Báo cáo khoa học: Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin⁄26S proteasome-mediated proteolysis

Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin⁄26S proteasome-mediated proteolysis Antony S. Fatimababy1, Ya-Ling Lin1,2,3, Raju Usharani1, Ramalingam Radjacommare1, Hsing-Ting Wang1, Hwang-Long Tsai1, Yenfen Lee1 and Hongyong Fu1,2,3

1 Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 2 Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University and Academia Sinica, Taipei, Taiwan 3 Graduate Institute of Biotechnology and Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan

Keywords RPN10; RPN13; ubiquitin receptor; ubiquitin recognition; UBL–UBA factors

Correspondence H. Fu, Institute of Plant and Microbial Biology, Academia Sinica, 128, Sec 2, Academia Road, Nankang, Taipei 115, Taiwan Fax/Tel: +886 2 2787 1183 E-mail: hongyong@gate.sinica.edu.tw

(Received 23 October 2009, revised 24 November 2009, accepted 2 December 2009)

doi:10.1111/j.1742-4658.2009.07531.x

Structured digital abstract l A list of the large number of protein-protein interactions described in this article is available

via the MINT article ID MINT-7307429

Abbreviations GST, glutathione S-transferase; LRR, leucine-rich repeat; PRU, Pleckstrin-like receptor of ubiquitin; RP, regulatory particle; UBA, ubiquitin- associated domain; UBL, ubiquitin-like domain; UIM, ubiquitin-interacting motif; UPP, ubiquitin ⁄ 26S proteasome-mediated proteolysis; Y2H, yeast two-hybrid analysis.

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The recognition of ubiquitylated substrates is an essential element of ubiqu- itin ⁄ 26S proteasome-mediated proteolysis (UPP), which is mediated directly by the proteasome subunit RPN10 and ⁄ or RPN13, or indirectly by ubiquitin receptors containing ubiquitin-like and ubiquitin-associated domains. By pull-down and mutagenesis assays, we detected cross-species divergence of the major recognition pathways. RPN10 plays a major role in direct recogni- tion in Arabidopsis and yeast based on the strong afﬁnity for the long and K48-linked ubiquitin chains. In contrast, both the RPN10 and RPN13 ho- mologs play major roles in humans. For indirect recognition, the RAD23 and DSK2 homologs (except for the human DSK2 homolog) are major receptors. The human RAD23 homolog is targeted to the 26S proteasome by the RPN10 and RPN13 homologs. In comparison, Arabidopsis uses UIM1 and UIM3 of RPN10 to bind DSK2 and RAD23, respectively. Yeast uses UIM in RPN10 and LRR in RPN1. Overall, multiple proteasome subunits are responsible for the direct and ⁄ or indirect recognition of ubiquitylated substrates in yeast and humans. In contrast, a single proteasome subunit, RPN10, is critical for both the direct and indirect recognition pathways in Arabidopsis. In agreement with these results, the accumulation of ubiquity- lated substrates and severe pleiotropic phenotypes of vegetative and repro- ductive growth are associated with the loss of RPN10 function in an Arabidopsis T-DNA insertion mutant. This implies that the targeting and proteolysis of the critical regulators involved are affected. These results sup- port a cross-species mechanistic and functional divergence of the major rec- ognition pathways for ubiquitylated substrates of UPP.

A. S. Fatimababy et al.

Cross-species divergence of ubiquitin receptors

Introduction

some and ubiquitylated substrates, respectively [4,15– 17]. It appears that multiple docking sites for various UBL–UBA factors are located on the base subcomplex of the regulatory particle, including RPN1 and the ubiquitin receptors, RPN10 and RPN13 [12,18]. The third class includes CDC48-based complexes, which are involved primarily in endoplasmic reticulum-associ- ated degradation [19,20].

Distinct ubiquitin-binding motifs ⁄ domains are used by the various ubiquitin receptors [13,21,22]. The ubiquitin-interacting motif (UIM), the Pleckstrin-like receptor of ubiquitin (PRU) and UBA are utilized by RPN10, RPN13 and UBL–UBA factors, respectively. Multiple ubiquitin-binding sites are associated with dif- ferent subunits of the CDC48 complexes, including the NPL4-zinc ﬁnger [23] and UBA [24] in NPL4 and p47, respectively, and the CDC48 ⁄ p97 N-domain fold in CDC48 and UFD1 [25].

Ubiquitin ⁄ 26S proteasome-mediated proteolysis (UPP) controls the half-life of numerous critical regulatory proteins and is an intimate regulatory component of many cellular processes, including cell division, tran- scription, DNA repair and signal transduction [1]. The proteasomal recognition of ubiquitylated substrates is an important mechanistic and regulatory component of UPP that connects the substrate of the conjugation machinery to the 26S proteasome. Although the pre- dominant step(s) in controlling substrate speciﬁcity are regulated by post-translational modiﬁcation or confor- mational changes in the substrates and by the associa- tion between the substrates and their conjugation enzymes [2,3], accumulating evidence indicates that an additional layer of substrate selectivity can be medi- ated by various ubiquitin receptors during the prote- asomal recognition of ubiquitylated substrates [4,5]. However, limited information is available on how this substrate speciﬁcity is determined by the ubiquitin receptors.

recognition of

in UPP,

The predominant targeting signal for 26S protea- some-mediated proteolysis appears to be the K48-linked ubiquitin chain, which has a minimum length of four ubiquitin units [6]. The hydrophobic patch comprised of L8, I44 and V70 in ubiquitin is the primary contact sur- face for ubiquitin receptors that mediate proteasomal degradation [7]. Like other types of linkage, the exact structural elements in the K48-linked chain that deter- mine the selectivity by various ubiquitin receptors remain largely undeﬁned. However, they are probably associated with the L8–I44–V70 hydrophobic surface. Although the K48-linked ubiquitin chain is the predomi- nant signal for the recognition of ubiquitylated sub- strates structural variants probably exist because there are abundant receptors in different spe- cies. Furthermore, ubiquitin chains that are linked at other positions, such as K11, K29 and K63, are compe- tent signals for proteasomal degradation [8–10].

To resolve the mechanistic details of the distinct proteasomal recognition pathways for the ubiquitylat- ed substrates of UPP, the structural determinants for several critical interfaces need to be resolved. These include interactions between various ubiquitin recep- tors and ubiquitin chains of various linkage types, pro- teasomal the UBL–UBA factors, interactions among the major ubiquitin receptors, and interactions between ubiquitin receptors and their asso- ciated regulators or speciﬁc substrates. Moreover, little is known regarding the biochemical properties of the major ubiquitin receptors from different species, in terms of their selectivity for linkage types and the lengths of the ubiquitin chains and their associated structural elements. An extensive survey of UBA-con- taining factors including several mammalian and yeast UBL–UBA factors revealed signiﬁcant differences with regard to the selectivity of the linkage type. However, these results were primarily acquired using isolated domains ⁄ motifs and could be substantially different if examined in the context of the full-length proteins [26]. Furthermore, the potential cross-species divergence of proteasomal docking and the associated structural determinants for the UBL–UBA factors have not yet been thoroughly examined.

for

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Three major classes of ubiquitin receptors for UPP that appear to be conserved among different species have been described. The ﬁrst class includes intrinsic 26S proteasome base subunits, such as RPN10 [11], RPN13 [12,13] and RPT5 [14], which directly recognize ubiquitylated substrates. The second class includes shuttle factors that contain ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, such as RAD23, DSK2 and DDI1, which require an additional prote- asomal docking step to target the ubiquitylated sub- strates to the 26S proteasome. The UBL–UBA factors contain one UBL and one or two UBAs in the N- and C-termini that are capable of binding the 26S protea- Using a cross-species comparison approach, we observed distinct ubiquitin chain binding properties and associated structural elements the major Arabdopsis, human and yeast ubiquitin receptors. Moreover, we also identiﬁed distinct proteasomal docking sites and divergent interfaces for the RAD23 and DSK2 homologs. Interestingly, whereas multiple proteasome subunits are involved in the direct and ⁄ or

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Cross-species divergence of ubiquitin receptors

(RPN10) in Arabdopsis. is most critical

dominant targeting signal for UPP [6], the K63-linked ubiquitin chain is the predominant signal for DNA repair, endocytosis and signal transduction [27–29]. The major ubiquitin receptors were expressed and puri- ﬁed as glutathione S-transferase (GST)-tagged wild- type or mutated variants (Table S1). The preferences of various ubiquitin receptors for particular chain types and lengths were examined using GST pull-down anal- ysis, in which the proﬁles of the pulled-down and input chains were compared by immunoblotting. indirect proteasomal recognition of ubiquitylated sub- strates in yeast and humans, a single proteasome sub- unit In agreement with these results, the accumulation of ubiq- uitylated substrates and severe pleiotropic phenotypes were observed in an Arabdopsis RPN10 knockout mutant. This implies that targeting and proteolysis of the relevant critical regulators are affected. Our results support a cross-species mechanistic and functional divergence of the major recognition pathways for the ubiquitylated substrates of UPP.

Results

Divergence of the ubiquitin binding properties of the major ubiquitin receptors

Whereas Arabdopsis and yeast RPN10 had signiﬁ- cantly stronger afﬁnities for long and K48-linked ubiqu- itin chains rather than the K63-linked chains, the human RPN10 homolog (S5a) showed strong afﬁnities for long ubiquitin chains of both linkage types (Table 1 and Fig. S1). The distinct chain-type preferences of RPN10 from different species were conﬁrmed by competitively pulling-down mixtures of tetra-ubiquitin chains contain- ing an equal amount of both linkage types, which could be distinguished by their distinct electrophoretic mobili- ties (data not shown). A similar, strong afﬁnity for either the K48- or K63-linked tetra-ubiquitin chain was also reported previously for S5a [26].

Table 1. Ubiquitin chain binding properties and associated structural domains of the major ubiquitin receptors from Arabidopsis, humans and yeast. DN, data not shown; NA, not applicable; ND, a potential novel domain is involved; PRU, Pleckstrin-like receptor of ubiquitin; TS, this study; UBA, ubiquitin-associated domain; UIM, ubiquitin-interacting motif.

Arabidopsis

Human

Yeast

Ubiquitin bindingb

K48 K63 Domainc Moded Ref

K48 K63 Domainc Moded

Ref

K48 K63 Domainc Moded Ref

Namea

RPN10

+++ +

UIM1

TS [30] +++ +++ UIM1

TS [20,31] +++ +

UIM

TS [30]

+ UIM2

)

+ + +++ +

PRU UBA1

+++ +++ PRU +++ +

UBA1

+++ +

NA UBA1

TS [12] TS

d TS I (N10) TS

TS, [12] D I (N10,N13) TS, [20]

d? I (N1)

& UBA2

+++ +

UBA

+++ ++ UBA

I (N10) DN

I (N10) TS [20]

RPN13 RAD23 (hHR23) DSK2 (PLIC1) DDI1

UBA

UBA*

TS [20]

a The names in parentheses are those for the human homologs. b The approximate binding afﬁnity for either the K48- or K63-linked ubiquitin chains is designated qualitatively by +++, ++, + and ) for strong, moderate and weak binding, and the absence of binding, respectively. c For those situations in which multiple domains are involved in the binding, & indicates that the involved domains contribute additively to the binding, and + indicates that both domains act cooperatively. Human UIM2 is more critical to the binding and is underlined. UBA of yeast DDI1 (marked with an asterisk) was determined by mutagenesis to have diverged residues at the interaction interface. d D ⁄ d and I ⁄ i indicate a direct or indirect role, respectively, in the recognition of ubiquitylated substrates. The upper/bold and lower cases indicate a major and a minor role, respectively, based on the binding afﬁnity for the K48-linked ubiquitin chains. N1, N10 and ⁄ or N13 (for RPN1, -10 and -13, respec- tively) in bold and parentheses are the docking subunits for either the RAD23 or DSK2 homologs from different species. A ? is added for yeast RPN13 and all DDI1 homologs as chain binding activity was not detected using yeast RPN13 and the docking site for DDI1 was not identiﬁed. Therefore, their roles in the recognition of ubiquitylated substrates are not clear.

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To examine potential differences in the substrate selec- tivity and structural divergence of the major ubiquitin receptors of UPP across species, we determined their ubiquitin chain binding properties and associated struc- tural elements ⁄ residues. We ﬁrst compared binding properties among Arabdopsis, human and yeast homo- logs of the major ubiquitin receptors RPN10, RPN13, RAD23, DSK2 and DDI1, with either K48- or K63- two to seven linked ubiquitin chains consisting of ubiquitin units (Table 1). Whereas a K48-linked ubiqu- itin chain of more than four ubiquitin units is the pre- The novel base subunit RPN13 was found to be a new proteasomal ubiquitin receptor [12,13]. Distinct ubiquitin chain binding properties were observed for the Arabdopsis, human and yeast RPN13 homologs. As shown in Table 1 and Fig. S2A, Arabdopsis RPN13

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Cross-species divergence of ubiquitin receptors

exhibited a weak afﬁnity for K48-linked chains of three and four ubiquitin units and for K63-linked chains of various lengths. By contrast, human RPN13 had a strong and approximately equivalent afﬁnity for both K48- and K63-linked chains, revealing that the binding is similar to that of human S5a (RPN10). It appears that human RPN13 prefers chains of more than three and four ubiquitin units for the K48- and K63-linked chains, respectively (compare the input and eluted chain proﬁles in Fig. S2A). Moreover, yeast RPN13 did not interact with either K48- or K63-linked chains. IAYAM) of

left),

substrates, we determined the involved structural ele- ments ⁄ residues of the major ubiquitin receptors (Table 1). RPN10 homologs from Arabdopsis, humans and yeast contain three, two and one UIMs, respec- tively, of which Arabdopsis uses the ﬁrst UIM (UIM1) for binding ubiquitin chains [30,31] (Table 1, and data not shown). Involvement of the UIMs of the human and yeast RPN10 homologs in binding to K48- and K63-linked ubiquitin chains was determined using sin- gle or double UIM mutations. Five critical hydropho- bic residues within UIM1 (216–220; LALAL) and ⁄ or UIM2 (287–291; the human RPN10 homolog (S5a) and UIM of yeast RPN10 (228–232; LAMAL) were replaced by asparagines. Mutation of UIM abolished the binding activity of yeast RPN10 to the ubiquitin chains of both linkage types (Fig. S1B, GST–Scrpn10–uim), indicating that UIM plays a criti- cal role in ubiquitin chain binding. Mutation of UIM2 of the human RPN10 homolog S5a abolished binding to the K48- and K63-linked chains almost completely, whereas mutation of UIM1 reduced binding to both signiﬁcantly (Fig. S1A, GST–S5a–uim2 chain types and GST–S5a–uim1). Double-site mutation abrogated the binding activity completely (GST–S5a–uim1_2), indicating that the two UIM sites of S5a are the pri- mary structural motifs for ubiquitin chain binding. The association of a stronger binding defect with the UIM2 mutation suggests a more critical role for UIM2. It is apparent that the amount of precipitation of the K48- and K63-linked ubiquitin chains associated with wild-type S5a cannot simply be attributed to an additive effect of the two single UIM-containing vari- ants (Fig. S1A), and this observation supports a coop- erative binding mode for the two UIMs of the human RPN10 homolog (S5a).

A signiﬁcant amount of high molecular mass ubiqui- tylated proteins from crude Arabdopsis extracts can be pulled-down readily by GST-fused Arabdopsis or human RPN13 (Fig. S2B, indicating a role for ubiquitylated substrate recognition. In agreement with stronger ubiquitin chain binding activity, a the relatively higher level of ubiquitylated proteins was associated with human RPN13. By contrast, no ubiqui- tylated proteins can be precipitated using yeast RPN13. As shown in Table 1 and Fig. S3, distinct ubiquitin chain binding properties were also detected with the Arabdopsis, human and yeast UBL–UBA ubiquitin receptors examined, except with the RAD23 homologs. Similar to Arabdopsis RPN10 (Fig. S1), RAD23 homo- logs from Arabdopsis, humans (hHR23b) and yeast had signiﬁcantly stronger afﬁnities for longer K48- linked chains than for K63-linked chains (Fig. S3A,B). However, the human DSK2 homolog (PLIC-1) had moderate afﬁnity but a clear preference for K63-linked chains (Fig. S3A). This contrasts to the preference for longer, K48-linked ubiquitin chains displayed by the Arabdopsis DSK2 homologs (Table 1 and data not shown). Furthermore, yeast DSK2 showed strong and nearly equivalent afﬁnities for the K48- and K63- linked ubiquitin chains (Fig. S3B). A similar preference for either K48- or K63-linked tetra-ubiquitin chains was observed previously for the isolated UBA of yeast DSK2 [26]. This was conﬁrmed by comparing the pull- down of tetra-ubiquitin chains of either linkage type (data not shown). In the case of DDI1 homologs, we observed weak afﬁnities for both K48- and K63-linked chains when using the human and yeast homologs (Table 1 and Fig. S3A,B). These results are similar to those obtained with Arabdopsis DDI1 (Table 1 and data not shown).

The divergent structural requirements of the major ubiquitin receptors for ubiquitin chain binding

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To examine the cross-species divergence of the struc- tural requirements for the recognition of ubiquitylated The residues of human RPN13 that are critically involved in ubiquitin binding have been identiﬁed by molecular docking, based primarily on the crystal structure of mammalian RPN13, and these residues are located within a novel ubiquitin-binding domain PRU [13]. In general, the corresponding residues are conserved in Arabdopsis RPN13, but they diverge sig- niﬁcantly in yeast RPN13 (Fig. S4). These ﬁndings are in agreement with the observation that yeast RPN13 is unable to bind both ubiquitin chains and conjugates. Several critical residues of mammalian RPN13, includ- ing L56, F76, D79 and F98, have been shown to be essential for ubiquitin binding using mutagenesis and in vitro pull-down assays [13]. Binding to both the K48- and K63-linked ubiquitin chains was affected drastically when the corresponding residues of Arabd- opsis and human RPN13 were mutated individually to A, R, Q (or N) and R, respectively (Figs S4–S5),

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Cross-species divergence of ubiquitin receptors

indicating that the same interfaces in Arabdopsis and human RPN13 appear to be critical for ubiquitin chain binding (Table 1).

ubiquitin chains. However, although it was signiﬁ- cantly reduced, we clearly detected binding of the UBA-mutated yeast DDI1 to K48-linked ubiquitin chains. This contrasts with the complete abrogation of binding associated with a similar UBA mutation of Arabdopsis DDI (Table 1 and data not shown). This indicates a possible variation in the chain- binding interface in yeast DDI1.

Structural divergence of proteasomal recognition of the RAD23 and DSK2 homologs by RPN10

With respect to the indirect recognition of ubiquity- lated substrates by UBL–UBA factors, is not known whether the same proteasomal docking sites are used in a given species and whether the docking sites and associated interfaces are conserved across species. Arabdopsis uses RPN10, but not RPN1 and RPT5, to receive RAD23 and DSK2 through separate sites (UIM3 and UIM1, respectively) (see below and Table 2). We determined the potential role and associ- the human and yeast ated domains ⁄ residues of RPN10 homologs in the recognition of UBL–UBA factors using pull-down assays (Table 2 and Figs 1–2). Human and yeast RPN10 homologs were expressed and puriﬁed as T7-tagged wild-type or mutated vari- ants; the UBL–UBA factors, including the RAD23, DSK2 and DDI1 homologs, were expressed and puri- ﬁed as GST-tagged wild-type or mutated variants (Table S1).

RAD23 homologs from Arabdopsis, humans or yeast contain two UBA domains (UBA1 and -2), in which each of the UBAs of Arabdopsis RAD23 contributes additively to the binding of K48-linked ubiquitin chains (Table 1 and data not shown). The roles of the UBAs in ubiquitin chain binding of human and yeast RAD23 homologs were determined using single- or double UBA mutations (Table 1 and Fig. S6A,B). Three conserved residues implicated in ubiquitin bind- ing within each UBA were replaced with alanines; these residues were 200–202 (MGY) and 376–378 (LGF) in the human homolog hHR23b and 158–160 (MGY) and 367–369 (LGF) in yeast RAD23. As shown in Fig. S6A, mutation of UBA1 or UBA2 of human hHR23b reduced the binding activity of K48- or K63-linked ubiquitin chains signiﬁcantly, indicating that both UBAs are critical for the binding of ubiqu- itin chains of both linkage types. It appears that the amount of K48-linked ubiquitin chain pulled-down by the wild-type protein is approximately the additive contribution of the two single UBA site-containing mutants. Mutation of both sites completely abrogated the chain binding activity (uba1_2) of both linkage types, indicating that the two UBA sites of human hHR23b are the primary structural motifs responsible for ubiquitin chain binding. For yeast RAD23, the mutation of UBA1, but not of UBA2, abrogated the binding activity to ubiquitin chains of either linkage type almost completely, indicating that UBA1 plays a major role in binding of the ubiquitin chain (Fig. S6B). Mutation of both sites also abrogated the binding activity for both linkage types completely (Fig. S6B, uba1_2).

As shown in Fig. 1A, the human RPN10 homolog (S5a) was pulled-down readily by the GST-fused RAD23 (hHR23b) or DSK2 (PLIC1) homolog, but not by DDI1. By contrast, yeast RPN10 was pulled- down by GST-fused DSK2, but not RAD23 and DDI1 (Fig. 1B). These results indicate that, as for Arabdopsis RPN10, the human RPN10 homolog (S5a) can function as a potential docking subunit for both the RAD23 and DSK2 homologs. However, yeast RPN10 can serve as a docking subunit for DSK2 but not for RAD23 (Table 2). structural motif

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The human DSK2 homolog (PLIC1) and yeast DSK2 and DDI1 each contain a single UBA; the role of these UBAs in ubiquitin chain binding was deter- mined (Table 1 and Fig. S6C). No sequence similar to the UBA was identiﬁed in human DDI1, suggest- ing that a potentially novel is involved in ubiquitin binding. Two conserved residues believed to mediate the interaction with ubiquitin within the various UBAs were replaced by alanines; these are 557–558 (MG) of the human DSK2 homo- log (PLIC1), 342–343 (MG) of yeast DSK2 and 401– 402 (LG) of yeast DDI1. Mutation of the UBA of human and yeast DSK2 homologs abolished binding to ubiquitin chains of both linkage types, as did the the Arabdopsis DSK2 equivalent UBA mutants of homologs (Table 1 and Fig. S6C). The UBA mutation of yeast DDI1 also abolished binding to K63-linked The involvement of the UIM in the human and yeast RPN10 homologs in recognition of the RAD23 and ⁄ or DSK2 homologs was determined (Table 2) using single and double UIM mutations similar to those used in the chain-binding analyses. Compared with wild-type human S5a, recovery of the UIM1 or UIM2 mutant by GST-fused hHR23b or PLIC1 was signiﬁcantly reduced or completely abolished, respec- tively (Fig. 1A, uim1 and uim2). This indicates that UIM1 and, in particular, UIM2 of S5a play a critical role in the recognition of hHR23b and PLIC1. The amount of wild-type S5a precipitated using GST-fused

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Cross-species divergence of ubiquitin receptors

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hHR23b or PLIC1 did not reﬂect additive contribu- tions from the two single UIM mutants. This indicates possible cooperation between UIM1 and UIM2 in S5a in the interaction with hHR23b and PLIC1 (Fig. 1A), in a way that is similar to their roles in ubiquitin chain binding (Fig. S1A). As expected, recovery of the dou- ble UIM mutant by either GST-fused hHR23b or PLIC1 was also abrogated completely (uim1_2). For yeast RPN10, the UIM mutation abolished its recov- ery by GST-fused DSK2. This indicates that it is criti- cal for DSK2 recognition, in addition to its role in ubiquitin chain binding (Fig. 1B, uim). Because the UIMs of RPN10 homologs

3 2 D A R

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U R P

. s t l u s e r

a e m a N

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s e m a N a

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are involved in recognition of the RAD23 and ⁄ or DSK2 homologs, it is rational to suggest that the potential hydrophobic patches in the UBLs of the RAD23 and DSK2 homologs, which are equivalent to the hydro- phobic patch containing L8, I44 and V70 in ubiqu- itin, are involved in the association with the RPN10 homologs. In general, residues that correspond to L8, I44 and V70 of ubiquitin in the UBLs of the RAD23 and DSK2 homologs from Arabdopsis, humans and yeast are conserved. However, we observed a clear divergence in the corresponding residues in the UBLs of the yeast RAD23 and DDI1 homologs (Fig. S7), supporting their inability to associate with the RPN10 homolog. We examined the role and poten-

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A

homologs and yeast DSK2 play a role in the associa- tion with the RPN10 homolog. Furthermore, we detected a clear structural divergence in the UBL interfaces of human hHR23B and yeast DSK2, com- pared with the corresponding homologs in Arabdopsis (Table 2).

B

Fig. 2. The UBLs of human and yeast RAD23 and ⁄ or DSK2 homo- logs are critical for association with the proteasomal subunit RPN10. The association with the RPN10 homolog from humans (S5a) or yeast (ScRPN10) was analyzed by pull-down using various GST-fused UBL variants of human hHR23b, PLIC1 (A) or yeast DSK2 (B). One-hundredth of the input RPN10 homolog (Inp) from humans or yeast and the pulled-down products were analyzed by immunoblotting against a-T7. One-tenth of the pulled-down prod- ucts (Baits 10·) was examined by staining with Brilliant Blue R to conﬁrm that the baits had been immobilized equally. The pulled- down products that were derived from GST alone were analyzed as a negative control. The asterisks indicate degradation products of the GST-fused yeast DSK2 variants. WT, wild-type.

Interestingly, whereas

We further examined the overall structural conserva- tion of the interfaces between RPN10 and RAD23 or DSK2 homologs using cross-species interaction analy- ses. Wild-type and single or double UIM variants of the RPN10 homolog from one species were tested using GST pull-down assays to assess their ability to interact with the RAD23 (Fig. 3A–C) or DSK2 homo- logs (Fig. 3D–F) from different species. As shown in Fig. 3A, the single-site mutation of UIM3 (uim3), but not of UIM1 or -2 (uim1 or uim2), abolished the asso- ciation of RPN10 with the RAD23 homolog from humans or Arabdopsis. Furthermore, the double UIM mutant (uim1_2) containing an intact UIM3 motif associated with the Arabdopsis and human RAD23 homologs, but the double UIM mutants (uim2_3 and uim1_3) containing intact UIM1 or UIM2 motifs, respectively, did not. Similarly, as shown in Fig. 3B, human S5a was capable of interacting in a cooperative manner with the RAD23 homolog from humans or Arabdopsis through UIM2 and, to a lesser extent, the Arabdopsis and UIM1. human RAD23 homologs were capable of interacting with yeast RPN10 through UIM (Fig. 3C), yeast RAD23 did not bind to yeast RPN10 or to the Arabd- opsis and human RPN10 homologs (Fig. 3A–C, upper). These results indicate that the interfaces of the RPN10–RAD23 interaction are conserved in Arabdop- sis and humans, and that UIM3 of Arabdopsis RPN10 and both UIM sites of human S5a play critical roles. However, whereas the UIM motif of yeast RPN10 is conserved through evolution for association with the RAD23 homologs from Arabdopsis and humans, the UBL of yeast RAD23 has diverged.

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tial divergence of hydrophobic patches in the UBLs of the RAD23 and ⁄ or DSK2 homologs from Arabd- opsis, humans and yeast in binding to RPN10. Two conserved residues that were equivalent to L8, I44 or V70 of ubiquitin in the UBLs of the RAD23 and DSK2 homologs from Arabdopsis and humans were replaced separately by alanine (Fig. S7). Replacement of the residue corresponding to L8 or I44 of ubiqu- itin in Arabdopsis RAD23 and I44 or V70 of ubiqu- itin in Arabdopsis DSK2 abrogated the RPN10 interaction (Table 2 and data not shown). Whereas similar replacements in the human RAD23 homolog (hHR23b; L8A) and the DSK2 homolog (PLIC1; I79A and V105A) abrogated the interaction with the human RPN10 homolog (S5a), replacement of the residue corresponding to I44 of ubiquitin in human hHR23b (I47A) did not (Fig. 2A). In yeast DSK2, replacement of the residue that corresponds to I44 or V70 of ubiquitin (I45 and V71, respectively) or their adjacent residue (L44 or L70, respectively) did not affect the association with RPN10 (Fig. 2B). Only the double-alanine mutation at positions 44–45 (LI) or 70–71 (LV) and a UBL deletion mutant (UBLD; residues 1–73 deleted) of yeast DSK2 abolished the association with RPN10 (Fig. 2B). These results indi- cate that the UBLs of the human RAD23 and DSK2 Using single and double UIM mutations, we found that RPN10s from Arabdopsis and yeast were capable of interacting with DSK2 homologs from other species through UIM1 and UIM, respectively, as seen with DSK2 from their own species (Fig. 3D,F). For the human RPN10 homolog (S5a), both UIM1 and UIM2 facilitated association with the Arabdopsis and yeast DSK2 homolog (Fig. 3E). Whereas UIM2 played a more critical role in the interaction with the DSK2 homolog of both humans and Arabdopsis, UIM1 played a more critical role in the interaction with yeast DSK2 (Fig. 3E). The latter probably evolved to cope with the divergent UBL interface detected in yeast DSK2 (Fig. 2B).

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D

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F

Fig. 3. Cross-species interaction analyses between the homologs of RPN10 and RAD23 or DSK2. Wild-type and single or double UIM mutants of the Arabdopsis (A,D), human (B,E), or yeast (C,F) RPN10 homologs were analyzed separately by pull- down using various GST-fused Arabdopsis, human and yeast RAD23 (A–C) or DSK2 (D–F) homologs. The various UIM mutations for Arabdopsis RPN10 are located at residues 226–230 for uim1 (LALAL ﬁ DDDDD), 286–290 for uim2 (LLDQA ﬁ NNDND) and 310–314 for uim3 (LALAL ﬁ NNNDN). One-ﬁftieth of the prey (Input) and pulled-down products were analyzed by immunoblotting against a-T7. One-ﬁfth of a set of eluted products (Baits 10·) was examined by staining with Brilliant Blue R to conﬁrm that the baits had been immobilized equally.

Structural divergence of the RPN13-mediated proteasomal recognition of the RAD23 and DSK2 homologs

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As reported recently, the base subunit RPN13 is also capable of binding to UBL–UBA factors [13]. We determined the roles and associated interfaces of Arabdopsis, human and yeast RPN13 homologs in the recognition of UBL–UBA factors (Table 2). Arabdop- sis, human and yeast RPN13 and RPN10 (for compar- ison) homologs were puriﬁed as T7-tagged proteins including the (Fig. 4A), and the UBL–UBA factors,

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D

Fig. 4. Interaction analyses of the RAD23, DSK2 and DDI1 homologs from Arabdopsis, humans and yeast with the proteasome subunit RPN13. (A) The input prey of the T7-tagged RPN13 and RPN10 homologs. One-ﬁfth of the input Arabdopsis, human and yeast RPN10 and RPN13 homologs (1 lg each) were visualized by staining with Brilliant Blue R. Puriﬁed recombinant yeast RPN13 was mobilized as a dou- blet, which is probably derived from different translational initiation sites. (B–D) The RPN10 and RPN13 homologs from Arabdopsis (B), humans (C) or yeast (D) were analyzed separately by pull-down using GST-fused RAD23, DSK2 or DDI1 homolog(s) from the respective spe- cies. The Arabdopsis RAD23 homologs examined include RAD23b–d. One-hundredth of the input RPN10 or RPN13 homologs (Input) and the pulled-down products were analyzed by immunoblotting against a-T7. One-tenth of the eluted products (Baits 10·) was examined by staining with Brilliant Blue R to conﬁrm that the baits had been immobilized equally. The pulled-down products that were derived from GST alone were analyzed as a negative control, and the RPN10 pull-down analyses were analyzed for comparison.

level

RAD23, DSK2 and DDI1 homologs, were puriﬁed as GST-tagged proteins. As shown in Fig. 4B,D, Arabd- opsis (AtRPN13) and yeast (ScRPN13) RPN13 homo- logs were recovered using GST-fused DSK2, but not RAD23 and DDI1, homologs from their respective species. However, recovery occurred at a signiﬁcantly compared with the RPN10 homolog lower pulled-down using GST-fused Arabdopsis RAD23 ho- mologs or DSK2a (Fig. 4B), or using GST-fused yeast DSK2 (Fig. 4D). Human RPN13 was recovered using GST-fused human hHR23b or PLIC1, but not DDI1, at a level slightly lower than that of S5a recovered using the respective GST-fusion (Fig. 4C). The data indicate that Arabdopsis or yeast RPN13, which has a minor role compared with RPN10, is capable of func- tioning as a recognition subunit for DSK2, and that human RPN13 is capable of serving as the recognition subunit for both hHR23b and PLIC1.

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It is logical to propose that the interfaces ⁄ residues in the PRU domains of Arabdopsis and human RPN13 responsible for ubiquitin chain binding (Fig. S5) are also involved in recognition of the UBL–UBA factors. The same single-residue mutants of RPN13 from Arabdopsis or humans as constructed for the ubiquitin- binding experiments were used to determine their role in the recognition of RAD23 and ⁄ or DSK2 homologs. A few residues, including E72 and F91 (corresponding to D79 and F98 in mammalian RPN13), are also con- served in the potential PRU domain in yeast RPN13 (Fig. S4). Therefore, we mutated E72 and F91 to Q72 and R91, respectively, and tested their role in the rec- ognition of yeast DSK2. In addition, we mutated L43 in yeast RPN13 at a position one residue away from F45 (corresponding to L56 of mammalian RPN13) to alanine, and tested this mutant also. All the tested resi- dues appear to be critical for the interaction between the RPN13 and DSK2 homologs from Arabdopsis, yeast and humans. When compared with wild-type proteins from Arabdopsis (Fig. 5A), yeast (Fig. 5B) and humans (Fig. 5C), the levels of the RPN13 vari- ants recovered using the GST-fused DSK2 homologs from the respective species were reduced drastically. However, apart from a slight reduction in the recovery of human RPN13 variant A56 using GST-fused

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Fig. 5. Cross-species interaction analyses between the RPN13 variants and the DSK2 or RAD23 homologs. The Arabdopsis (A), yeast (B) or human (C) RPN13 variants were analyzed by pull-down using GST-fused Arabdopsis, human or yeast DSK2 homo- logs. Human RPN13 variants were also ana- lyzed using GST-fused Arabdopsis (RAD23b–d), human or yeast RAD23 homo- logs (D). The RPN13 variants that were examined include the wild-type and single- residue mutants L47A, F67R, E70Q and F88R for Arabdopsis (A); L43A, E72Q and F91R for yeast (B); and L56A, F76R, D79N and F98R for humans (C,D). The mutage- nized residues correspond to L56, F76, D79 or F98 in the PRU domain of mammalian RPN13 [13] (Fig. S4). One-hundredth of the input RPN13 variants (Input) and one-hun- dredth (1·) or one-tenth (10·) of the pulled-down products were analyzed by immunoblotting against a-T7. One-tenth of the eluted products (Baits 10·) was examined by staining with Brilliant Blue R to conﬁrm that the baits had been immobilized equally. The pulled-down products that were derived from GST alone were analyzed as a negative control. The asterisks in (C) indi- cate an unspeciﬁc pull-down product.

equivalent to wild-type RPN13

hHR23b, human RPN13 variants were recovered at a level (Fig. 5D, Human). These observations indicate that Arabdopsis, human and yeast DSK2 homologs might be recognized by RPN13 homologs from the respective species using the conserved interfaces in the PRU domains [12,13], which are also critical for ubiquitin binding. Interest- ingly, the interface of the PRU of human RPN13, which is critical for the interaction with ubiquitin and PLIC1, is not required to bind hHR23b.

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Because the conserved residues of the PRU domain of Arabdopsis, human and yeast RPN13 homologs have roles in the interaction with the DSK2 homolog from the respective species, we decided to test the hydrophobic patches within the UBLs of the DSK2 homologs for their involvement in the interaction with RPN13. The potential involvement of the hydrophobic patch in the UBL of hHR23b was also examined. The UBL variants constructed for analysis of the associa- tion of RPN10 with human hHR23b and the DSK2 homologs from Arabdopsis, humans and yeast were examined for their association with RPN13. Whereas the recovery of human RPN13 was abrogated when the GST-fused I79A or V105A PLIC1 variant was used (Fig. 6B), recovery of Arabdopsis RPN13 was abrogated using the GST-fused Arabdopsis I61A DSK2a variant, but not the V87A variant (Fig. 6A). All the residues tested in yeast DSK2 affected RPN13 recognition. However, I45 and L44 appear to be more critical for the interaction with RPN13 than V71 and L70 (Fig. 6C). Recovery of yeast RPN13 was abol- ished when the GST-fused I45A or L44A DSK2 single

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interface in the human RPN13 PRU is not critical for association with hHR23b (Fig. 5D), and that novel interfaces are probably involved.

B

C

Fig. 6. The role of the hydrophobic patches in the UBLs of the DSK2 homologs and human hHR23b in association with RPN13. The Arabdopsis (AtRPN13), human (HsRPN13) or yeast (ScRPN13) RPN13 were analyzed by pull-down using various GST-fused UBL variants of Arabdopsis DSK2 (A), human hHR23b or PLIC1 (B) or yeast DSK2 (C). The UBL variants of human hHR23b and PLIC1, and yeast DSK2, are the same as those used for association with the RPN10 homologs (Fig. 2). For the UBL variants of Arabdopsis DSK2, two of the conserved residues, which were equivalent to I44 and V70 of ubiquitin, were individually mutagenized to alanine (I61A and V87A). One-hundredth of the input Arabdopsis, human or yeast RPN13 homologs (Input) and the pulled-down products were analyzed by immunoblotting against a-T7. One-tenth of the pulled- down products (Baits 10·) was examined by staining with Brilliant Blue R to conﬁrm that the baits had been immobilized equally. The pulled-down products that were derived from GST alone were ana- lyzed as a negative control.

We examined the overall structural conservation of the interfaces between the RPN13 and DSK2 homo- logs further using cross-species interaction analyses. Arabdopsis RPN13 was recovered at a reduced efﬁ- ciency using the GST-fused human and yeast DSK2 that was homologs when compared with the level using GST-fused Arabdopsis DSK2a recovered (Fig. 5A). Mutation of critical residues in the Arabdop- sis RPN13 PRU disrupted this interaction (Fig. 5A). Human RPN13 was recovered at a similar level using GST-fused human or yeast DSK2 homologs (Fig. 5C). The mutation of critical residues in the PRU of human RPN13 also disrupted the interaction (Fig. 5C). Yeast RPN13 was recovered only when using GST-fused yeast DSK2, and not when using GST-fused Arabdop- sis or human DSK2 homologs (Fig. 5B). Our results suggest that the overall structure of the RPN13–DSK2 interface is conserved across species. However, the lack of cross-species interaction between yeast RPN13 and either the human or Arabdopsis DSK2 homologs is in agreement with the observation of a greater divergence at critical positions on the PRU interface for yeast RPN13 (Fig. S4).

Interestingly, human RPN13 and its single-mutation variants were recovered using GST-fused Arabdopsis or yeast RAD23 homologs at a level similar to the level observed when using GST-fused recovery hHR23b (Fig. 5D). This suggests that the interface of hHR23b responsible for its interaction with human RPN13 is conserved in the Arabdopsis and yeast RAD23 homologs. It also suggests that the interfaces of the Arabdopsis and yeast RPN13s, which corre- spond to the interface of human RPN13 that mediates the interaction with the RAD23 homologs, are diver- gent or have been deleted. In agreement with this sug- the Arabdopsis nor yeast RPN13 gestion, neither homolog was recovered when using GST-fused hHR23b (data not shown).

Yeast RAD23 is recognized by the base subunit RPN1

mutant, or the LI–AA (44–45) double mutant, was used. The recovery of yeast RPN13 was reduced signif- icantly when using GST-fused V71A, L70A or LV–AA (70–71) DSK2 mutants. As expected, the recovery of yeast RPN13 was abolished using the GST-fused DSK2 UBL deletion mutant (Fig. 6C, UBLD). These results indicate clearly that structural divergence exists at the interfaces of the UBLs of Arabdopsis, human and yeast DSK2 homologs for RPN13 association.

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Human RPN13 was recovered at a similar level using the GST-fused hHR23b or hHR23b variants (Fig. 6B; L8A and I47A), indicating that the hydro- phobic patch in the UBL of hHR23b is nonessential for RPN13 association. This corroborates the afore- the ubiquitin-binding mentioned observation that As reported previously, RPN1 is the primary docking subunit for RAD23 in yeast [18]. Yeast DSK2 also com- petes slightly for the interaction between RAD23 and the 26S proteasome, indicating that DSK2 can also be recognized by RPN1 [18]. However, a direct interaction between Arabdopsis RPN1 and UBL–UBA factors, including the RAD23 and DSK2 homologs and DDI1, was not detected (data not shown). To conﬁrm the pos-

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sible role of yeast RPN1 and RPN10 in the recognition of RAD23 and DSK2, respectively, and to explore whether other potential proteasome subunits are impor- tant for the recognition of RAD23, DSK2 and DDI1, we searched for direct binding partners from the subun- its of the regulatory particle (RP) of the 26S proteasome using yeast two-hybrid (Y2H) analyses.

RAD23 and RPN1 (Fig. S8D). An interaction between RAD23 and RPT6 was also detected. However, the inter- actions between RAD23 and RPN1 and RAD23 and RPT6 were weak, and only one of the two Y2H reporters (HIS3) was activated. These results support the observa- tion that the recognition of RAD23 is mediated by RPN1 in yeast [18]. In addition, the stronger Y2H interaction also suggests that the recognition of DSK2 could poten- tially be mediated by RPN10.

RPN10 is critical for both vegetative and reproductive growth in Arabdopsis

Based on in vitro interaction analyses, Arabdopsis RPN10 appears to play a critical role in both the As shown in Fig. S8A, yeast RAD23, DSK2 and 17 RP subunits, including RPT1–6, RPN1–3 and RPN5–12, were analyzed as C-terminal GAL4-BD fusions and as C- and N-terminal GAL4–AD fusions. DDI1 was tested as N- and C-terminal GAL4–AD fusions. After analyzing all possible BD ⁄ AD fusion combinations between the RP subunits and UBL–UBA factors (Fig. S8A), we detected interactions between DSK2 and RPN10, and between

A

B

C

D

Fig. 7. RPN10 is essential for Arabdopsis growth and development. A T-DNA insertion knockout Arabdopsis mutant, rpn10-2, was character- ized. (A) The T-DNA insertion site in the fourth intron of RPN10 (At4g38630) for rpn10-2 is indicated schematically (large triangle, not to scale). Exons and introns are indicated using boxes and lines, respectively. The positions of the primers that were used to detect the T-DNA insert, the endogenous RPN10 gene and the transcript are indicated. The primers that were used include GABI-LB4, RPN10-5¢b (5¢b), RPN10-3¢B (3¢B), cRPN10–Sma (cN10-Sma) and cRPN10–Sst (cN10-Sst) (see Experimental procedures). (B) Transgene genotyping and tran- script expression of the rpn10-2 and complementation lines. (Left) The presence of endogenous RPN10 (eN10), T-DNA insertion (tDNA) and the complemented RPN10 (cN10) coding region were examined by PCR using genomic DNA that was isolated from Col-0, rpn10-2 and rpn10-2 expressing the RPN10 coding region driven by the CaMV 35S promoter (two lines, line-1 and -2), respectively. (Right) RT-PCR shows that the RPN10 transcripts were not detected in rpn10-2. (C) The expression of RPN10 was knocked-out and ubiquitylated proteins accumu- lated in rpn10-2. The expression of RPN10 and the accumulation of ubiquitylated proteins and free di-ubiquitin (diUB) were examined by immunoblotting against Arabdopsis RPN10 (aRPN10) or human ubiquitin antibodies (aUB) in crude protein extracts that were prepared from Col-0 (Col), rpn10-2 and two complementation lines. The expression of CSN5 was examined using the Arabdopsis CSN5 antibody (aCSN5) to conﬁrm equal loading. (D) The growth of Col-0, rpn10-2 and two complementation lines was followed at different stages (21, 50 and 80 days after germination).

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Discussion

a cross-species

in which a C-terminal truncated. This

direct and indirect recognition of ubiquitylated sub- strates of UPP. In agreement with this idea, rpn10-1, a reported T-DNA insertion mutant, has been shown to have pleiotropic phenotypes including a reduction in stamen number, genetic germination, growth rate, transmission through the male gametes, hormone- induced cell division and seed set, as well as increased sensitivity to abscisic acid [32]. A slight accumulation of ubiquitylated substrates and a speciﬁc and drastic stabilization of ABI5 were observed with rpn10-1, indicating a defect in substrate targeting [32]. The RPN10 protein in rpn10-1 was expressed as a NPT-II fragment containing fusion, UIMs was fusion protein was expressed at a very low level compared with that of RPN10 in wild-type. However, the chimeric RPN10 was assembled into the 26S proteasome in rpn10-1 at near wild-type levels [32].

Using comparison approach, we observed distinct ubiquitin chain binding properties requirements among the and associated structural major Arabdopsis, human and yeast ubiquitin recep- tors (Table 1 and Fig. 8). Moreover, we also observed distinct proteasomal docking sites and interfaces for homologs of the major UBL–UBA factors (Table 2 and Fig. 8) in different species. Our results support the mechanistic divergence across species of the major recognition pathways for ubiquitylated substrates of UPP. Interestingly, Arabdopsis RPN10 plays a major role in both the direct and indirect recognition of ubiquitylated substrates, and this is in agreement with the in accumulation of ubiquitylated conjugates T-DNA-inserted Arabdopsis RPN10 mutant lines and the associated pleiotropic phenotypes for both vegeta- tive and reproductive growth reported here and previ- ously [32].

Arabdopsis RPN10 plays a major role in both the direct and indirect recognition of ubiquitylated substrates of UPP

complementation analyses and the

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As determined by the strong afﬁnity for long K48- linked ubiquitin chains, which is the primary signal for targeting to the 26S proteasome [6], humans use RPN10 (S5a) and RPN13 as major receptors for the direct recognition of ubiquitylated substrates, whereas both yeast and Arabdopsis use RPN10 as the major receptor (Fig. 8). Weak or absent binding afﬁnity for either K48- or K63-linked ubiquitin chains suggests that RPN13 in Arabdopsis and yeast is likely to play a minor role in direct substrate recognition. Although it is possible that the recombinant Arabdopsis or yeast RPN13 was not folded properly, their interaction with the DSK2 homologs through PRU makes this less likely (Fig. 5A,B). Alternatively, the conformation and chain-binding properties of RPN13 in Arabdopsis and yeast may be altered when they are assembled into the 26S proteasome, or the RPN13 homologs may be reg- ulated by association with additional regulatory fac- tor(s). Other subunits of the 26S proteasome RP are not likely to participate as major receptors in direct substrate recognition, because an extensive yeast two- hybrid analysis (Y2H) between yeast ubiquitin and RP subunits did not detect any novel interaction. Further- more, Arabdopsis RPT5 and RPN1 did not bind to ubiquitin chains (data not shown) although these sub- units have been suggested as candidates for substrate recognition because ubiquitin- or UBL-binding activity was observed in other species [14,18]. We predicted that more severe phenotypes would be associated with a complete loss of RPN10 func- tion. A new T-DNA insertion mutant line of RPN10 in a Col-0 background was obtained, and is desig- nated here as rpn10-2. In this line, the T-DNA inser- tion was located in the fourth intron (Fig. 7A). A homozygous T-DNA insertion line was obtained by segregating T2 plants (Fig. 7B, left); neither the wild- type RPN10 transcript (Fig. 7B, right) nor its protein (Fig. 7C, upper) was detected, indicating that rpn10-2 is a null mutant. As expected, rpn10-2 showed more severe pleiotropic phenotypes than rpn10-1. Pheno- types included: reduced growth rate; larger, thicker, lanceolar, serrated rosette and cauline leaves; delayed ﬂowering time; reduced axillary inﬂorescences; longer increased increased length of pedicels; internodes; accumulation of anthocyanin; abnormal ﬂower organ number; larger ﬂower organs; larger petal cells; pro- longed life cycle; delayed leaf senescence; increased plant height; defective male and female gametophytes; and infertility (Fig. 7D and data not shown). We also detected the accumulation of ubiquitylated sub- strates and free di-ubiquitin (Fig. 7C, middle). Except for the gametophyte phenotypes, all the phenotypes (including conjugate accumulation) can be comple- mented when a wild-type RPN10 coding region that is driven by the CaMV 35S promoter was reintro- duced into rpn10-2 (Fig. 7C,D and data not shown). Part of the reason for the lack of complementation of the gametophyte phenotypes is probably because of an absence of 35S promoter expression in the anthers [33]. The detailed phenotypes of the rpn10-2 are plants described in a separate study (Y.L. Lin and H. Fu, unpublished results).

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B

C

Fig. 8. The major recognition pathways for the ubiquitylated substrates of UPP are divergent across species. Schematic diagrams show the direct and indirect recognition pathways of ubiquitylated substrates in humans (A), yeast (B) and Arabdopsis (C). For the direct recognition of ubiquitylated substrates (CONJUGATES), humans use RPN10 (N10 ⁄ S5a) and RPN13 (N13) as the major receptors, whereas yeast and Arabdopsis use RPN10 (N10) as the major receptor (indicated by the wide, red double-arrowhead lines). Arabdopsis RPN13 (N13) plays a minor role (the thin, red double-arrowhead line). Whereas a single UIM motif of Arabdopsis (UIM1 ⁄ U1) or yeast RPN10 (UIM ⁄ U) is required for substrate recognition, UIM1 (U1) and UIM2 (U2) of human S5a act cooperatively (bracketed). Of the two, UIM2 is more critical for sub- strate binding (colored in red and yellow for UIM2 and UIM1, respectively). For the human and Arabdopsis RPN13 homologs, the PRU domains are required. Except for the human DSK2 homolog (DSK) (indicated by the thin, black double-arrowhead lines), both the RAD23 (RAD) and DSK2 homologs could serve as major receptors for indirect recognition (as indicated by the wide, black double-arrowhead lines). (A) The docking of the human RAD23 and DSK2 homologs is mediated by both RPN10 (S5a) and RPN13. Docking by RPN10 (S5a) is medi- ated by UIM1 (U1) and UIM2 (U2) in a mode that is similar to substrate binding. By contrast, the docking of the human RAD23 and DSK2 homologs by RPN13 is mediated by a novel domain that has not yet been deﬁned (DN) and by PRU, respectively. Although both UBLs (the red subregions) of the RAD23 and DSK2 homologs are involved in binding RPN10 (S5a), the UBL of the DSK2 homolog and a novel unidenti- ﬁed domain (the green-colored subregion, DN) of the RAD23 homolog are involved in RPN13 binding. The role of human RPN1 (N1) in the recognition of UBL–UBA factors has not been determined (marked ? in the ﬁgure). (B) Docking of the yeast RAD23 and DSK2 homologs is mediated primarily by RPN1 and RPN10, respectively. RPN1 and RPN13 also play a minor role in DSK2 docking (the thin, black double-arrow- head lines). The interaction of RPN1 and RAD23 or DSK2 is mediated by LRR in RPN1 and by the UBLs in RAD23 ⁄ DSK2; residues that are critically involved in the UBLs of RAD23 ⁄ DSK2 have not been determined (marked by asterisks in the ﬁgure). The interaction between DSK2 and RPN10 ⁄ RPN13 is mediated by the UBL in DSK2, and by UIM and PRU, respectively, in RPN10 and RPN13. (C) Docking of the Arabdopsis RAD23 and DSK2 homologs is mediated primarily by UIM3 and UIM1, respectively, in the same base subunit, RPN10. UIM2 (shown in yellow) also plays a minor role in binding submembers of the RAD23 family (data not shown; the thin, black double-arrowhead lines). Via PRU, Arabdopsis RPN13 also plays a minor role in docking DSK2 (the thin, black double-arrowhead lines). Arabdopsis RPN1 (depicted in gray) is not involved in the recognition and is marked with an X. For the involvement of the UBLs in proteasomal docking, the conserved residues (corresponding to those located in the hydrophobic patch of ubiquitin) are generally critical. However, divergent interfaces have been detected. The residues with altered importance are designated with the corresponding binding proteasome subunit indicated in parentheses.

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Based on the strong afﬁnity for long K48-linked ubiquitin chains, the RAD23 and DSK2 homologs may be used as major receptors for indirect recogni- tion in the species examined (Fig. 8). The exception is the human DSK2 homolog, which probably plays a minor role because of its weak afﬁnity for K48-linked ubiquitin chains. Other UBL–UBA factors such as DDI1 and NUB1 also probably play a minor role, or

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they act beyond UPP because they have weak or absent afﬁnity for K48-linked ubiquitin chains (Fig. S3 and data not shown).

Pleiotropic phenotypes of loss of function support a major role for Arabdopsis RPN10 in the direct and indirect recognition of ubiquitylated substrates

Interestingly, critical roles of

The multiplicity of major recognition pathways of ubiquitylated substrates was mediated in yeast and humans through separate proteasomal subunits. A non- essential role was observed for the major yeast ubiqu- itin receptors RPN10, RAD23 and DSK2 [11,16], suggesting their functional redundancy. However, it can be predicted that simultaneous loss of the ubiqu- itin chain-binding activity of RPN10 and the proteaso- mal docking activity of RPN1 (Fig. 8) would have severe consequences in yeast. By contrast, the major recognition pathways (both direct and indirect) in Arabdopsis are all mediated by RPN10, suggesting an essential role in growth and development. In agree- ment with this idea, Arabdopsis RPN10 was shown to be essential for both vegetative and reproductive growth, as reported previously [32] and here through the examination of a new T-DNA inserted null mutant. the RPN10 homolog in vivo were also observed in several other species, including Drosophila [34], mouse [35], Physc- omitrella patens [36] and Caenorhabditis elegans [37].

Based on the strength of the interaction, multiple RP base subunits play a major role in proteasomal docking of the RAD23 and DSK2 homologs in yeast (RPN1 and RPN10) and humans (RPN10 and RPN13), whereas a single base subunit RPN10 plays a major role in Arabd- opsis (Fig. 8). In yeast, RPN1 plays a major role in the recognition of RAD23 and probably also plays a minor role in the recognition of DSK2, as described previously [18] and conﬁrmed using Y2H (Fig. S8). Based on the observation that the Y2H reporter activity derived from the RPN10–DSK2 interaction is stronger than that from the RPN1–RAD23 interaction, and also on observation of the strong interaction detected by the pull-down assay, RPN10 probably plays a major role in DSK2 rec- ognition (Figs 1B and S8). With its relatively weak bind- ing compared with RPN10, yeast RPN13 probably plays a minor role in DSK2 recognition (Fig. 4D). In humans, both RPN10 and RPN13 play a major role in the recognition of RAD23 and DSK2 homologs (Figs 1A and 4C). The role of human RPN1 in the rec- ognition of UBL–UBA factors has not been examined. Uniquely for Arabdopsis, the proteasomal docking of the RAD23 and DSK2 homologs is mediated primarily by RPN10 through separate sites (Fig. 3). However, a minor role in DSK2 recognition is mediated by RPN13 (Fig. 4B).

recognition is mediated by a

recognition of

The observed pleiotropic phenotypes associated with rpn10-1 [32] and rpn10-2 (Fig. 7D and data not shown) support defective proteolysis of the critical regulatory factors involved. Accordingly, accumulation of ubiqui- tylated substrates and free di-ubiquitin was clearly observed; the latter is probably derived from active deubiquitylation activities that exist in vivo. However, RPN10 is a protein that has multiple activities. The N-terminal vWA domain is critical for the stable asso- ciation between the lid and base subcomplexes of RP [38], UIM1 is critical for both the direct and indirect recognition of ubiquitylated sub- (through DSK2) strates, and UIM3 is important for indirect (through RAD23) recognition of ubiquitylated substrates. The null-mutation of rpn10-2 will allow analyses to corre- late the observed phenotypes with separate RPN10 activities by complementation with site-mutagenized RPN10 mutants. It can also be tested whether the direct and indirect recognition that are mediated by UIM1 and the indirect recognition that is mediated by UIM3 are redundant.

Functional roles of major ubiquitin receptors beyond UPP

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Except during docking by the human RPN10 homolog (S5a), the proteasomal recognition sites of the RAD23 and DSK2 homologs are separated struc- turally. Here, recognition of the DSK2 homolog over- laps structurally with the recognition of ubiquitylated substrates (Fig. 8). For example, all the PRU domains of the RPN13 homologs are involved in the recogni- tion of the ubiquitin chains and DSK2, except for yeast RPN13. UIM1 of Arabdopsis RPN10 and the UIM of yeast RPN10 are also involved. By contrast, RAD23 separate motif(s) ⁄ domain(s) that includes UIM3 and, to a les- ser extent, UIM2 of Arabdopsis RPN10 (Fig. 3 and data not shown), LRR of yeast RPN1 [18] and a novel domain in human RPN13 (Figs 5D and 6B). However, the RAD23 and DSK2 homologs by human S5a is mediated by the two UIMs using a very similar mode to the recognition of ubiquitin chains (Fig. 1A). The structural separation or overlap between the ubiquitin-binding and prote- asomal recognition sites of different UBL–UBA fac- tors is probably a critical element in the mechanistic relay or for regulation during proteasomal recognition of ubiquitylated substrates. Human S5a ⁄ RPN10, RPN13 and yeast DSK2 have strong afﬁnities, not only for K48-, but also for

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ubiquitin chains using a single UIM site (Fig. S1). Dis- tinct substrate-binding properties are also associated with the RPN13 and DSK2 homologs from different species (Figs S2A and S3).

is it

K63-linked ubiquitin chains. This suggests that addi- tional ubiquitin recognition functions beyond UPP may be associated with these ubiquitin receptors. How- ever, ubiquitin chains of other linkage types, such as K11, K29 and K63, may still serve as competent sig- nals for proteasomal degradation [8,9]. The afﬁnity of the major ubiquitin receptors for in vivo physiological substrates, which probably have highly extended ubiquitin chains at multiple sites, could be modulated through the interaction with associated factors or after assembly into the 26S proteasome. Further differentia- tion of the biochemical properties of the major ubiqu- itin receptors may yet be discovered when more extended ubiquitin chains or physiological substrates are analyzed.

Second, structural divergence for ubiquitin chain binding was detected with major ubiquitin receptors. the human and Arabdopsis RAD23 For example, homologs bind ubiquitin chains using both UBAs additively, whereas yeast RAD23 binds chains using a single UBA1 (Table 1 and Fig. S6A,B, and data not shown). Because the UBA was not detected in human DDI1, likely that a novel ubiquitin-binding domain is involved. Also, divergence of the interface of the UBA in yeast DDI1 was detected by mutagene- sis (Fig. S6C, left). Moreover, divergence of the PRU interface was detected in yeast RPN13, and this obser- vation agrees with the lack of afﬁnity for ubiquitin chains and conjugates (Fig. S4).

the

divergent interfaces were

DDI1 homologs from various species have weak for either K48- or K63-linked ubiquitin afﬁnities chains. Weak ubiquitin chain binding is reﬂected by the low afﬁnity for endogenous ubiquitylated sub- strates observed with Arabdopsis DDI1 (data not shown). In addition, the potential proteasomal docking site for DDI1 has not been detected in Arabdopsis, humans or yeast (Figs 1, 4 and S8, and data not shown). These results do not favor a role for DDI1 homologs as major receptors for ubiquitylated sub- strates during UPP. However, the involvement of yeast DDI1 in the recognition of speciﬁc substrates, such as Ho endonuclease and the F-box protein of the E3 complex SCFUfo1, has been described [5,17]. The human DSK2 homolog (PLIC1) has a clear preference and moderate afﬁnity for K63-linked ubiquitin chains (Fig. S3), indicating that PLIC1 plays a minor, if any, role in the recognition of ubiquitylated substrates dur- ing UPP. The possible involvement of PLIC1 in the sequestration of UIM-containing endocytic compo- nents has been described previously [39].

Structural divergence supports mechanistic differentiation of the major recognition pathways across species

species recognition pathways across

example, whereas

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Although conserved domains ⁄ motifs are used by vari- ous ubiquitin receptors for substrate binding and pro- teasomal docking, a clear structural divergence of the major exists (Tables 1 and 2 and Fig. 8). First, distinct substrate- binding properties are associated with major ubiquitin indicating structural receptors from different species, differentiation. For the human RPN10 homolog binds to both the K48- and K63- linked ubiquitin chains with high afﬁnities using the two UIM sites in a cooperative manner, Arabdopsis and yeast homologs preferentially bind to K48-linked Third, structural divergence was detected in the interaction interfaces between the proteasomal docking subunits and the UBL–UBA factors in different spe- structural conservation was cies. Although overall detected of RPN10–RAD23, interfaces for RPN10–DSK2 and RPN13–DSK2 (when examined using cross-species interaction analyses; Figs 3 and 5), detected nevertheless, (Fig. 8). The most obvious example of a divergent interface is the UBL of yeast RAD23 (Fig. S7). This divergence leads to a loss of binding to RPN10 (Fig. 3A–C, upper). However, yeast RPN10 retains the ability to bind the UBLs of the RAD23 homologs from Arabdopsis and humans (Fig. 3C). Uniquely, yeast RPN1 acquired the LRR for binding with the UBL of RAD23, which appears to be divergent in Arabdopsis. Functional divergence of the conserved residues in the UBL interfaces of human RAD23 (I47; Fig. 2A) and yeast DSK2 (L44, I45, L70 and V70; Fig. 2B) for association with the RPN10 homolog were detected. Cross-species interaction analysis between human S5a and the yeast DSK2 homolog also sup- ports a divergent UBL in yeast DSK2. Although both UIM1 and UIM2 of human RPN10 ⁄ S5a are important for the interaction with yeast DSK2, UIM1 is more critical. This is different from the interaction of S5a with the human or Arabdopsis DSK2 homolog, in which UIM2 is more critical (Fig. 3E). We also detected divergence in the UBL interfaces of Arabdop- sis DSK2 (V87, Fig. 6A) and yeast DSK2 (L44, L70 and V71; Fig. 6C) in their association with RPN13. The novel interfaces between the human RPN13 and RAD23 homologs appear to be partly divergent in other species. RAD23 homologs from Arabdopsis and interacting with human yeast are still capable of

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0.05% Tween 20 and stratiﬁed them for 3 days on plates at 4 (cid:2)C in the dark. The seeds were germinated on half- strength MS solid medium (0.8% agar, pH 5.8) supple- mented with 1% sucrose. Two-week-old seedlings were transferred to soil (equal parts of humus, vermiculite and per- lite) and grown using a 16 h light ⁄ 8 h dark photoperiod with a light intensity of (cid:2) 120 lmolÆm)2Æs)1 at 22 (cid:2)C.

their

35S-Fw-T ⁄ RPN10-3¢B,

For genotyping, a single rosette leaf was mashed in 600 lL of extraction buffer using a SH-48 homogenizer (J&H Technology, Taipei, Taiwan) and the genomic DNA was extracted as described [42]. For RT-PCR, total RNA was isolated from 30-day-old rosette leaves using TRIzol according to manufacturer’s instructions (Invitrogen, Carls- bad, CA, USA). RNA was quantiﬁed using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). Three micrograms of total RNA were used for ﬁrst- strand cDNA synthesis in a 20 lL reaction using poly(T), random primers and SuperScript III reverse transcriptase (Invitrogen). Sterile water was added to achieve a ﬁnal vol- ume of 150 lL, and 2.5 lL was used as the template for each PCR. The PCR primer pairs used to detect the endog- enous RPN10, T-DNA insertion, RPN10 complementation construct, RPN10 coding region and a UBQ10 cDNA frag- ment were RPN10-5¢b ⁄ RPN10-3¢B, RPN10-3¢B ⁄ GABI- LB4, cRPN10-Sma ⁄ cRPN10-Sst and UBQ10-5¢ ⁄ UBQ10-3¢, respectively. The sequences of the primers used are listed in 5¢ to 3¢ direction as follows:

interaction indicating that RPN13 (Fig. 5D), interfaces (which do not involve UBLs, Figs 5D and 6B) are still conserved when compared with the human RAD23 homolog. By contrast, RPN13 homologs from Arabdopsis and yeast did not interact with the human RAD23 homolog, indicating that their binding inter- faces had diverged (data not shown).

Mechanistic differentiation across species, supported by divergent structural requirements, may potentially lead to distinct functions for the different recognition pathways, as exempliﬁed here by Arabdopsis RPN10, and different in vivo regulation (such as the association with distinct regulators). Because the speciﬁcity of UPP can be modulated at the ubiquitylated substrate recognition step [4,5,17], additional structural elements or associated factors probably play roles in determin- ing substrate speciﬁcity. Taking these suggestions together, it is likely that more subtle mechanistic com- ponents are involved in the proteasomal recognition of ubiquitylated substrates, and these may have diverged in the major recognition pathways across species. Fur- ther structural analyses and comparisons are required to resolve the divergence detected in this study to pro- vide a solid basis for mechanistic insight. In vivo stud- ies, including loss- and gain-of-function analyses and structure ⁄ function correlations, are required to deter- mine the functional roles associated with the various recognition pathways. Alternatively, identiﬁcation of speciﬁc substrates using proteomic approaches may insights into the various also contribute functional recognition pathways.

Experimental procedures

RPN10-3¢a, CACCCGTGAATCACGGTGTGCTGGA AG; RPN10-5¢b, GAGTTTGACATCAATTTGCTACTTG CGTC; RPN10-3¢B, CTGCGGCCGCTGCAGCAGCTG CCGCAG; GABI-LB2, GCTGATCCATGTAGATTTCCC CGGACATG; GABI-LB4, CACGGATGATCTCGCGGA GGGTAG; 35S-Fw-T, CTCGGATTCCATTGCCCAGCT ATCTG; cRPN10–Sma, TCCCCGGGATGGTTCTCGAG GCGACTATG; cRPN10–Sst, ATGAGCTCTCACTTCT TCTCATCCTCGCC; UBQ10-5¢, GTGGTGGTTTCTAAA TCTCGTCTCTG; UBQ10-3¢, GAAGAAGTTCGACTTG TCATTAGAAAG.

Characterization of the Arabdopsis T-DNA insertion line rpn10-2

(to detect

To prepare crude protein extracts for the detection of RPN10, CSN5 and the ubiquitylated conjugates by immu- noblotting, we ground rosette leaves in liquid nitrogen using an equal volume of pull-down binding buffer (see below) supplemented with 1· protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) or 2 vol of the ubiquitylated conjugates). sample buffer Samples were dissolved by vortexing, incubated on ice for 5 min, centrifuged at 16 000 g and 4 (cid:2)C, and ﬁltered using 0.45 lm ﬁlters (Millipore Corp., Bedford, MA, USA). For pulling-down endogenous Arabdopsis ubiquitin conjugates, we prepared the crude Arabdopsis extract from the upper

The GABI-Kat T-DNA insertion line GK-734B02 (in the Col-0 background) designated here as rpn10-2 was ordered from the European Arabdopsis Stock Center (University of Nottingham, Loughborough, UK). The T-DNA insertion site was determined by sequencing a PCR fragment ampli- ﬁed from the junction using the primer pair GABI-LB2 and RPN10-3¢a. To complement the rpn10-2 phenotypes, the Arabdopsis RPN10 coding region was ampliﬁed from ﬁrst- strand cDNAs using the primer pair cRPN10–Sma and cRPN10–Sst (these added SmaI and SstI restriction sites, respectively) and cloned into pBI121, downstream of the CaMV 35S promoter [40]. This construct was mobilized into Agrobacterium GV3101 using a freeze–thaw method. Arabdopsis was transformed as described previously [41]. To grow Col-0, rpn10-2 or complemented rpn10-2 Arabdop- sis, we surface sterilized the seeds using 20% bleach and

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Preparation of crude protein extracts from Arabdopsis

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parts of 1-month-old plants using pull-down binding buffer supplemented with 1· protease inhibitor cocktail (Roche Diagnostics), 50 lm proteasome inhibitor MG132 (Biomol International, Plymouth Meeting, PA, USA) and 50 lm N- ethylmaleimide (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined using Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA, USA).

GST pull-down analyses and immunoblotting

The coding sequences for wild-type, site-mutagenized and deletion variants of Arabdopsis, yeast and human ubiquitin receptors were generated by PCR and inserted into either pET42a or pET28a (Novagen, Madison, WI, USA) to yield constructs that encoded for GST ⁄ HIS- or T7 ⁄ HIS-tagged proteins, respectively (Table S1). Site-directed mutagenesis was performed using a QuickChange Kit with PfuTurbo and paired primers that were centered at the mutation sites according to the manufacturer’s protocols (Stratagene, La Jolla, CA, USA). Double-site mutants were generated by sequential mutagenesis. The coding sequences for human S5a (U51007), hHR23b (D21090), PLIC1 (BC010066), DDI1 (NM032341) and RPN13 (NM175573) were ampli- ﬁed using PCR from ﬁrst-strand cDNAs prepared from human HeLa S3 cells (Stratagene). The coding sequences for yeast RAD23, DSK2 and DDI1 were PCR-ampliﬁed or subcloned (DSK2) from Y2H constructs (see the Doc. S1 and Table S1). The coding sequence for yeast RPN13 (U20939) was isolated from genomic DNA prepared from the DF5 strain [11]. All expression constructs were con- ﬁrmed by DNA sequencing using an ABI Prism 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Protein expression constructs

cultures were

(Novagen). Bacterial

GST pull-down experiments were performed using immobi- lized glutathione according to the manufacturer’s instruc- tions (Pierce, Rockford, IL, USA). Brieﬂy, the glutathione resin (25 lL ﬁnal bed volume for each reaction) was washed ﬁve times with 400 lL of binding buffer (50 mm Tris ⁄ HCl, pH 7.5, 25 (cid:2)C, 100 mm NaCl, 1 mm EDTA and 0.1% NP-40). GST-fused baits were then individually immobilized on the resin in 400 lL of binding buffer for 2 h on ice (gently mixed by inverting). The immobilized baits were washed ﬁve times with 400 lL of binding buffer. A speciﬁc prey was then added, and the resin complexes were incubated for 2 h on ice, with gentle mixing by invert- ing. After ﬁve washes with 400 lL of binding buffer, the pulled-down products were boiled for 5 min in sample buf- fer, analyzed by SDS ⁄ PAGE and detected by immunoblot- ting using chemiluminescence (Perkin–Elmer, Shelton, CT, USA) or color development. The NaCl concentration in the binding buffer was increased to 150 mm when analyzing the interactions between the UBL–UBA factors and the RPN10 or RPN13 variants. To detect the ubiquitin chains or conjugates, the proteins were transferred onto Hybond ECL nitrocellulose membranes (0.45 lm; Amersham Biosciences, Freiburg, Germany) following separation by SDS ⁄ PAGE. The membrane was then sandwiched between two glass plates and autoclaved for 20 min in transfer buf- fer to assist epitope exposure. For other immunoblotting assays, poly(vinylidene diﬂuoride) membranes were used (Perkin–Elmer, Boston, MA, USA). To visualize the pulled- down ubiquitin chains, conjugates and T7-tagged recombi- nant proteins, we used either rabbit anti-human ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-T7 primary serum (Novagen). Horseradish per- oxidase-conjugated goat anti-(rabbit IgG) serum or anti- (mouse IgG) serum (Santa Cruz Biotechnology) were used as secondary antibodies. To detect RPN10, CSN5 and the conjugates from the Arabdopsis crude extracts, we used pri- mary rabbit polyclonal antibodies raised against Arabdopsis RPN10, CSN5 (custom-made by Genesis Biotech, Taipei, Taiwan or purchased from Biomol, respectively), or human ubiquitin (Santa Cruz Biotechnology). These were used in conjunction with an alkaline phosphatase-labeled goat anti-(rabbit IgG) serum (Santa Cruz Biotechnology) and the substrates Nitro blue tetrazolium and 5-bromo-4- chloro-3-indolyl phosphate (Sigma-Aldrich). The K48- and K63-linked ubiquitin chains (Ub2–7) and tetra-ubiquitin were purchased from BostonBiochem (Cambridge, MA, USA).

Recombinant protein puriﬁcation from Escherichia coli

The presence of UIM, UBA and other protein domains was detected using available web programs from the ExPASy proteomics server (Swiss Institute of Bioinformatics,

The various recombinant proteins were expressed in BL21 (DE3) cells using the pET28a or pET42a expression plas- mids induced at D600 = 0.6 for protein expression with 1 mm isopropyl thio- b-d-galactoside and incubated at 16, 30 or 37 (cid:2)C depending on the construct. Escherichia coli cells were resuspended in 1· GST- or HIS-binding buffer (Novagen), supplemented with 200 lgÆmL)1 lysozyme, 0.1% NP-40, 10% glycerol and 1· protease inhibitor cocktail (Roche Diagnostics). Resus- pended cells were incubated for 15 min at room temperature and sonicated on ice for 5–10 min (15% power, 30-s pulses with 30-s intermittent pauses; Misonix XL2020, Farming- dale, NY, USA). His- and GST-tagged recombinant pro- teins were puriﬁed by immobilized metal- or glutathione- afﬁnity chromatography, respectively (Novagen) using buf- fers and procedures recommended by the manufacturer. Puriﬁed proteins were dialyzed and concentrated in GST pull-down binding buffer (see below) using Microcon Ultra- cel YM-30 ﬁlter units (Millipore Corp.).

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Sequence analyses

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http://www.expasy.ch/). Routine sequence analyses were per- formed using version 10.3 of the wisconsin gcg package (Accelrys, San Diego, CA, USA).

Acknowledgements

9 Johnson ES, Ma PCM, Ota IM & Varshavsky A (1995) A proteolytic pathway that recognizes ubiquitin as a degradation signal. J Biol Chem 270, 17442–17456. 10 Hofmann RM & Pickart CM (2001) In vitro assembly and recognition of Lys-63 polyubiquitin chains. J Biol Chem 276, 27936–27943.

11 van Nocker S, Sadis S, Rubin D, Glickman M, Fu H, Coux O, Wefes I, Finley D & Vierstra R (1996) The multiubiquitin-chain-binding protein Mcb1 is a compo- nent of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-speciﬁc role in protein turnover. Mol Cell Biol 16, 6020–6028.

12 Husnjak K, Elsasser S, Zhang N, Chen X, Randles L, Shi Y, Hofmann K, Walters KJ, Finley D & Dikic I (2008) Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453, 481–488.

13 Schreiner P, Chen X, Husnjak K, Randles L, Zhang N, Elsasser S, Finley D, Dikic I, Walters KJ & Groll M (2008) Ubiquitin docking at the proteasome through a novel pleckstrin–homology domain interaction. Nature 453, 548–552.

14 Lam YA, Lawson TG, Velayutham M, Zweier JL & Pickart CM (2002) A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763–767.

15 Saeki Y, Sone T, Toh-e A & Yokosawa H (2002)

We thank Drs Michael H. Glickman, Richard D. Vier- stra and Shu-Hsing Wu for critical reading of the manuscript, Dr Michael H. Glickman for the Y2H bait constructs BD-UB and BD-UB5, and for the Y2H constructs of yeast RAD23, DSK2 and DDI1. We also thank Tzuning Ho and Ting-Ting Yu for technical assistance. R. Radjacommare is the recipient of a post- doctoral fellowship from Academia Sinica (2005– 2006). Y.L. Lin is supported by a graduate fellowship from the Taiwan International Graduate Program (2003-2006; National Chung-Hsing University and Academia Sinica). This work was supported by grants from the National Science Council (NSC 88-2311- B001-127, NSC 89-2311-B001-125, and NSC 89-2311- B001-044, NSC 95-2311-B-001-045-MY3) and from Academia Sinica (AS-94-TP-B08 and AS-97-TP-B03), Taipei, Taiwan.

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This following supplementary material is available: Fig. S1. Ubiquitin chain binding properties and the rele- vant domains of the human and yeast RPN10 homologs. Fig. S2. Ubiquitin chain binding properties of the RPN13 homologs. Fig. S3. Ubiquitin chain binding properties of the human and yeast RAD23, DSK2 and DDI1 homologs. Fig. S4. Sequence similarity among the RPN13 homo- logs. Fig. S5. Involvement of PRU in the Arabdopsis and human RPN13 homologs in the binding of ubiquitin chains. Fig. S6. Involvement of the UBAs of the RAD23, DSK2 and DDI1 homologs in the interaction with ubiquitin chains. Fig. S7. Sequence similarity of ubiquitin and the UBLs of the UBL–UBA factors. Fig. S8. The Y2H search for the interacting RP sub- unit(s) of the UBL–UBA factors. Doc. S1. Supplementary experimental procedures. The methods used for the Y2H analyses. Supplementary references.

A. S. Fatimababy et al.

Cross-species divergence of ubiquitin receptors

Table S1. Expression constructs. This supplementary material can be found in the online version of this article.

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Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing ﬁles) should be addressed to the authors.

Báo cáo khoa học: Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin⁄26S proteasome-mediated proteolysis

Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin⁄26S proteasome-mediated proteolysis Antony S. Fatimababy1, Ya-Ling Lin1,2,3, Raju Usharani1, Ramalingam Radjacommare1, Hsing-Ting Wang1, Hwang-Long Tsai1, Yenfen Lee1 and Hongyong Fu1,2,3

Introduction

Results

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Discussion

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Experimental procedures

Acknowledgements

References

Supporting information

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