuclear envelope.23

Whereas Uch2 is a subunit of the 26 Sproteasome,13,14 the UBL domain protein Ubp6

were precipitated using IgG Sepharose beads from either withe precipitates was revealed using antibodies specific for Uc

is probably only loosely associated with theproteasome.17 To determine how large a fractionof Ubp6 is associated with 26 S proteasomes inwild-type cells we separated S. pombe extracts bycentrifugation in a glycerol gradient. When theresulting fractions were analysed by SDS-PAGE andWestern blotting we found Mts4/Rpn1 (Figure 4a)and Uch2/Uch37 (Figure 4b) exclusively present inthe high molecular mass fractions. Ubp6 was alsoabundant in the high molecular mass fractions(Figure 4c), though a small amount was stilldetectable in low molecular mass fractions. Thisindicates that most of the Ubp6 in wild-typeS. pombe cells is bound to the 26 S proteasome,whereas the remainder exists in an unbound form.

Characterisation of uch2 and ubp6 null-mutants

To gain further insight to the function of theseproteasome-associated deubiquitinating enzymes,null-mutants of Uch2/Uch37 and Ubp6/Usp14were generated by PCR mutagenesis (Figure 5).Both the uch2D and ubp6D strain were viable anddid not display the pleiotropic phenotypes, such assensitivity to heat shock or high temperatures,normally associated with defects in the ubiquitin/proteasome pathway (not shown). Nor didubiquitin–protein conjugates appear to be stabilisedin the mutant strains (not shown). However, whenthese null-mutants were crossed to various strainswhere the activity of the 26 S proteasome had beencompromised by mutations, severe growth defectswere observed (Table 1). As the proteasomesubunits are encoded by essential genes, theproteasome mutants are all conditional tempera-ture-sensitive mutants, at 25 8C the mutant strainsare viable, whereas at 36 8C the cells arrest anddisplay their mutant phenotype.24 As listed in theTable, Ubp6 displayed synthetic lethality withmutants in the Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12 proteasome subunits when grown at thepermissive temperature of 25 8C. However, thesephenotypes were not due to a general proteolysisdefect in these cells, as ubp6Dmts4-1, ubp6Dmts8-1,ubp6Dpus1D and ubp6Dpad1-1 double mutants were

Figure 2. Direct interactionsbetween 26 S proteasome subunitsand Uch2 and Ubp6. a, Beadscontaining GST-Uch2/Uch37,GST-Ubp6/Usp14 or GST wereincubated with preparations ofMts4/Rpn1 or Pus1/Rpn10 asindicated. Subsequent Westernblot analysis of bound proteinsusing antibodies specific for Pus1or Mts4 revealed that Ubp6/Usp14 interacts with Mts4/Rpn1while Uch2/Uch37 interacts withPus1/Rpn10. b, Pad1/Rpn11 pro-tein A-tagged 26 S proteasomes

ld-type or pus1D strains. The presence of Uch2/Uch37 inh2.

Figure 3. Subcellular localisation of deubiquitinating enzymes in S. pombe. a, Immunofluorescence microscopy offormaldehyde-fixed S. pombe cells revealed that Uch2/Uch37 (red) and Pad1/Rpn11 (green) co-localise (yellow) in apunctate pattern at the nuclear periphery. b, Similarly, Ubp6 (red) co-localises with Mts4/Rpn1 (green) in an identicalpattern (yellow) at the nuclear rim. DAPI staining (blue) was used to detect the nucleus.

Figure 4. Glycerol gradient cen-trifugation analyses. Wild-typeS. pombe extracts were separatedby centrifugation on a 10%–40%glycerol gradient. Every secondfraction was collected and sub-jected to SDS-PAGE and Westernblotting probing with antibodiesspecific for: a, Mts4; b, Uch2; andc, Ubp6 as indicated.


Figure 5. Null mutants in uch2C

and ubp6C. Extracts from wild-type(wt), uch2D and ubp6D were separ-ated on SDS/polyacrylamide gels,blotted and probed with antibodiesagainst Uch2/Uch37, Ubp6/Usp14and as control Mts4/Rpn1. Thelatter was used to ensure equalamounts were loaded.


viable at 25 8C. Together these data indicate that theMts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12 subunitsare likely to function in unknown pathways thatparallel the Ubp6 activity, whereas the Mts4/Rpn1,Mts8/b1, Pus1/Rpn10 and Pad1/Rpn11 subunitsare not. In contrast to Ubp6/Usp14, the activity ofUch2/Uch37 was not needed for viability of any ofthe proteasome mutants (Table 1). Moreover, Uch2/Uch37 and Ubp6/Usp14 did not appear tocooperate in vivo, since no synthetic effects wereobserved in a uch2Dubp6D double null (Table 1). Wealso tested whether either of the Uch2/Uch37 orUbp6/Usp14 enzymes displayed any syntheticeffects with a null-mutant in the gene encodingisopeptidase T; however, we observed no evidencefor such genetic interactions (not shown).

Previously, double mutants between Pad1/Rpn11 and Ubp6 have been found to be syntheti-cally lethal in budding yeast.12 However, we did notobserve any obvious synthetic phenotypes of thepad1-1ubp6 double mutant, this is probably becausethe pad1-1 mutant is not defective in its deubiqui-tination activity.

To study the genetic interactions we observedbetween the ubp6D and mts1-1, mts2-1 and mts3-1mutants more closely, we generated a proteolyti-cally dead Ubp6-C109A point mutant, where theactive-site cysteine of the Ubp6 protease is replacedwith alanine. When this Ubp6 mutant strain wascrossed with the proteasome mutant mts2-1, thecells proved to be viable (Table 2), revealing that theobserved synthetic lethality between the ubp6D andproteasome mutants is independent of Ubp6’s

Table 1. Phenotypes of the mutants

Strain Phenotype

uch2D Viableubp6D Viableuch2D, mts1-1 Temperature-sensitiveuch2D, mts2-1 Temperature-sensitiveuch2D, mts3-1 Temperature-sensitiveuch2D, mts4-1 Temperature-sensitiveuch2D, mts8-1 Temperature-sensitiveuch2D, pad1-1 Temperature-sensitiveuch2D, pus1D Viableubp6D, mts1-1 Inviableubp6D, mts2-1 Inviableubp6D, mts3-1 Inviableubp6D, mts4-1 Temperature-sensitiveubp6D, mts8-1 Temperature-sensitiveubp6D, pad1-1 Temperature-sensitiveubp6D, pus1D Viableubp6D, uch2D Viable

enzymatic activity. Instead we then tested whetherthe synthetic lethality was due to the UBL domainin Ubp6. Indeed cells expressing a truncated form ofUbp6 that lacks the N-terminal UBL domain(Ubp6UBLD) displayed synthetic lethality with themts2-1 proteasome mutant (Table 2). This indicatesthat the observed synthetic lethality is due tostructural proteasome stabilisation and not catalyticeffects of Ubp6.

Deubiquitinating activity of S. pombeproteasomes

Next we crossed the uch2 and ubp6 null-mutantsto the protein A-tagged pus1C strain and precipitatedthe Uch2- and/or Ubp6-deficient 26 S proteasomesfrom these strains. When the deubiquitinatingactivity of the 26 S proteasomes was assessed usingubiquitin–aminomethylcoumarin (AMC) as asubstrate, only Uch2/Uch37 appeared to contributesignificantly to the activity (Figure 6a). Next wedetermined whether similar conclusions could bedrawn when the deubiquitinating activity of themutant proteasomes instead was assayed using asubstrate composed of a branched chain of fourubiquitin-moieties linked through lysine 48(Figure 6b). Though some monoubiquitin is gener-ated by the Uch2-deficient proteasomes (Figure 6b),the activity of Uch2 again appears to be significantlygreater than that of Ubp6.

This reveals that Ubp6/Usp14 probably prefersa chain of ubiquitin over ubiquitin–AMC as asubstrate. Moreover, the results indicate thatwhereas Ubp6/Usp14 is the major deubiquitinatingenzyme associated with the proteasome in buddingyeast,17 the deubiquitinating enzyme that is respon-sible for the majority of the activity at the 26 Sproteasome is Uch2 in fission yeast and probablyUch37 in human cells.

In conclusion Uch2/Uch37 is responsible for themajority of the deubiquitination activity associatedwith the 26 S proteasome in fission yeast, whereasUbp6/Usp14 appears to play a more elusiveregulatory or structural role in cooperation with

Table 2. Effect of ubp6C mutations for lethality with mts2-1

Strain Containing Phenotype

ubp6D, mts2-1 pREP1 Inviableubp6D, mts2-1 pREP1-ubp6C Temperature-sensitiveubp6D, mts2-1 pREP1-ubp6C109A Temperature-sensitiveubp6D, mts2-1 pREP1-ubp6UBLD Inviable

Figure 6. Deubiquitinating activity of Uch2 and Ubp6 null mutants. a, The deubiquitinating activity of purified 26 Sproteasomes from wild-type, uch2D, ubp6D and uch2Dubp6D cells was measured using ubiquitin–AMC as a substrate.The activity is displayed as averagesGSEM in percentage of the wild-type, nZ5. b, The deubiquitinating activity ofpurified 26 S proteasomes from wild-type (lane 2), uch2D (lane 3), ubp6D (lane 4) and uch2Dubp6D (lane 5) cells wasassayed by following the disassembly of K48-linked tetraubiquitin chains (lane 1) by SDS-PAGE and Western blottingusing ubiquitin-specific antibodies. The presence of mono-, di- and tetraubiquitin species is indicated. We neverobserved any triubiquitin intermediates.


the proteasome subunits Mts1/Rpn9, Mts2/Rpt2and Mts3/Rpn12.

Discussion

In order to be degraded by the 26 S proteasomemost proteins must carry a ubiquitin chaincomposed of at least four ubiquitin moieties.3

When the proteasome degrades such ubiquitin-tagged substrates, the ubiquitin chains becomedetached. Ubiquitin C-terminal hydrolases orubiquitin-specific proteases associated with 19 Sparticle of the 26 S proteasome presumably mediatethis deubiquitination event and hence proteindegradation and deubiquitination seem to betightly connected.8–11

The relatively small fission yeast genome encodesat least 18 different putative deubiquitinatingenzymes and higher eukaryotes contain manymore. Some of these deubiquitinating enzymestrim ubiquitin chains sequentially from the distalend. This ubiquitin chain editing process poten-tially regulates the substrate’s degradation by the26 S proteasome, and such an isopeptidase hasindeed been shown to rescue certain proteins from

proteolysis by disassembling the ubiquitin chainsbefore degradation commences.9

Here, we compared the roles of two deubiquiti-nating enzymes, Uch2/Uch37 and Ubp6/Usp14,associated with the 26 S proteasome in fission yeast.To this end we utilised a combination of bio-chemical and genetic approaches.

As expected both Uch2/Uch37 and Ubp6/Usp14co-purify with 26 S proteasomes. By in vitroco-precipitation analyses we show that Ubp6directly interacts with the Mts4/Rpn1 subunit ofthe 26 S proteasome. This is in accordance withresults from budding yeast17 although Ubp6 frombudding yeast and fission yeast are only about 40%identical. The results are also in agreement withdata on other UBL domain-containing proteins suchas Rhp23/Rad23 and Udp7.21,22,25 With respect toUch2/Uch37 we found that Uch2 can directlyinteract with the 26 S proteasome subunit Pus1/Rpn10.

Uch2/Uch37 is most likely an integral subunit ofthe 26 S proteasome and may therefore be expectedto interact with several proteasome subunits. Thus,it is not surprising that 26 S proteasomes purifiedfrom a pus1D strain still contain Uch2/Uch37,indicating that Uch2/Uch37 interacts with other


subunits in addition to Pus1/Rpn10. PreviouslyUch2/Uch37 has been shown to interact withMts3/Rpn1226 and to localise to the “hinge region”,between the base and lid subcomplexes of the 19 Sregulatory complex of the 26 S proteasome inDrosophila melanogaster.13 This is corroborated byour observation of an interaction between Uch2/Uch37 and Pus1/Rpn10, as Pus1/Rpn10 has alsobeen reported to localise to this area of the 19 Sparticle.27

From our immuno-localisation studies, bothUbp6/Usp14 and Uch2/Uch37 appear to be associ-ated with 26 S proteasomes in vivo. Previously Uch2has been shown to co-localise with the 26 Sproteasome,14 an observation that is in agreementwith Uch2/Uch37 being a proteasome subunit inD. melanogaster.13 However, the co-localisation ofUbp6/Usp14 with the 26 S proteasome indicatesthat in vivo a significant amount of Ubp6/Usp14 isassociated with proteasomes. This is supported byour density-gradient centrifugation analyses, whichreveal that the majority of intracellular Ubp6 isassociated with the 26 S proteasome. This resultmay be somewhat surprising considering that Ubp6is a UBL domain protein and therefore mightcompete with other UBL domain proteins likeRhp23/Rad23 and Udp7 for 26 S proteasomeassociation. However, the 26 S proteasome is highlyabundant, accounting for about 1% of the total cellprotein,28 and therefore is probably in excess to thesum of the cellular UBL domain proteins. Moreover,recent data from budding yeast suggest that Ubp6/Usp14 surprisingly does not compete with Rhp23/Rad23 for proteasome binding,22 indicating that the26 S proteasome subunit Mts4/Rpn1 contains morethan one UBL binding site.29

Next we constructed null-mutants ofUch2/Uch37 and Ubp6/Usp14. Curiously none ofthese mutants displayed any obvious phenotypesand did not accumulate ubiquitin–protein conju-gates (not shown), indicating that in vivo otherdeubiquitinating enzymes have overlappingactivities. Most likely such activity is provided bydeubiquitinating enzymes associated with the 26 Sproteasome, e.g. Pad1/Rpn11. However, in mice,mutation of the gene encoding Ubp6/Usp14 causesataxia.30 In budding yeast loss of Ubp6 causeshypersensitivity to a variety of toxic compounds,an effect that is rescued by overexpression ofubiquitin.31–33

We observed that when the fission yeast Ubp6null-mutant was crossed to the mts1-1, mts2-1 ormts3-1 strains where the activity of the Mts1/Rpn9,Mts2/Rpt2 and Mts3/Rpn12 proteasome subunits,respectively, had been compromised by mutation,24

the hybrids proved non-viable.Though this at first indicates that the 26 S

proteasome itself may remedy for the lackingUbp6/Usp14 activity, this is unlikely, since lethalitywas not observed when the subunits Mts4/Rpn1,Pus1/Rpn10, Mts8/b1 and Pad1/Rpn11 weremutated. Accordingly, overexpression of a proteo-lytically dead Ubp6 mutant (Ubp6-C109A) rescued

the synthetic lethality of the ubp6Dmts2-1 strain.However, UBL-dependent proteasome associationof Ubp6 proved to be necessary for rescue of theubp6Dmts2-1 double mutant. Hence, a more likelyexplanation for these genetic interactions betweenubp6D and the proteasome subunits is that associ-ation of Ubp6 with the mutant 26 S proteasomesstabilises these otherwise labile proteasomes.

Recent observations in budding yeast, haverevealed that whereas ubiquitin in wild-type cellsis metabolically stable, budding yeast mutants inDoa4 or Ubp6/Usp14 degrade ubiquitin.17,31–34

Perhaps because the mutant cells fail to releaseubiquitin from the substrate, which thereforeprobably drags ubiquitin with it into the proteolytic20 S particle. The data presented here, revealing theviable phenotype of the ubp6C109Amts2-1 strain,indicate that the synthetic lethality between ubp6Dand the mts1-1, mts2-1 and mts3-1 proteasomemutants is also not connected with ubiquitindegradation, but probably reflects a structural effectof Ubp6 binding.

Recently, a study showed that Ubp6/Usp14 isresponsible for most deubiquitinating activityassociated with the 26 S proteasome in buddingyeast.17 However, as this organism, unlike mostother eukaryotes, does not contain an orthologue ofUch2/Uch37, we analysed the relative activities ofUch2/Uch37 and Ubp6/Usp14 in fission yeast.Surprisingly and in contrast with the results frombudding yeast, the deubiquitinating activity of 26 Sproteasomes purified from strains lacking Uch2/Uch37 and/or Ubp6/Usp14 showed that onlyUch2/Uch37 appeared to actively disassembleubiquitin–AMC conjugates. When deubiquitinationinstead was monitored using branched tetraubiqui-tin chains as substrate, Uch2 again contributed tothe majority of the activity. In this respect it iscurious that the phenotype of the uch2-null mutantis not more severe, but indicates that in vivo otherdeubiquitinating enzymes may compensate for thelacking Uch2/Uch37 subunit.

It is of course possible that the reason for the lowlevel of activity of the Ubp6/Usp14 associated withour proteasome preparations is due to the lack ofcertain activating co-factors. Recently anotherdeubiquitinating enzyme, Ubp3, was found torequire the presence of Bre5 to efficiently removeubiquitin chains from certain proteins.35 However,if a similar co-factor is required for the activity ofUbp6/Usp14 one would expect it to be present inour proteasome preparations and also be conservedto budding yeast, where proteasome-associatedUbp6/Usp14 is highly active.17 In budding yeastUbp6/Usp14 was found to be strongly stimulatedupon proteasome binding,17 a result that does notexplain the low level of activity of proteasome-associated Ubp6/Usp14 in fission yeast, but revealsthat in this respect the 26 S proteasome may beconsidered a co-factor for Ubp6/Usp14 activity.

In conclusion, our results on ubiquitin–AMC andtetraubiquitin disassembly indicate that in fissionyeast 26 S proteasomes, Uch2/Uch37 is responsible


for the majority of the deubiquitination activity,whereas Ubp6/Usp14 appears to play a structurallymore important role in cooperation with variousproteasome subunits, such as Mts1/Rpn9,Mts2/Rpt2 and Mts3/Rpn12, but not withMts4/Rpn1, Mts8/b1, Pus1/Rpn10 and the newlyidentified metallo-deubiquitinating subunitPad1/Rpn11.10,11

Finally, as the human deubiquitinating enzymeUch37 is conserved to fission yeast, but not tobudding yeast, S. pombe may provide a moresuitable genetic system for studying proteasome-associated deubiquitination than the more widelyused S. cerevisiae.

Materials and Methods

S. pombe strains and techniques

Fission yeast strains used in this study (wt, pus1Tpus1ProtA, pad1Tpad1HA, pad1Tpad1ProtA, uch2D, ubp6D,mts1-1, mts2-1, mts3-1, mts4-1, mts8-1, pad1-1, pus1D,uch2Dubp6D, uch2Dmts1-1, uch2Dmts2-1, uch2Dmts3-1,uch2Dmts4-1, uch2Dmts8-1, uch2Dpad1-1, uch2Dpus1D,ubp6Dmts1-1, ubp6Dmts2-1, ubp6Dmts3-1, ubp6Dmts4-1,ubp6Dmts8-1, ubp6Dpad1-1, ubp6Dpus1D, pus1Tpus1ProtAuch2D, pus1Tpus1ProtAubp6D, pus1Tpus1ProtAuch2Dubp6D, pad1Tpad1ProtApus1D) are derivatives of thewild-type heterothallic strains 927hK and 927hC. Theproteasome mutants mts2-1, mts3-1, mts4-1, pad1-1 andpus1D have been described,24,36–39 whereas mts1-1 andmts8-1 are novel temperature-sensitive proteasomemutants in subunits Mts1/Rpn9 and Mts8/b1,respectively (C.G., unpublished results). Standard geneticmethods and media were used and S. pombe transform-ations were performed using the lithium acetateprocedure.40 The PCR mutagenesis was performedaccording to a previously published procedure.41

Plasmids

To generate Uch2 (SPBC409.06) and Ubp6(SPAC6G9.08) constructs, the full-length cDNAs wereamplified from an S. pombe cDNA library preparation andsubcloned into pGEX-KG (Amersham Biosciences). Forexpression in S. pombe cells, cDNAs were subcloned intothe pREP1 plasmid. The C109A site-directed mutagenesisof Ubp6 was performed using Quickchange (Stratagene).Plasmids were integrated into the S. pombe genome bylinearisation with MluI followed by transformation andselection.

Purification of GST-fusion proteins

Fusion proteins were expressed in Escherichia coli BL21(DE3) pLysS and bound to glutathione Sepharose 4 beads(Amersham Biosciences) as described by themanufacturer.

Binding and wash steps were performed in buffer A(50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2,5 mM DTT, 10% (v/v) glycerol, 1% (v/v) Triton X-100,1 mM PMSF and Completee protease inhibitors (Roche)).The bound proteins were eluted using thrombin protease(Amersham Biosciences), as described by the manufac-turer and utilised for the generation of polyclonalantibodies or in co-precipitation experiments.

Purification of 26 S proteasomes from S. pombe

Cultures of cells containing the pus1Tpus1ProtA orpad1Tpad1ProtA tag were grown to late logarithmic phaseand harvested. The cells were washed once in water andonce in buffer B (25 mM Tris–HCl (pH 7.5), 50 mM NaCl,10 mM MgCl2, 5 mM ATP, 5 mM DTT, 10% glycerol, 0.1%Triton X-100, 1 mM PMSF and Completee proteaseinhibitors (Roche)). Cell breakage was performed inbuffer B using a RyboLyser (Hybaid) and cell debriswas removed by centrifugation. The 26 S proteasomeswere then precipitated from the cleared lysate byincubation for four hours at 4 8C with IgG Sepharosebeads (Amersham Biosciences). The 26 S proteasomeswere either assayed directly on the beads or first elutedwith rTEV protease (Invitrogen).

Co-precipitation experiments

About 5 mg of purified Mts4 or Pus1 was incubated in1 ml of buffer A with GST-Uch2 or GST-Ubp6 coupled toglutathione Sepharose beads at 4 8C overnight. The beadswere then washed three times before the bound proteinswere eluted with SDS sample buffer and separated on12% (w/v) polyacrylamide SDS-PAGE gels. The boundproteins were identified in subsequent Western blottingusing polyclonal antibodies specific for Mts4 or Pus1.

Glycerol gradient centrifugation

Extracts from wild-type S. pombe cells were separatedon 12 ml 10%–40% glycerol gradients in buffer B bycentrifugation according to a previously publishedprocedure.23

Deubiquitination assays

For ubiquitin-7-amino-4-methylcoumarin (ubiquitin–AMC) (Affiniti Research Products Ltd) deubiquitinationassays, samples were incubated for 30 minutes at 30 8C in200 ml of 0.5 mM ubiquitin–AMC, 50 mM Tris–HCl (pH8.0), 5 mM DTT, 0.1% Tween-20. After addition of 100 mlof 10% (w/v) SDS and 1.5 ml of 0.1 M sodium borate(pH 9.1), the fluorescence was determined at an excitationwavelength of 380 nm and an emission of 460 nm.

For tetraubiquitin disassembly assays, 1 mg of purifiedpreparations of proteasomes from various strains wereincubated with 1 mg of K48-tetraubiquitin (AffinitiResearch Products Ltd) at 30 8C for six hours in a buffercontaining 50 mM Tris–HCl (pH 8.0), 5 mM DTT, 2 mMEDTA, 0.1% Tween-20 and 50 mM the proteasomeinhibitor Z-Ile-Glu(OtBu)-Ala-Leu-aldehyde (Bachem).After addition of SDS sample buffer, deubiquitinationwas assessed by SDS-15% PAGE and Western blotting,probing with polyclonal rabbit antibodies towardsubiquitin (a gift from Klavs B. Hendil).

Immunostaining

Immunocytochemistry was performed according to apreviously published procedure23 using affinity purifiedpolyclonal antibodies against Uch2, Ubp6, Mts4 andHA-tag. DNA was stained using 4 0,6-diamidino-2-phenylindole dihydrochloride (DAPI).


Acknowledgements

The authors thank Dr Andrew Carothers andProfessor Nick Hastie for helpful comments andencouragement. This work was supported by aMedical Research Council funding to C.G. as well asby a grant from the Wellcome Trust to R.H.-P.

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Edited by R. Huber

(Received 22 April 2004; received in revised form 1 September 2004; accepted 9 September 2004)

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Uch2/Uch37 is the Major Deubiquitinating Enzyme Associated with the 26 S Proteasome in Fission Yeast