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MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function

MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and... Background: Three macromolecular assemblages, the lid complex of the proteasome, the COP9- Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function. Results: We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. Conclusions: The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity. covalently attached to a polyubiquitin chain via a cascade Background Many regulatory proteins are removed from the cell in a of ubiquitinating enzymes. This ubiquitination process is timely and specific manner by a large multi subunit en- reversible. Specific cysteine proteases known as DUBs zyme called the proteasome [1,2]. For proteins to be rec- (deubiquitinating enzymes) can hydrolyze the amide ognized by the proteasome, they are usually first bond between the Carboxy-terminus of ubiquitin and an Page 1 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 amino group on the substrate [3,4]. Proteolysis takes ized profile method [23]. An improved MPN domain pro- place within the 20S core particle (CP) of the proteasome, file detected a large number of novel significant matches while the 19S regulatory particle (RP) binds polyubiquiti- including some prokaryotic proteins from archaeal and nated substrates, unfolds, and translocates them into the eubacterial origins, which until now have not been 20S CP for proteolysis. The discovery that the 19S regula- known to contain MPN domains. In addition to being tory particle of the proteasome (RP) can be separated into structurally related to the published eukaryotic MPN do- two discrete subcomplexes, the lid and the base, suggests mains, all prokaryotic MPN domains contain an addition- that they have distinct roles in preparing a substrate for al pattern of five polar amino acids, which are conserved degradation [5]. The base contains six ATPase subunits, in a highly correlated fashion. This motif is embedded Rpt1-6, as well as the two largest non-ATPase subunits within the conventional MPN domain, and is also con- Rpn1 and Rpn2, and plays a role in anchoring the sub- served in some – but not all – eukaryotic MPN proteins. strate, unfolding it and gating the channel leading into the We therefore term it the MPN+ motif. The polar nature 20S CP [6–9]. The lid complex consists of eight non-AT- and coordinated conservation of the MPN+ residues sug- Pase subunits whose functions have not been defined. All gest a catalytic and/or metal-binding function. Since subunits of the lid subcomplex contain one of two struc- Rpn11 is one of the eukaryotic MPN domain proteins har- tural motifs: six contain a PCI domain (Proteasome, boring the MPN+ motif, we used mutational analysis to COP9, eIF3), while the other two (Rpn8 and Rpn11) con- assess the importance of these conserved amino acids for tain an MPN domain (Mpr1, Pad1 N-terminal) [5,10,11]. the function of Rpn11 in S. cerevisiae. Rpn11 also contains These domains are found in members of two other eu- a highly conserved cysteine residue that is not part of the karyotic macromolecular assemblages as well: the COP9 MPN+ motif but is present in a number of its close para- signalosome (CSN) and the eukaryotic translation initia- logs such as the COP9 signalosome subunit, Csn5. It has tion factor 3 (eIF3). The functions of these domains are been suggested that this may correspond to the active site not known, but they are necessary for proper interactions cysteine of a catalytic DUB motif [14]. Therefore, we also between subunits of these complexes [12,13]. The lid ap- analyzed mutants of Cys116, and compared them to mu- pears to be required for the degradation of polyubiquiti- tations in the MPN+ residues of Rpn11. nated substrates but not for hydrolysis of unstructured or short polypeptides [5]. Thus, one possibility is that the lid Results is required in one way or another for proper interactions Extending the scope of the MPN domain with polyubiquitinated chains. In order to identify distantly related members of the MPN domain family, we constructed generalized profiles of At 66% identity between the human and yeast forms, the previously established MPN proteins. Included in the pro- MPN domain protein Rpn11 is the most highly conserved file construction were the proteasome lid components non-ATPase subunit of the 19S RP, on par only with the Rpn8 and Rpn11, the COP9 signalosome components highly conserved ATPase subunits, suggesting that it too Csn5 and Csn6, and the translation initiation factor 3 may play an enzymatic role within the RP [14,15]. Muta- components eIF3f and eIF3h, all from various eukaryotic tions in RPN11 cause cell cycle and mitochondrial defects, species. After scaling of the profile [24], significant hits temperature sensitivity, and sensitivity to DNA damaging were found in protein database, including the STAM-in- reagents such as UV or MMS, underscoring the impor- teracting protein AMSH [25], and the uncharacterized hu- tance of this subunit in proteasome function [16–18]. man proteins C6.1A and KIAA1915. Subsequently, Rpn11 is one of a minority of proteasome subunits that iterative profile refinement [24] was used to make the pro- exhibit dominant phenotypes upon overexpression. High file searches more sensitive. For that purpose, the newly dosage of human or S. pombe Rpn11 orthologs confer identified MPN proteins were included in the profile con- multidrug and UV resistance [19,20]. These effects may be struction process. After four iteration cycles, a stable set of linked to the stabilization of c-Jun observed upon overex- significantly matching bona fide MPN proteins was identi- pression of the Rpn11 subunit in Schistosoma, SmPOH1 fied. Representative members of this superfamily are [21]. In another case, however, overexpression of Rpn11 shown in Figure 1. can suppress an srp1 mutation, the yeast homolog of im- portin alpha, and enhance degradation of a proteasome In addition to the previously known MPN proteins, a substrate [22], illustrating that effects of Rpn11 are pleio- number of prokaryotic members were identified, includ- tropic. Together, these results may suggest that Rpn11 em- ing the phage tail assembly protein K from the bacteri- bodies an intrinsic enzymatic activity. ophage lambda and its closely related homologs from other phages and prophages. Even archaeal members of In order to gain insight into the functions and evolution- the MPN domain family were found, including predicted ary history of MPN domain proteins such as Rpn11, we proteins from Archaeoglobus fulgidus, Methanobacter ther- performed extended database searches using the general- moautotrophicum and from various Pyrococcus species (Fig. Page 2 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 1 The MPN+ motif. Sequence alignment of representative MPN domain protein, only two conserved blocks containing the MPN+ motif are shown. The proteins are grouped in seven categories: A, bacterial; B, archaeal; C, proteasome lid compo- nents; D, CSN components; E, eIF3 components; F, Prp8-like proteins; G other eukaryotic MPN proteins. In the eukaryotic groups, representative sequences from human (HS), Arabidopsis (AT), S. pombe (SP) and S. cerevisiae (SC) are shown. Prokary- otic species shown are lambda phage (BPL), Yersinia pestis (YP), Synechocystis sp. (SS), Mycobacterium tuberculosum (MyT), Pseu- domonas aeruginosa (PA), Pyrococcus horikoshii (PH), Archaeoglobus fulgidus (AF), Methanobacter thermoautotrophicum (MT). Residues invariant or conservatively substituted in at least 50% of all sequences are shown on black and grey background, respectively. The MPN+ motif residues are shown in red. The top line indicates the amino acids constituting the MPN+ motif; the rightmost column indicates whether a protein is considered an MPN+ protein or a plain MPN protein. Page 3 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 1). These proteins are the shortest MPN protein identified Mutants in the MPN+ motif of Rpn11 exhibit severe so far and most likely correspond to the structural core re- growth defects In order to assess the importance of the MPN+ residues for gion of the domain. Since the finding of prokaryotic MPN proteins was highly unexpected, the validity of the assign- efficient Rpn11 function, we performed site-directed mu- ment was confirmed by profile searches starting from the tagenesis of His111, Ser119, Asp122, which are part of the bacteriophage proteins that resulted in the same stable set conserved HxH–S–D sequence that defines the MPN+ of significantly matching proteins. motif. In addition, Cys116, which is not part of the MPN+ motif but is highly conserved between Rpn11 and Csn5 Definition of the MPN+ motif orthologs might be a candidate for an active site residue, Overall, there are no residues that are invariant through- was mutagenized too. As seen in Fig. 2, alanine substitu- out the MPN domain superfamily. However, while ana- tions in the MPN+ motif of Rpn11 cause severe growth de- lyzing the alignment of the newly identified proteins, it fects, temperature sensitivity, and sensitivity to amino became apparent that there are a number of polar residues acid analogs such as canavanine. Substitution mutations that are conserved in a highly coordinated fashion in a at different locations in the MPN+ motif display similar, subset of MPN domains (highlighted in Red in Fig. 1). but slightly distinct phenotypes. Specifically, the These amino acids form a pattern, referred to herein as the His111Ala mutant is extremely slow growing at 25C and 'MPN+ motif', which is part of the conventional MPN do- lethal when plated at elevated temperatures, even as low main and embedded within it. The MPN+ motif contains as 30C. The Asp122Ala and Ser119Ala mutants are viable a well-defined pattern of 'H-x-H-x[7]-S-x[2]-D', where x[7] but slow growing at 25C and 30C, and lethal when shift- and x[2] indicate stretches of seven and two non-con- ed to 37C or when exposed to amino acid analogs. By served residues, respectively. In addition to this conserved contrast, substitutions of the conserved Cys at position arrangement of four polar residues, there is an additional 116 showed no growth defect, and the cells appeared nor- glutamate residue in a more N-terminal region of the do- mal under all conditions tested. main, whose conservation is perfectly correlated with the occurrence of the motif (Figure 1). An aromatic residue, Stress conditions such as exposure to elevated tempera- preferentially a tryptophane, is found two positions up- ture or amino acid analogs are known to promote accu- stream of the conserved serine in most but not all MPN+ mulation of damaged proteins, which must be removed proteins. Thus, it should not be considered a part of the by the proteasome [26]. Since defects in the MPN+ motif core MPN+ motif. of Rpn11 cause heightened sensitivity to such conditions, it is reasonable to assume that proteasome function is With the possible exception of Prp8, all MPN domain pro- jeopardized in these mutants. We conclude that the teins shown in Figure 1 can unambiguously be classified MPN+ motif is critical for the proper function of Rpn11 as either belonging to the MPN+ or to the 'plain' MPN within the proteasome, but that Cys116 does not appear class. The validity of this distinction is underscored by the to be an essential residue in Rpn11. fact that invariably all observed orthologs of MPN+ pro- teins also belong to the MPN+ class. For instance, the pro- Multiubiquitinated proteins accumulate in MPN+ motif teasome lid complex from all eukaryotes contains one mutants MPN+ protein (Rpn11/S13) and one plain MPN protein In order to test whether proteasome function is indeed (Rpn8/S12). The same is true for the analogous CSN com- hampered in these rpn11 mutants, we checked the effects plex in multicellular eukaryotes, where the MPN+ protein of mutations on the ubiquitination pattern of cellular pro- is Csn5 and the plain MPN protein is Csn6. In fission teins. Whole cell extracts from rapidly growing WT or yeast, Csn6 appears to be absent. The same is true for the MPN+ motif mutants were separated by SDS PAGE and recently identified CSN-like complex of budding yeast; it immunoblotted with anti-Ub antibody (Fig. 3). High mo- contains only Csn5 but not Csn6 (Hofmann and Glick- lecular weight polyubiquitin-conjugates are not detected man; submitted for publication). The two MPN proteins at appreciable levels in extracts from yeast containing nor- of the eIF3 complex are both of the plain MPN type in mal proteasomes since they are rapidly turned over. High multicellular eukaryotes and fission yeast, while in bud- molecular weight polyubiquitinated proteins do accumu- ding yeast they appear to be missing altogether. Notably, late, however, in rpn11 MPN+ mutants, indicating defec- all newly identified prokaryotic MPN proteins are of the tive proteasome activity. In this assay, all mutations in MPN+ type; the same is true for the remaining unassigned MPN+ residues behave similarly. His111 mutants also ac- 'orphaned' MPN proteins in eukaryotes (Fig. 1). It should cumulate polyubiquitinated proteins when grown at be stressed that the MPN+ motif is not a stand-alone motif 25C, but since this strain is extremely slow growing it was to be found independently of the MPN domain; thus, all difficult to prepare extracts for comparison (not shown). MPN proteins can be classified as either MPN+ or plain No such accumulation is seen in cells containing Rpn11 MPN proteins. with Cys116 substitutions. Page 4 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 2 Growth of rpn11 Mutants under Different Growth Conditions. WT and mutant strains were streaked on YPD at 25, 30 and 37C. Cells were also plated on complete minimal media containing 1 g/ml of the amino acid analog canavinine instead of arginine. Plates were photographed after 3–5 days. Three single amino acid substitutions in the MPN+ motif of Rpn11 were studied: his111ala, Ser119ala, and asp122ala. For comparison, a substitution of a highly conserved cysteine residue (cys116ala) that is not part of the MPN+ motif (see Fig. 1) was included as well. At 25C, all MPN+ mutations are viable but slow growing. Hiss111 is extremely temperature sensitive, showing no appreciable growth even when shifted to 30C, or in the presence of even 1 g/ml of the amino acid analog canavinine. Asp122 and Ser119 substitutions show lethality under elevated temperature (37C) or exposure to canavanine. In comparison, Cys116 substitutions show no growth defects under these conditions. It appears that the MPN+ motif defines the role of Rpn11 short-lived proteins were measured in WT and in a repre- in the proteasome but that the conserved Cys116 residue sentative rpn11 mutant strain. In order to estimate the is not critical for proteasome function. The steady state generality of the effect, two different substrates were used: levels of polyubiquitin-protein conjugates are influenced a protein that is ubiquitinated by enzymes of the UFD by the rate of ubiquitination on the one hand, and by ubiquitination pathway [27], and a protein that is ubiqui- rates of deubiquitination or proteasome proteolysis on tinated by enzymes of the N-end rule pathway [28]. WT the other. Accumulation of polyubiquitinated proteins in Cells, or those harboring the S119A substitution in the rpn11 mutants could therefore be due to a slowdown in ei- MPN+ motif of Rpn11 were transformed with plasmids ther proteasome associated deubiquitination or proteas- expressing Arg--galactosidase (an N-end rule substrate) ome-dependent proteolysis. or Ub-pro--galactosidase (A UFD substrate). Arg--gal and Ub-Pro--gal are short-lived in wild-type yeast with Stabilization of short-lived proteasome substrate half-lives of ~2 and ~6 minutes, respectively [29]. Steady In order to test whether the accumulation of polyubiqui- state levels of these proteins were compared to the levels tinated conjugates is directly correlated with a defect in of a stable, long-lived protein, Met--galactosidase. As ex- proteasome function, we checked whether mutations in pected, WT cells accumulated high levels of the stable the MPN+ motif of Rpn11 bring about stabilization of Met-gal but only low levels of the rapidly degraded Arg- proteasome substrates. The steady state levels of known -gal and Ub-Pro--gal (Fig. 4). In contrast, the S119A Page 5 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 3 Accumulation of multiubiquitinated proteins in MPN motif mutants. Total cell extracts from WT yeast and from strains containing the MPN+ motif substitutions were separated on an 8% SDS gel and blotted with anti-ubiquitin antibodies. Cells were rapidly lysed in presence of 12% TCA in order to inhibit post lysis enzymatic activity. Accumulation of high MW polyubiquitinated proteins is detected in the Ser119 and Asp122 mutants, but not in WT or in the Cys116 mutants. His111 mutants accumulate polyubiquitinated proteins as well (not shown). A protein band migrating at around 20 kDa that is detected with the anti Ub antibody is used as an internal loading control. mutation in rpn11 lead to dramatic stabilization and accu- the proteasome. The importance of Rpn11 is independent mulation of both these short-lived proteins, such that the of the ubiquitination pathway. steady state levels of all three substrates were similar (Fig. 4). Stabilization of these short lived proteins was noted The possibility that these deficiencies in proteasome de- also in the D122A mutant (not shown). From these re- pendent proteolysis were caused by improper incorpora- sults we conclude that the MPN+ motif of Rpn11 is essen- tion of mutated Rpn11 into the lid, or that proteasome tial for proper proteolysis of ubiquitinated substrates by structure was hampered in rpn11 mutants was addressed Page 6 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 4 Stabilization of short-lived proteasome substrates. WT and the S119A mutant strains containing the Ub-Met-, Ub-Arg- , and Ub-Pro-gal constructs on multi-copy plasmids under the GAL promotor were tested for LacZ activity after galactose- induction. LacZ activity is indicative of steady-state levels of the reporter protein. WT cells accumulate high levels of the stable Met-gal, but rapidly degrade Arg-gal and UB-Pro-gal (left panel), whereas the MPN+ mutation shows dramatic stabilization and accumulation of both short-lived fusion proteins (right panel). by native gel electrophoresis. Cell extract from WT and ical for the proteins in which they are found. This idea is rpn11 mutants was resolved by nondenaturing PAGE and corroborated by the polar nature of the conserved MPN+ no gross structural changes were observed (Fig. 5). Overall residues: glutamate, histidines, serine and aspartate are all levels and proteolytic intensities of proteasomes from amino acids frequently found in the active site of enzymes MPN+ mutants were indistinguishable from WT, indicat- or as the coordinating ligands in metal-binding proteins. ing that the substitutions do not alter the structure of Certainly, these two possibilities are not mutually exclu- peptidase function of the proteasome. In addition, we sive. Several classes of enzymes, particularly metal con- found no evidence for natural abundance WT Rpn11 out- taining hydrolases and proteases, harbor bound metal 2+ side of the proteasome (data not shown), indicating that ions such as Zn as part of their catalytic center [30]. the phenotypes associated with mutated rpn11 are unlike- However, while the motif does bear some resemblance to ly to be due to unincorporated protein. Since peptidase ac- that found in metalloproteases, the specific organization tivity of mutant proteasomes was similar to WT (Fig. 5), of E–HxH–S–D residues does not correspond to the metal the effects of rpn11 mutations is most likely due to a defect ligands in any of the known Zinc proteases [30], thus pos- in proteolysis of ubiquitinated substrates (Fig. 4). itive identification of Rpn11 as a metalloprotease awaits definite proof. Discussion We have identified an arrangement of five residues, which The proteasome complex from a number of sources is are perfectly conserved in a subclass of MPN domains, known to contain a ubiquitin hydrolyzing activity [31– while none of them shows appreciable conservation out- 39], while the CSN complex is known to cause the hydro- side of this subclass. The highly correlated conservation of lytic removal of the Nedd8/Rub1 ubiquitin-like molecule these residues suggests that they participate in a common from the cullin subunit of the SCF ubiquitin ligase (E3) structural element and/or a common function that is crit- complex [40–42]. Thus, the MPN+ containing subunits of Page 7 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 5 Migration pattern of proteasome from WT and rpn11 mutants by non-denaturing PAGE. Cell extracts were pre- pared from Logarithmically growing WT or the rpn11 MPN+ mutants that were brought to an identical OD600 level. Extracts were clarified by centrifugation, and identical amounts of total protein were separated on native gel. The gel was then incu- bated for 10 minutes with the fluorescent peptide LLVY-AMC at 30C, and photographed under UV light (380/440 nm). Fluo- rescent bands indicate migration of Doubly capped and Singly capped 26S proteasome forms [14]. There is no noticeable change in migration pattern or overall levels of proteasomes from the different rpn11 mutants, indicating that mutated Rpn11 is properly incorporated into the lid and no gross structural changes in proteasome composition or amounts are due to the mutations. the proteasome lid (Rpn11) and the COP9 signalosome Single site substitutions in various Rpn11 MPN+ residues (Csn5) would be prime candidates for such a hydrolytic exhibit similar phenotypes supporting the identification function. The architecturally related eIF3 complex, for of MPN+ as a discrete functional motif. Due to the severe which no such enzymatic activity has been described, is growth phenotypes and attenuated ability of the proteas- conspicuously devoid of MPN+ proteins. That the CSN ome to proteolyze polyubiquitinated substrates in these from yeast lacks the plain MPN protein (Csn6) retaining mutants, it appears that the MPN+ motif defines the role only the MPN+ subunit (Csn5), while the plain MPN pro- carried out by Rpn11 in the lid. As we show, these defects teins found in eIF3 from eukaryotes appear to be missing arise from an intrinsic activity of Rpn11 within the context altogether in the yeast complex, emphasizes that the of the proteasome, and not due to a gross structural effect MPN+ residues are likely to be the catalytic residues, with upon incorporation of mutated Rpn11. As mentioned plain MPN subunit playing a redundant structural role in above, the lid, where Rpn11 is situated, is critical for pro- complexes in which they are found. Even though purified teolysis of polyubiquitinated substrates. So far, and some- recombinant Rpn11 does not appear to exhibit DUB capa- what surprisingly, all ubiquitin binding activity has been bilities (data not shown), it is possible that once incorpo- mapped to the Base of the 19S RP. Two subunits in the rated into their respective complexes, Rpn11 and Csn5 base can interact directly with ubiquitin chains, Rpt5 and confer the documented hydrolase activities onto the 19S Rpn10 [5,7,43,44]. A number of proteins, such as Rad23 RP and CSN. In this case, Rpn11 would belong to a and Dsk2, can also bind ubiquitin and interact with the unique class of enzymes, as all other known DUBs are proteasome, presumably with the base, thus they are cysteine proteases. Of note, the conserved cysteine residue thought to serve as shuttles of polyubiquitinated sub- common to both Rpn11 and Csn5, which is not part of strates to the proteasome [45,46]. It is possible that the lid the MPN+ motif, is not important for the function of rather than bind ubiquitin, serves to cleave or trim polyu- Rpn11. biquitin chains once attached to the Base. Page 8 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 While this manuscript was under review, two independ- Interestingly, all identified prokaryotic MPN domains, ent papers substantiated our findings by characterizing a both from archaea and from eubacteria, also contain the novel deubiquitinating activity in the proteasome, and MPN+ motif. It thus appears likely that the ancestral MPN showing that the MPN+ motif residues of Rpn11 are large- domain was of the MPN+ type, and that the extant 'plain' ly responsible for this activity [47,48]. These independent MPN proteins have lost this motif later on. It is possible studies identified DUB activity associated with the 19S RP that the plain MPN proteins play a structural role in com- of the proteasome, which is lacking upon lethal substitu- plexes that additionally contain a MPN+ subunit. So far tions in Rpn11. Interestingly, all substitution mutations all known MPN proteins are incorporated into complexes that we studied in the MPN+ motif of Rpn11 are viable. that also contain PCI proteins [51,52]. Interestingly, sev- Quite possibly, Rpn11 is not the sole proteasome-associ- eral of the prokaryotic organisms harboring MPN+ pro- ated DUB, and a number of DUBs play partially overlap- teins appear to lack PCI domain proteins and therefore ping functions. For instance, Doa4/Ubp4 interacts weakly probably could not form large PCI/MPN complexes relat- and substoichiometrically with the proteasome and may ed to the lid and CSN particles. An interesting case is the serve to release ubiquitin and regenerate the proteasome tail assembly protein 'K' of the bacteriophage lambda for the next catalytic cycle [37]. The Ubiquitin-like do- (vtak) and its many homologs from other phages and main (UBL) containing deubiquitinating enzyme, USP14, prophages (Fig. 1). Little is known about the specific func- has been found to interact with the proteasome from tion of the protein but none of the other tail assembly fac- mammalian sources [38]. Ubp6, the budding yeast ho- tors contain a PCI or MPN domain (K.H unpublished molog of USP14, interacts with the proteasome as well results). Our work suggests that the K-protein plays an en- [49], and plays a role in proteasome-associated deubiqui- zymatic role in tail assembly rather than a merely structur- tination [39]. It has also been reported that the Drosophila al one, although whether the analogy to Rpn11 is relevant DUB p37a [31] and its homologs UCH37 (H. sapiens) and to vtak is still enigmatic. Uch2 (S. pombe), may be responsible for the polyubiqui- tin chain editing function associated with purified protea- A similar situation exists for the orphan MPN proteins of somes [31,34,35]. However, as budding yeast lacks an eukaryotes. It is noteworthy that complex eukaryotic or- obvious ortholog of UCH37/p37a, other DUBs must play ganisms have far more orphan MPN protein than orphan a greater role in proteasomes from this organism. PCI proteins. However, the known PCI/MPN complex particles have stoichiometries that require more PCI com- Finding MPN+ motif proteins in prokaryotes will help ponents than MPN components. This apparent paradox- elucidate the origins of proteasome evolution and the on can be resolved by a number of different but non- function of Rpn11 in particular. The proteasome probably exclusive assumptions. i) Additional PCI proteins have evolved from self-compartmentalized macromolecular eluded detection due to high sequence divergence allow- proteases found in prokaryotes. Thus, proteasomal subu- ing for more than three PCI/MPN complexes to exist, ii) nits, or proteins with motifs related to proteasomal subu- alternative MPN proteins can interact with existing PCI nits, are present in archaea and certain eubacteria. These complexes, iii) some MPN proteins have (enzymatic) "lidless" prototypes include the 20S CP subunits, and ho- functions outside of PCI/MPN complexes. Currently, mologs of the base ATPases (Rpt subunits) [50]. However, there is no data available supporting the first two possibil- proteins relating to the ubiquitination process, such as ities, although there is some promiscuity in the PCI pro- ubiquitin itself, ubiquitin activating conjugating or ligat- tein interactions [52,53]. The third possibility is likely to ing enzymes, and even lid subunits, are missing from be true in prokaryotes and could also explain why all eu- prokaryotes. Identification of a motif from Rpn11 – a pro- karyotic orphan MPN proteins belong to the MPN+ class. tein that is linked to processing of polyubiquitinated pro- This could also explain how supra-stochiometric amounts teins – in prokaryotic proteins is an interesting of Rpn11 exhibit dominant phenotypes; possibly it can development. It is possible that during evolution, the pro- function outside of the proteasome as well. teasome recruited an existing enzyme as the lid was form- ing into a regulatory module of the proteasome; likewise Conclusions for the analogous CSN complex. Studying the prokaryotic The MPN+ motif is abundant in certain MPN-domain MPN+ motif proteins should aid in elucidating what this proteins, including newly identified proteins of eukaryo- motif does. The fact that these proteins are the shortest tes, bacteria and archaea thought to act outside of the tra- MPN proteins and correspond to the structural core re- ditional large PCI/MPN complexes. The putative catalytic gion of the domain should greatly aid in enzymatic and nature of the MPN+ motif makes it a good candidate for a structural studies, especially in comparison to members of pivotal enzymatic function, possibly a proteasome-associ- the family such as Rpn11 and Csn5 that are naturally ated deubiquitinating activity and a CSN-associated found only incorporated into complexes. Nedd8/Rub1-removing activity. The importance of the MPN+ motif for the efficient function of the proteasome Page 9 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 component Rpn11 is compatible with this idea, though Stabilization of known proteasome substrates the fact that none of the single amino acid substitutions Steady-state levels of the Met-, Arg-, and Ub-Pro-gal fu- are lethal indicates that either the function of Rpn11 is not sion proteins were measured by testing LacZ activity [27]. strictly essential for proteasome function, or more likely, Wild type and rpn11 cells harboring multi-copy URA3- it is partially redundant with other subunits. marked plasmids containing each construct were grown to late log in minimal media containing raffinose for carbon Methods source, and induced with 2% Galactose for 4 hrs. The cells Bioinformatics were then lysed and LacZ activity upon introduction of the All database searches were performed with a nonredun- substrate ONPG was calculated when taking into account dant data set constructed from current releases of Swiss- the time, total protein concentration and amount of prod- Prot, TrEMBL, and GenPept [54,55]. Generalized profile uct obtained (measured by 405 nm absorbance). [23] construction and searches were run locally using the pftools package, version 2.1. (program available from the Non-denaturing PAGE and peptidase activity detection URL [ftp://ftp.isrec.isb-sib.ch/sib-isrec/pftools/]). Profiles Yeast cells were lysed in buffer A (10% glycerol, 50 mM , 1 mM EDTA) using glass were constructed using the BLOSUM45 substitution ma- tris7.4, 1 mM ATP, 1 mM MgCl trix [56] and default penalties of 2.1 for gap opening and beads, and clarified by centrifugation at 20  g. Non-dena- 0.2 for gap extension. The statistical significance of profile turing gel was run as previously described [14]. The gel matches was derived from the analysis of the score distri- was then incubated for 10 minutes in 10 ml of buffer A bution of a randomized database as described [24]. Data- and 0.1 mM of the fluorescent peptide LLVY-AMC at base randomization was performed by individually 30C. Under UV light, appearing bands show peptidase inverting each protein sequence, using SwissProt 34 as the activity indicate the migration pattern of the 26S proteas- data source. ome. Single amino acid substitutions Abbreviations Haploid yeast strain with a chromosomal knock out of RP: 19S regulatory particle of the proteasome rpn11 was constructed, in which a single copy URA- marked plasmid expressing the RPN11 gene complements CP: 20S core particle of the proteasome the chromosomal knock out (MY71). The heterozygote RPN11/rpn11 diploid was purchased from EUROSCAF CSN: COP9 signalosome and transformed with a single copy CEN plasmid with URA3 selection (ycplac33) expressing RPN11 from its eIF3: eukaryotic initiation of translation factor 3 own promotor (M82). Growth of MY71 was identical to the isogenic WT strain from EUROSCARF. Plasmids ex- Rpn: regulatory particle non-ATPase subunit pressing the single site substitutions in Rpn11 were gener- ated using PCR site directed mutagenesis on a similar CEN Rpt: Regulatory particle triple-A ATPase subunit plasmid with the LEU2 marker for selection (ycplac111). In this manner the following plasmids were constructed Ub: ubiquitin RPN11 (M134), rpn11-C116A (M134), rpn11-H111A (M143), rpn11-S119A (M144), rpn11-D122A (M145). DUB: deubiquitinating enzyme Those plasmids were then transformed into the above yeast strains, and the URA3-marked WT rescue plasmid -gal: -galactosidase was then forced out of the cells by the presence of 5'FOA in the medium. FOA shuffling was done at 25C as most Authors' contributions rpn11 mutants were severely temperature sensitive. In this Kay Hofmann and Michael Glickman initiated the project manner we got viable yeast rpn11 mutant strains. and participated in its design. Kay Hofmann performed bioinformatic characterization of the MPN+ motif (fig. 1). Phenotypes and mutant characterization Noa Reis and Vered Maytal designed and constructed the Single colonies grown on YPD at 25C were streaked onto MPN+ amino acid substitutions. Vered Maytal carried out YPD and shifted to various temperatures. Plates were pho- all characterization of Rpn11 mutants shown in Figures tographed after 3–5 days. For canavinine sensitivity, 2,3,4,5. All authors read and approved the manuscript. plates containing complete minimal media containing 1 mg/ml canavinine in replace of arginine were used and Acknowledgments We thank Allen Taylor for anti-Ub antibodies. This work was supported by growth measured at 25C. the German Israel foundation for scientific research (GIF), the Israel Sci- ence Foundation (ISF), The Israel Cancer Research Foundation (ICRF), a grant for promotion of research at the Technion, a Technion Vice presiden- tial grant, and the Wolfson fund for study of ubiquitin in health and disease. Page 10 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 23. Bucher P, Karplus K, Moeri N, Hofmann K: A flexible motif search References technique based on generalized profiles. Comput Chem 1996, 1. Pickart CM: Mechanisms underlying ubiquitination Annu Rev Bi- 20:3-23 ochem 2001, 70:503-533 24. Hofmann K: Sensitive protein comparisons with profiles and 2. 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MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function

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Springer Journals
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Copyright © 2002 by Maytal-Kivity et al; licensee BioMed Central Ltd.
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Life Sciences; Biochemistry, general; Life Sciences, general
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1471-2091
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Abstract

Background: Three macromolecular assemblages, the lid complex of the proteasome, the COP9- Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function. Results: We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. Conclusions: The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity. covalently attached to a polyubiquitin chain via a cascade Background Many regulatory proteins are removed from the cell in a of ubiquitinating enzymes. This ubiquitination process is timely and specific manner by a large multi subunit en- reversible. Specific cysteine proteases known as DUBs zyme called the proteasome [1,2]. For proteins to be rec- (deubiquitinating enzymes) can hydrolyze the amide ognized by the proteasome, they are usually first bond between the Carboxy-terminus of ubiquitin and an Page 1 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 amino group on the substrate [3,4]. Proteolysis takes ized profile method [23]. An improved MPN domain pro- place within the 20S core particle (CP) of the proteasome, file detected a large number of novel significant matches while the 19S regulatory particle (RP) binds polyubiquiti- including some prokaryotic proteins from archaeal and nated substrates, unfolds, and translocates them into the eubacterial origins, which until now have not been 20S CP for proteolysis. The discovery that the 19S regula- known to contain MPN domains. In addition to being tory particle of the proteasome (RP) can be separated into structurally related to the published eukaryotic MPN do- two discrete subcomplexes, the lid and the base, suggests mains, all prokaryotic MPN domains contain an addition- that they have distinct roles in preparing a substrate for al pattern of five polar amino acids, which are conserved degradation [5]. The base contains six ATPase subunits, in a highly correlated fashion. This motif is embedded Rpt1-6, as well as the two largest non-ATPase subunits within the conventional MPN domain, and is also con- Rpn1 and Rpn2, and plays a role in anchoring the sub- served in some – but not all – eukaryotic MPN proteins. strate, unfolding it and gating the channel leading into the We therefore term it the MPN+ motif. The polar nature 20S CP [6–9]. The lid complex consists of eight non-AT- and coordinated conservation of the MPN+ residues sug- Pase subunits whose functions have not been defined. All gest a catalytic and/or metal-binding function. Since subunits of the lid subcomplex contain one of two struc- Rpn11 is one of the eukaryotic MPN domain proteins har- tural motifs: six contain a PCI domain (Proteasome, boring the MPN+ motif, we used mutational analysis to COP9, eIF3), while the other two (Rpn8 and Rpn11) con- assess the importance of these conserved amino acids for tain an MPN domain (Mpr1, Pad1 N-terminal) [5,10,11]. the function of Rpn11 in S. cerevisiae. Rpn11 also contains These domains are found in members of two other eu- a highly conserved cysteine residue that is not part of the karyotic macromolecular assemblages as well: the COP9 MPN+ motif but is present in a number of its close para- signalosome (CSN) and the eukaryotic translation initia- logs such as the COP9 signalosome subunit, Csn5. It has tion factor 3 (eIF3). The functions of these domains are been suggested that this may correspond to the active site not known, but they are necessary for proper interactions cysteine of a catalytic DUB motif [14]. Therefore, we also between subunits of these complexes [12,13]. The lid ap- analyzed mutants of Cys116, and compared them to mu- pears to be required for the degradation of polyubiquiti- tations in the MPN+ residues of Rpn11. nated substrates but not for hydrolysis of unstructured or short polypeptides [5]. Thus, one possibility is that the lid Results is required in one way or another for proper interactions Extending the scope of the MPN domain with polyubiquitinated chains. In order to identify distantly related members of the MPN domain family, we constructed generalized profiles of At 66% identity between the human and yeast forms, the previously established MPN proteins. Included in the pro- MPN domain protein Rpn11 is the most highly conserved file construction were the proteasome lid components non-ATPase subunit of the 19S RP, on par only with the Rpn8 and Rpn11, the COP9 signalosome components highly conserved ATPase subunits, suggesting that it too Csn5 and Csn6, and the translation initiation factor 3 may play an enzymatic role within the RP [14,15]. Muta- components eIF3f and eIF3h, all from various eukaryotic tions in RPN11 cause cell cycle and mitochondrial defects, species. After scaling of the profile [24], significant hits temperature sensitivity, and sensitivity to DNA damaging were found in protein database, including the STAM-in- reagents such as UV or MMS, underscoring the impor- teracting protein AMSH [25], and the uncharacterized hu- tance of this subunit in proteasome function [16–18]. man proteins C6.1A and KIAA1915. Subsequently, Rpn11 is one of a minority of proteasome subunits that iterative profile refinement [24] was used to make the pro- exhibit dominant phenotypes upon overexpression. High file searches more sensitive. For that purpose, the newly dosage of human or S. pombe Rpn11 orthologs confer identified MPN proteins were included in the profile con- multidrug and UV resistance [19,20]. These effects may be struction process. After four iteration cycles, a stable set of linked to the stabilization of c-Jun observed upon overex- significantly matching bona fide MPN proteins was identi- pression of the Rpn11 subunit in Schistosoma, SmPOH1 fied. Representative members of this superfamily are [21]. In another case, however, overexpression of Rpn11 shown in Figure 1. can suppress an srp1 mutation, the yeast homolog of im- portin alpha, and enhance degradation of a proteasome In addition to the previously known MPN proteins, a substrate [22], illustrating that effects of Rpn11 are pleio- number of prokaryotic members were identified, includ- tropic. Together, these results may suggest that Rpn11 em- ing the phage tail assembly protein K from the bacteri- bodies an intrinsic enzymatic activity. ophage lambda and its closely related homologs from other phages and prophages. Even archaeal members of In order to gain insight into the functions and evolution- the MPN domain family were found, including predicted ary history of MPN domain proteins such as Rpn11, we proteins from Archaeoglobus fulgidus, Methanobacter ther- performed extended database searches using the general- moautotrophicum and from various Pyrococcus species (Fig. Page 2 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 1 The MPN+ motif. Sequence alignment of representative MPN domain protein, only two conserved blocks containing the MPN+ motif are shown. The proteins are grouped in seven categories: A, bacterial; B, archaeal; C, proteasome lid compo- nents; D, CSN components; E, eIF3 components; F, Prp8-like proteins; G other eukaryotic MPN proteins. In the eukaryotic groups, representative sequences from human (HS), Arabidopsis (AT), S. pombe (SP) and S. cerevisiae (SC) are shown. Prokary- otic species shown are lambda phage (BPL), Yersinia pestis (YP), Synechocystis sp. (SS), Mycobacterium tuberculosum (MyT), Pseu- domonas aeruginosa (PA), Pyrococcus horikoshii (PH), Archaeoglobus fulgidus (AF), Methanobacter thermoautotrophicum (MT). Residues invariant or conservatively substituted in at least 50% of all sequences are shown on black and grey background, respectively. The MPN+ motif residues are shown in red. The top line indicates the amino acids constituting the MPN+ motif; the rightmost column indicates whether a protein is considered an MPN+ protein or a plain MPN protein. Page 3 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 1). These proteins are the shortest MPN protein identified Mutants in the MPN+ motif of Rpn11 exhibit severe so far and most likely correspond to the structural core re- growth defects In order to assess the importance of the MPN+ residues for gion of the domain. Since the finding of prokaryotic MPN proteins was highly unexpected, the validity of the assign- efficient Rpn11 function, we performed site-directed mu- ment was confirmed by profile searches starting from the tagenesis of His111, Ser119, Asp122, which are part of the bacteriophage proteins that resulted in the same stable set conserved HxH–S–D sequence that defines the MPN+ of significantly matching proteins. motif. In addition, Cys116, which is not part of the MPN+ motif but is highly conserved between Rpn11 and Csn5 Definition of the MPN+ motif orthologs might be a candidate for an active site residue, Overall, there are no residues that are invariant through- was mutagenized too. As seen in Fig. 2, alanine substitu- out the MPN domain superfamily. However, while ana- tions in the MPN+ motif of Rpn11 cause severe growth de- lyzing the alignment of the newly identified proteins, it fects, temperature sensitivity, and sensitivity to amino became apparent that there are a number of polar residues acid analogs such as canavanine. Substitution mutations that are conserved in a highly coordinated fashion in a at different locations in the MPN+ motif display similar, subset of MPN domains (highlighted in Red in Fig. 1). but slightly distinct phenotypes. Specifically, the These amino acids form a pattern, referred to herein as the His111Ala mutant is extremely slow growing at 25C and 'MPN+ motif', which is part of the conventional MPN do- lethal when plated at elevated temperatures, even as low main and embedded within it. The MPN+ motif contains as 30C. The Asp122Ala and Ser119Ala mutants are viable a well-defined pattern of 'H-x-H-x[7]-S-x[2]-D', where x[7] but slow growing at 25C and 30C, and lethal when shift- and x[2] indicate stretches of seven and two non-con- ed to 37C or when exposed to amino acid analogs. By served residues, respectively. In addition to this conserved contrast, substitutions of the conserved Cys at position arrangement of four polar residues, there is an additional 116 showed no growth defect, and the cells appeared nor- glutamate residue in a more N-terminal region of the do- mal under all conditions tested. main, whose conservation is perfectly correlated with the occurrence of the motif (Figure 1). An aromatic residue, Stress conditions such as exposure to elevated tempera- preferentially a tryptophane, is found two positions up- ture or amino acid analogs are known to promote accu- stream of the conserved serine in most but not all MPN+ mulation of damaged proteins, which must be removed proteins. Thus, it should not be considered a part of the by the proteasome [26]. Since defects in the MPN+ motif core MPN+ motif. of Rpn11 cause heightened sensitivity to such conditions, it is reasonable to assume that proteasome function is With the possible exception of Prp8, all MPN domain pro- jeopardized in these mutants. We conclude that the teins shown in Figure 1 can unambiguously be classified MPN+ motif is critical for the proper function of Rpn11 as either belonging to the MPN+ or to the 'plain' MPN within the proteasome, but that Cys116 does not appear class. The validity of this distinction is underscored by the to be an essential residue in Rpn11. fact that invariably all observed orthologs of MPN+ pro- teins also belong to the MPN+ class. For instance, the pro- Multiubiquitinated proteins accumulate in MPN+ motif teasome lid complex from all eukaryotes contains one mutants MPN+ protein (Rpn11/S13) and one plain MPN protein In order to test whether proteasome function is indeed (Rpn8/S12). The same is true for the analogous CSN com- hampered in these rpn11 mutants, we checked the effects plex in multicellular eukaryotes, where the MPN+ protein of mutations on the ubiquitination pattern of cellular pro- is Csn5 and the plain MPN protein is Csn6. In fission teins. Whole cell extracts from rapidly growing WT or yeast, Csn6 appears to be absent. The same is true for the MPN+ motif mutants were separated by SDS PAGE and recently identified CSN-like complex of budding yeast; it immunoblotted with anti-Ub antibody (Fig. 3). High mo- contains only Csn5 but not Csn6 (Hofmann and Glick- lecular weight polyubiquitin-conjugates are not detected man; submitted for publication). The two MPN proteins at appreciable levels in extracts from yeast containing nor- of the eIF3 complex are both of the plain MPN type in mal proteasomes since they are rapidly turned over. High multicellular eukaryotes and fission yeast, while in bud- molecular weight polyubiquitinated proteins do accumu- ding yeast they appear to be missing altogether. Notably, late, however, in rpn11 MPN+ mutants, indicating defec- all newly identified prokaryotic MPN proteins are of the tive proteasome activity. In this assay, all mutations in MPN+ type; the same is true for the remaining unassigned MPN+ residues behave similarly. His111 mutants also ac- 'orphaned' MPN proteins in eukaryotes (Fig. 1). It should cumulate polyubiquitinated proteins when grown at be stressed that the MPN+ motif is not a stand-alone motif 25C, but since this strain is extremely slow growing it was to be found independently of the MPN domain; thus, all difficult to prepare extracts for comparison (not shown). MPN proteins can be classified as either MPN+ or plain No such accumulation is seen in cells containing Rpn11 MPN proteins. with Cys116 substitutions. Page 4 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 2 Growth of rpn11 Mutants under Different Growth Conditions. WT and mutant strains were streaked on YPD at 25, 30 and 37C. Cells were also plated on complete minimal media containing 1 g/ml of the amino acid analog canavinine instead of arginine. Plates were photographed after 3–5 days. Three single amino acid substitutions in the MPN+ motif of Rpn11 were studied: his111ala, Ser119ala, and asp122ala. For comparison, a substitution of a highly conserved cysteine residue (cys116ala) that is not part of the MPN+ motif (see Fig. 1) was included as well. At 25C, all MPN+ mutations are viable but slow growing. Hiss111 is extremely temperature sensitive, showing no appreciable growth even when shifted to 30C, or in the presence of even 1 g/ml of the amino acid analog canavinine. Asp122 and Ser119 substitutions show lethality under elevated temperature (37C) or exposure to canavanine. In comparison, Cys116 substitutions show no growth defects under these conditions. It appears that the MPN+ motif defines the role of Rpn11 short-lived proteins were measured in WT and in a repre- in the proteasome but that the conserved Cys116 residue sentative rpn11 mutant strain. In order to estimate the is not critical for proteasome function. The steady state generality of the effect, two different substrates were used: levels of polyubiquitin-protein conjugates are influenced a protein that is ubiquitinated by enzymes of the UFD by the rate of ubiquitination on the one hand, and by ubiquitination pathway [27], and a protein that is ubiqui- rates of deubiquitination or proteasome proteolysis on tinated by enzymes of the N-end rule pathway [28]. WT the other. Accumulation of polyubiquitinated proteins in Cells, or those harboring the S119A substitution in the rpn11 mutants could therefore be due to a slowdown in ei- MPN+ motif of Rpn11 were transformed with plasmids ther proteasome associated deubiquitination or proteas- expressing Arg--galactosidase (an N-end rule substrate) ome-dependent proteolysis. or Ub-pro--galactosidase (A UFD substrate). Arg--gal and Ub-Pro--gal are short-lived in wild-type yeast with Stabilization of short-lived proteasome substrate half-lives of ~2 and ~6 minutes, respectively [29]. Steady In order to test whether the accumulation of polyubiqui- state levels of these proteins were compared to the levels tinated conjugates is directly correlated with a defect in of a stable, long-lived protein, Met--galactosidase. As ex- proteasome function, we checked whether mutations in pected, WT cells accumulated high levels of the stable the MPN+ motif of Rpn11 bring about stabilization of Met-gal but only low levels of the rapidly degraded Arg- proteasome substrates. The steady state levels of known -gal and Ub-Pro--gal (Fig. 4). In contrast, the S119A Page 5 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 3 Accumulation of multiubiquitinated proteins in MPN motif mutants. Total cell extracts from WT yeast and from strains containing the MPN+ motif substitutions were separated on an 8% SDS gel and blotted with anti-ubiquitin antibodies. Cells were rapidly lysed in presence of 12% TCA in order to inhibit post lysis enzymatic activity. Accumulation of high MW polyubiquitinated proteins is detected in the Ser119 and Asp122 mutants, but not in WT or in the Cys116 mutants. His111 mutants accumulate polyubiquitinated proteins as well (not shown). A protein band migrating at around 20 kDa that is detected with the anti Ub antibody is used as an internal loading control. mutation in rpn11 lead to dramatic stabilization and accu- the proteasome. The importance of Rpn11 is independent mulation of both these short-lived proteins, such that the of the ubiquitination pathway. steady state levels of all three substrates were similar (Fig. 4). Stabilization of these short lived proteins was noted The possibility that these deficiencies in proteasome de- also in the D122A mutant (not shown). From these re- pendent proteolysis were caused by improper incorpora- sults we conclude that the MPN+ motif of Rpn11 is essen- tion of mutated Rpn11 into the lid, or that proteasome tial for proper proteolysis of ubiquitinated substrates by structure was hampered in rpn11 mutants was addressed Page 6 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 4 Stabilization of short-lived proteasome substrates. WT and the S119A mutant strains containing the Ub-Met-, Ub-Arg- , and Ub-Pro-gal constructs on multi-copy plasmids under the GAL promotor were tested for LacZ activity after galactose- induction. LacZ activity is indicative of steady-state levels of the reporter protein. WT cells accumulate high levels of the stable Met-gal, but rapidly degrade Arg-gal and UB-Pro-gal (left panel), whereas the MPN+ mutation shows dramatic stabilization and accumulation of both short-lived fusion proteins (right panel). by native gel electrophoresis. Cell extract from WT and ical for the proteins in which they are found. This idea is rpn11 mutants was resolved by nondenaturing PAGE and corroborated by the polar nature of the conserved MPN+ no gross structural changes were observed (Fig. 5). Overall residues: glutamate, histidines, serine and aspartate are all levels and proteolytic intensities of proteasomes from amino acids frequently found in the active site of enzymes MPN+ mutants were indistinguishable from WT, indicat- or as the coordinating ligands in metal-binding proteins. ing that the substitutions do not alter the structure of Certainly, these two possibilities are not mutually exclu- peptidase function of the proteasome. In addition, we sive. Several classes of enzymes, particularly metal con- found no evidence for natural abundance WT Rpn11 out- taining hydrolases and proteases, harbor bound metal 2+ side of the proteasome (data not shown), indicating that ions such as Zn as part of their catalytic center [30]. the phenotypes associated with mutated rpn11 are unlike- However, while the motif does bear some resemblance to ly to be due to unincorporated protein. Since peptidase ac- that found in metalloproteases, the specific organization tivity of mutant proteasomes was similar to WT (Fig. 5), of E–HxH–S–D residues does not correspond to the metal the effects of rpn11 mutations is most likely due to a defect ligands in any of the known Zinc proteases [30], thus pos- in proteolysis of ubiquitinated substrates (Fig. 4). itive identification of Rpn11 as a metalloprotease awaits definite proof. Discussion We have identified an arrangement of five residues, which The proteasome complex from a number of sources is are perfectly conserved in a subclass of MPN domains, known to contain a ubiquitin hydrolyzing activity [31– while none of them shows appreciable conservation out- 39], while the CSN complex is known to cause the hydro- side of this subclass. The highly correlated conservation of lytic removal of the Nedd8/Rub1 ubiquitin-like molecule these residues suggests that they participate in a common from the cullin subunit of the SCF ubiquitin ligase (E3) structural element and/or a common function that is crit- complex [40–42]. Thus, the MPN+ containing subunits of Page 7 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 Figure 5 Migration pattern of proteasome from WT and rpn11 mutants by non-denaturing PAGE. Cell extracts were pre- pared from Logarithmically growing WT or the rpn11 MPN+ mutants that were brought to an identical OD600 level. Extracts were clarified by centrifugation, and identical amounts of total protein were separated on native gel. The gel was then incu- bated for 10 minutes with the fluorescent peptide LLVY-AMC at 30C, and photographed under UV light (380/440 nm). Fluo- rescent bands indicate migration of Doubly capped and Singly capped 26S proteasome forms [14]. There is no noticeable change in migration pattern or overall levels of proteasomes from the different rpn11 mutants, indicating that mutated Rpn11 is properly incorporated into the lid and no gross structural changes in proteasome composition or amounts are due to the mutations. the proteasome lid (Rpn11) and the COP9 signalosome Single site substitutions in various Rpn11 MPN+ residues (Csn5) would be prime candidates for such a hydrolytic exhibit similar phenotypes supporting the identification function. The architecturally related eIF3 complex, for of MPN+ as a discrete functional motif. Due to the severe which no such enzymatic activity has been described, is growth phenotypes and attenuated ability of the proteas- conspicuously devoid of MPN+ proteins. That the CSN ome to proteolyze polyubiquitinated substrates in these from yeast lacks the plain MPN protein (Csn6) retaining mutants, it appears that the MPN+ motif defines the role only the MPN+ subunit (Csn5), while the plain MPN pro- carried out by Rpn11 in the lid. As we show, these defects teins found in eIF3 from eukaryotes appear to be missing arise from an intrinsic activity of Rpn11 within the context altogether in the yeast complex, emphasizes that the of the proteasome, and not due to a gross structural effect MPN+ residues are likely to be the catalytic residues, with upon incorporation of mutated Rpn11. As mentioned plain MPN subunit playing a redundant structural role in above, the lid, where Rpn11 is situated, is critical for pro- complexes in which they are found. Even though purified teolysis of polyubiquitinated substrates. So far, and some- recombinant Rpn11 does not appear to exhibit DUB capa- what surprisingly, all ubiquitin binding activity has been bilities (data not shown), it is possible that once incorpo- mapped to the Base of the 19S RP. Two subunits in the rated into their respective complexes, Rpn11 and Csn5 base can interact directly with ubiquitin chains, Rpt5 and confer the documented hydrolase activities onto the 19S Rpn10 [5,7,43,44]. A number of proteins, such as Rad23 RP and CSN. In this case, Rpn11 would belong to a and Dsk2, can also bind ubiquitin and interact with the unique class of enzymes, as all other known DUBs are proteasome, presumably with the base, thus they are cysteine proteases. Of note, the conserved cysteine residue thought to serve as shuttles of polyubiquitinated sub- common to both Rpn11 and Csn5, which is not part of strates to the proteasome [45,46]. It is possible that the lid the MPN+ motif, is not important for the function of rather than bind ubiquitin, serves to cleave or trim polyu- Rpn11. biquitin chains once attached to the Base. Page 8 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 While this manuscript was under review, two independ- Interestingly, all identified prokaryotic MPN domains, ent papers substantiated our findings by characterizing a both from archaea and from eubacteria, also contain the novel deubiquitinating activity in the proteasome, and MPN+ motif. It thus appears likely that the ancestral MPN showing that the MPN+ motif residues of Rpn11 are large- domain was of the MPN+ type, and that the extant 'plain' ly responsible for this activity [47,48]. These independent MPN proteins have lost this motif later on. It is possible studies identified DUB activity associated with the 19S RP that the plain MPN proteins play a structural role in com- of the proteasome, which is lacking upon lethal substitu- plexes that additionally contain a MPN+ subunit. So far tions in Rpn11. Interestingly, all substitution mutations all known MPN proteins are incorporated into complexes that we studied in the MPN+ motif of Rpn11 are viable. that also contain PCI proteins [51,52]. Interestingly, sev- Quite possibly, Rpn11 is not the sole proteasome-associ- eral of the prokaryotic organisms harboring MPN+ pro- ated DUB, and a number of DUBs play partially overlap- teins appear to lack PCI domain proteins and therefore ping functions. For instance, Doa4/Ubp4 interacts weakly probably could not form large PCI/MPN complexes relat- and substoichiometrically with the proteasome and may ed to the lid and CSN particles. An interesting case is the serve to release ubiquitin and regenerate the proteasome tail assembly protein 'K' of the bacteriophage lambda for the next catalytic cycle [37]. The Ubiquitin-like do- (vtak) and its many homologs from other phages and main (UBL) containing deubiquitinating enzyme, USP14, prophages (Fig. 1). Little is known about the specific func- has been found to interact with the proteasome from tion of the protein but none of the other tail assembly fac- mammalian sources [38]. Ubp6, the budding yeast ho- tors contain a PCI or MPN domain (K.H unpublished molog of USP14, interacts with the proteasome as well results). Our work suggests that the K-protein plays an en- [49], and plays a role in proteasome-associated deubiqui- zymatic role in tail assembly rather than a merely structur- tination [39]. It has also been reported that the Drosophila al one, although whether the analogy to Rpn11 is relevant DUB p37a [31] and its homologs UCH37 (H. sapiens) and to vtak is still enigmatic. Uch2 (S. pombe), may be responsible for the polyubiqui- tin chain editing function associated with purified protea- A similar situation exists for the orphan MPN proteins of somes [31,34,35]. However, as budding yeast lacks an eukaryotes. It is noteworthy that complex eukaryotic or- obvious ortholog of UCH37/p37a, other DUBs must play ganisms have far more orphan MPN protein than orphan a greater role in proteasomes from this organism. PCI proteins. However, the known PCI/MPN complex particles have stoichiometries that require more PCI com- Finding MPN+ motif proteins in prokaryotes will help ponents than MPN components. This apparent paradox- elucidate the origins of proteasome evolution and the on can be resolved by a number of different but non- function of Rpn11 in particular. The proteasome probably exclusive assumptions. i) Additional PCI proteins have evolved from self-compartmentalized macromolecular eluded detection due to high sequence divergence allow- proteases found in prokaryotes. Thus, proteasomal subu- ing for more than three PCI/MPN complexes to exist, ii) nits, or proteins with motifs related to proteasomal subu- alternative MPN proteins can interact with existing PCI nits, are present in archaea and certain eubacteria. These complexes, iii) some MPN proteins have (enzymatic) "lidless" prototypes include the 20S CP subunits, and ho- functions outside of PCI/MPN complexes. Currently, mologs of the base ATPases (Rpt subunits) [50]. However, there is no data available supporting the first two possibil- proteins relating to the ubiquitination process, such as ities, although there is some promiscuity in the PCI pro- ubiquitin itself, ubiquitin activating conjugating or ligat- tein interactions [52,53]. The third possibility is likely to ing enzymes, and even lid subunits, are missing from be true in prokaryotes and could also explain why all eu- prokaryotes. Identification of a motif from Rpn11 – a pro- karyotic orphan MPN proteins belong to the MPN+ class. tein that is linked to processing of polyubiquitinated pro- This could also explain how supra-stochiometric amounts teins – in prokaryotic proteins is an interesting of Rpn11 exhibit dominant phenotypes; possibly it can development. It is possible that during evolution, the pro- function outside of the proteasome as well. teasome recruited an existing enzyme as the lid was form- ing into a regulatory module of the proteasome; likewise Conclusions for the analogous CSN complex. Studying the prokaryotic The MPN+ motif is abundant in certain MPN-domain MPN+ motif proteins should aid in elucidating what this proteins, including newly identified proteins of eukaryo- motif does. The fact that these proteins are the shortest tes, bacteria and archaea thought to act outside of the tra- MPN proteins and correspond to the structural core re- ditional large PCI/MPN complexes. The putative catalytic gion of the domain should greatly aid in enzymatic and nature of the MPN+ motif makes it a good candidate for a structural studies, especially in comparison to members of pivotal enzymatic function, possibly a proteasome-associ- the family such as Rpn11 and Csn5 that are naturally ated deubiquitinating activity and a CSN-associated found only incorporated into complexes. Nedd8/Rub1-removing activity. The importance of the MPN+ motif for the efficient function of the proteasome Page 9 of 12 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/28 component Rpn11 is compatible with this idea, though Stabilization of known proteasome substrates the fact that none of the single amino acid substitutions Steady-state levels of the Met-, Arg-, and Ub-Pro-gal fu- are lethal indicates that either the function of Rpn11 is not sion proteins were measured by testing LacZ activity [27]. strictly essential for proteasome function, or more likely, Wild type and rpn11 cells harboring multi-copy URA3- it is partially redundant with other subunits. marked plasmids containing each construct were grown to late log in minimal media containing raffinose for carbon Methods source, and induced with 2% Galactose for 4 hrs. The cells Bioinformatics were then lysed and LacZ activity upon introduction of the All database searches were performed with a nonredun- substrate ONPG was calculated when taking into account dant data set constructed from current releases of Swiss- the time, total protein concentration and amount of prod- Prot, TrEMBL, and GenPept [54,55]. Generalized profile uct obtained (measured by 405 nm absorbance). [23] construction and searches were run locally using the pftools package, version 2.1. (program available from the Non-denaturing PAGE and peptidase activity detection URL [ftp://ftp.isrec.isb-sib.ch/sib-isrec/pftools/]). Profiles Yeast cells were lysed in buffer A (10% glycerol, 50 mM , 1 mM EDTA) using glass were constructed using the BLOSUM45 substitution ma- tris7.4, 1 mM ATP, 1 mM MgCl trix [56] and default penalties of 2.1 for gap opening and beads, and clarified by centrifugation at 20  g. Non-dena- 0.2 for gap extension. The statistical significance of profile turing gel was run as previously described [14]. The gel matches was derived from the analysis of the score distri- was then incubated for 10 minutes in 10 ml of buffer A bution of a randomized database as described [24]. Data- and 0.1 mM of the fluorescent peptide LLVY-AMC at base randomization was performed by individually 30C. Under UV light, appearing bands show peptidase inverting each protein sequence, using SwissProt 34 as the activity indicate the migration pattern of the 26S proteas- data source. ome. Single amino acid substitutions Abbreviations Haploid yeast strain with a chromosomal knock out of RP: 19S regulatory particle of the proteasome rpn11 was constructed, in which a single copy URA- marked plasmid expressing the RPN11 gene complements CP: 20S core particle of the proteasome the chromosomal knock out (MY71). The heterozygote RPN11/rpn11 diploid was purchased from EUROSCAF CSN: COP9 signalosome and transformed with a single copy CEN plasmid with URA3 selection (ycplac33) expressing RPN11 from its eIF3: eukaryotic initiation of translation factor 3 own promotor (M82). Growth of MY71 was identical to the isogenic WT strain from EUROSCARF. Plasmids ex- Rpn: regulatory particle non-ATPase subunit pressing the single site substitutions in Rpn11 were gener- ated using PCR site directed mutagenesis on a similar CEN Rpt: Regulatory particle triple-A ATPase subunit plasmid with the LEU2 marker for selection (ycplac111). In this manner the following plasmids were constructed Ub: ubiquitin RPN11 (M134), rpn11-C116A (M134), rpn11-H111A (M143), rpn11-S119A (M144), rpn11-D122A (M145). DUB: deubiquitinating enzyme Those plasmids were then transformed into the above yeast strains, and the URA3-marked WT rescue plasmid -gal: -galactosidase was then forced out of the cells by the presence of 5'FOA in the medium. FOA shuffling was done at 25C as most Authors' contributions rpn11 mutants were severely temperature sensitive. In this Kay Hofmann and Michael Glickman initiated the project manner we got viable yeast rpn11 mutant strains. and participated in its design. Kay Hofmann performed bioinformatic characterization of the MPN+ motif (fig. 1). Phenotypes and mutant characterization Noa Reis and Vered Maytal designed and constructed the Single colonies grown on YPD at 25C were streaked onto MPN+ amino acid substitutions. Vered Maytal carried out YPD and shifted to various temperatures. Plates were pho- all characterization of Rpn11 mutants shown in Figures tographed after 3–5 days. For canavinine sensitivity, 2,3,4,5. 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