The armadillo repeats of the Ufd4 ubiquitin ligase recognize ubiquitin-fusion proteins

FEBS Letters 581 (2007) 265–270

The armadillo repeats of the Ufd4 ubiquitin ligase recognize ubiquitin-fusion proteins Donghong Ju, Xiaogang Wang, Haiming Xu, Youming Xie* Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 E Warren Avenue, Detroit, MI 48201, USA Received 13 November 2006; revised 7 December 2006; accepted 11 December 2006 Available online 19 December 2006 Edited by Gianni Cesareni

Abstract Armadillo (ARM) repeats have been identified in proteins regulating cell junction assembly, nuclear transport and transcription activation. Recent studies showed that a large number of ubiquitin (Ub) ligases (or E3s) also contain ARM repeats. The function of the ARM repeats in these E3s, however, remains unknown. Here we report that the ARM repeats of Ufd4, the E3 component of the UFD (Ub fusion degradation) pathway, recognize the Ub degradation signal of the UFD substrates. Disruption of the ARM repeats abolishes the ubiquitylating activity of Ufd4. This study uncovers a new role of the ARM repeats in protein ubiquitylation.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ubiquitin; Protein ubiquitylation; Ubiquitin ligase; Armadillo repeats; Ufd4; UFD pathway

1. Introduction Protein ubiquitylation involves consecutive reactions catalyzed by multiple enzymes including Ub-activating enzyme (E1), Ub-conjugating enzyme (E2) and Ub-ligase (E3). The specificity of protein ubiquitylation is mainly controlled by the degradation signal (degron) of the substrate and the activity of the cognate E3 [1]. In line with the large number of E3s, the substrates of the Ub-system are very diverse, and the degrons recognized by different E3s seldom share consensus sequences. So far, relatively few degrons have been characterized. The UFD pathway is responsible for ubiquitylation of the fusion proteins that bear a ‘‘non-removable’’ N-terminal Ub moiety, which serves as a degron [2]. The UFD pathway is conserved as the N-terminal Ub degron functions in both yeast and mammalian cells [3]. Previous work has identified the key ubiquitylating enzymes of the UFD pathway in Saccharomyces cerevisiae, including Ubc4 and Ubc5 E2s, the HECT E3 Ufd4, and the Ub-chain elongation factor Ufd2 (also called E4) [2,4]. However, how Ufd4 recognizes the N-terminal Ub degron is not understood. In this report, we demonstrate that the ARM repeats of Ufd4 interact with the N-terminal Ub degron of the UFD substrates, and that this interaction is essential for multiubiquitylation of the UFD substrates. Our work reveals a novel activity * Corresponding author. Fax: +1 313 831 7518. E-mail address: [email protected] (Y. Xie).

of the ARM repeats in protein ubiquitylation. The potential mechanism of the Ufd4 ARM repeats in Ub-chain synthesis is discussed.

2. Materials and methods 2.1. Strains and plasmids The S. cerevisiae strain used was YXY36(MATa ufd4D::LEU2 trp1D63 ura3-52 his3-D200 leu2–3, 112 lys2–801). E. coli strain BL21(DE3) was used to express Ub-fusions, GST fusions, and truncated Ufd4 proteins. pET-11d vector (Novagen) was used to express Ub-GST fusion and N-terminally hemaglutinin (ha)-tagged Ufd4 fragments in BL21(DE3). Ub-GST was constructed by adding a mouse UbG76V moiety to the N-terminus of GST. Plasmids pGEX4T-3-UFD4(202–437) and pGEX6P-1-RAD23 expressed GST fusions with residues 202–437 of Ufd4 and Rad23, whereas plasmids pT7-UbMeKDHFRhis6 (kindly provided by Varshavsky Lab, see Ref. [5]) expressed Ub-DHFRhis6 fusion in BL21(DE3). The plasmid encoding GST-Rpt6 was previously described [14]. The detailed protocols for plasmid construction are available on request. All mutants carrying point mutations and/or deletion were generated by PCR-mediated mutagenesis and confirmed by DNA sequencing. N-terminally ha-tagged Ufd4 and the internal deletion mutant Ufd4D263–335 were expressed from the induced CUP1 promoter in the low-copy vector pRS314. The plasmid expressing UbV76V-bgal from the GAL1 promoter was previously described [2]. 2.2. Pulse-chase and immunoprecipitation analysis S. cerevisiae cells from 10 ml cultures (OD600 of 0.8–1.0) in galactose medium containing 0.1 mM CuSO4 and essential amino acids were harvested. The cells were resuspended in 0.3 ml of the same medium supplemented with 0.15 mCi of [35S]-methionine/cysteine (EXPRESS [35S] Protein Labeling Mix, Perkin–Elmer), and incubated at 30 C for 5 min. The cells were then pelleted and resuspended in the same medium with cycleheximide (0.2 mg/ml) and excessive cold L -methionine/L -cysteine (2 mg/ml L -methionine and 0.4 mg/ml L -cysteine), and chased at 30 C. Equal volumes of samples were withdrawn at each time point. Labeled cells were harvested and lysed in equal volume of 2· SDS buffer (2% SDS, 30 mM dithiothreitol, 90 mM Na–HEPES, pH 7.5) by incubated at 100 C for 3 min. The supernatants were diluted 20-fold with buffer A (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 50 mM Na–HEPES, pH 7.5) before applied to immunoprecipitation with anti-bgal antibody (Promega) or anti-ha antibody (Sigma) with Protein A agarose (Calbiochem). The volumes of supernatants used in immunoprecipitation were adjusted to equalize the amounts of 10% trichloroacetic acid-insoluble 35S. The immunoprecipitates were washed three times with buffer A, and resolved by SDS–PAGE (6% gel), followed by autoradiography and quantitation with a PhosphorImager (Molecular Dynamics). 2.3. GST pulldown assays The procedure of pulldown assays was previously described [6], with minor modifications. BL21(DE3) cells transformed with plasmids encoding N-terminally ha-tagged Ufd4 fragments, Ub-DHFRhis6, Ub-GST, GST fusions and GST were induced with 1 mM IPTG at 28 C for 3 h. Cells were harvested, resuspended in buffer B (150 mM

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.12.024

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NaCl, 50 mM HEPES, pH 7.5) plus protease inhibitor mix (Roche Diagnostics), and lysed by sonication. The supernatants were collected after centrifugation for pulldown assays. Ub-DHFRhis6 fusion was further purified by Ni-NTA chromatography according to the manufacturer’s instructions (Qiagen). For each pulldown assay, 1 lg GST or GST fusions were pre-loaded on glutathione-agarose. Bacterial extracts or purified Ub-DHFRhis6 or a mix of Ub and K48-linked Ub-chains (generously provided by Dr. C. Pickart) were incubated with the loaded agarose in buffer B with 0.02% Triton X-100 at 4 C for 2 h. The beads were then washed three times with the same buffer, and the retained proteins were separated by SDS–PAGE, followed by immunoblotting with monoclonal anti-ha antibody (Sigma) or anti-Ub antibody (Covance), and detection with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) or the Odyssey infrared imaging system (Li-Cor Biosciences).

3. Results Since the UFD pathway is conserved from yeast to humans, we suspected that Ufd4 and its homologs in higher eukaryotes

might share a highly conserved domain that recognizes the common N-terminal Ub degron. We then blasted the NCBI (National Center for Biotechnology Information) database using full-length Ufd4 (1483 amino acids) as query, and identified several entries from higher eukaryotes that share significant sequence homology with Ufd4. The most noticeable homology other than the consensus C-terminal HECT domain is a segment of 200 amino acids near the N-terminus, consisting of four ARM repeats (Figs. 1a and 2a). ARM repeats are tandem repeated sequences, originally identified in the Drosophila segment polarity gene product Armadillo [7–9]. Each ARM repeat consists of approximately 40 amino acids forming three a helices, designated H1, H2 and H3. Successive ARM repeats may pack together to form an elongated superhelical structure, or ‘‘solenoids’’. Interestingly, the Ub-binding domains UBA and CUE, although unrelated at primary sequence level, display high structural similarity, folding into a three-helix bundle (Fig. 1b) [10–13]. We won-

a

b

Fig. 1. Sequence alignment of ARM repeats and Ub-binding domains. (a) A multiple sequence alignment of the ARM repeats from Ufd4 and its homologs. Residues identical in all the compared sequences are printed on black background, whereas similar or nearly invariant residues are highlighted in grey. The a helices predicted by the PredictProtein program (see Ref. [20]) are displayed at the top. For each sequence, the species, protein name and the positions of the repeats are indicated. Asterisks mark the conserved residues essential for the binding of the Ufd4 ARM repeats to Ub-GST. Species abbreviations: Sc, Saccharomyce cerevisiae; At, Arabidopsis thaliana; Os, Oryza sativa; Hs, Homo sapiens. Similar ARM repeats were also identified in the relatives of human TRIP12 (thyroid receptor-interacting protein 12) in other species (data not shown). (b) Sequence alignment of UBA and CUE domains from S. cerevisiaea Dsk2, Cue2 (Cue2-1 and Cue2-2) and human HR23A (UBA1 and UBA2). The secondary structures of Dsk2UBA1, HR23AUBA1 and CUE2-1 [10–13] are also shown at the top.

D. Ju et al. / FEBS Letters 581 (2007) 265–270

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Fig. 2. The ARM repeats of Ufd4 recognize Ub-GST. (a) Diagram of Ufd4 with the C-terminal HECT domain and the ARM repeats. Amino acids 1–200 are required for interaction with the proteasome [14]. (b) The ARM repeats 2 and 3 of Ufd4 constitute a binding site for Ub-GST. Various Nterminally ha-tagged fragments of Ufd4 were expressed form bacteria and used in pulldown assays with Ub-GST (lanes 3, 7, 9 and 12) or GST (lanes 2, 6, 8 and 11). The retained proteins were resolved by SDS–PAGE, followed by immunoblotting with anti-ha antibody (upper panels). 0.5% of the input extracts used in pulldown were included in the immunoblotting as control (lanes 1, 4, 5 and 10). It is unclear why haUfd4202–335 and haUfd4300–437 were so different in mobility on SDS–PAGE even though their calculated molecule masses are similar (lanes 4 and 5). Coomassie blue staining verified that comparable amounts of Ub-GST and GST were used (lower panels). (c) Mutations of the conserved amino acids in repeats 2 and 3 (see Fig. 1a) disrupted the interaction of haUfd4202–714 with Ub-GST. The same pulldown procedure as (b) was applied.

dered if the ARM repeats of Ufd4 could act as a binding site for the N-terminal Ub degron of the UFD substrates. To test this hypothesis, we generated a model substrate, Ub fused to the N-terminus of GST (Ub-GST). Ub-GST was expressed and purified from bacteria and therefore allowed us to assess the interaction between the ARM repeats of Ufd4 and the N-terminal Ub degron using pulldown assays. An N-terminally ha-tagged Ufd4 fragment with residues 202–437 (haUfd4202–437) consisting of the four ARM repeats was expressed from bacteria and applied to the pulldown assay. As shown in Fig. 2b (left panel), haUfd4202 437 was pulled down by Ub-GST (lane 3). The binding was specific to the N-terminal Ub because haUfd4202–437 was not retained by GST (lane 2). Comparable amounts of GST and Ub-GST were used in the pulldown assay, which was verified by Coomassie blue staining (lower panel). Thus, the ARM repeats of Ufd4 constitute a binding site for the N-terminal Ub degron. We then examined if all ARM repeats in haUfd4202–437 are involved in the interaction with Ub-GST. To this end, we expressed several smaller fragments carrying different ARM repeats in bacteria, and tested their ability to bind Ub-GST. haUfd4202–335 containing repeats 1–3 was pulled down by Ub-GST but not GST (Fig. 2b, middle panel, lanes 6 and 7). By contrast, haUfd4300–437 bearing repeat 4 and the last two helices of repeat 3 was not retained by Ub-GST under the same conditions (Fig. 2b, middle panel, lanes 8 and 9). Together, these data suggest that the Ub-binding site of Ufd4 is located in repeats 1–3. Since the stretch of residues 263–335 represents

the most conserved sequence between Ufd4 and its relatives (Fig. 1a), we tested if this subdomain consisting exclusively of repeat 3 and helices H2-2 and H2-3 is sufficient for binding to Ub-GST. As shown in Fig. 2b (right panel), haUfd4263–335 was indeed specifically pulled down by Ub-GST (lanes 11 and 12). These experiments conclude that the ARM repeats 2 and 3 of Ufd4 constitute the binding site for Ub-GST. We also assessed the requirement of several highly conserved amino acids in repeats 2 and 3, specifically in helices H2-3, H31 and H3-3 (Fig. 1a) for the binding to Ub-GST. Four mutants were generated from haUfd4202–714 that carried simultaneous point mutations E281A/Q282A/L284A, E288A/I290A/S291A, I298A/L299A, and Q319A/R320A, respectively. In contrast to wildtype haUfd4202–714, these mutant proteins lost the ability to interact with Ub-GST (Fig. 2c, compare lanes 7–10 and 6), indicating that the conserved amino acids are indeed critical for the binding activity of the ARM repeats. Although it remains to be determined whether these residues are in direct contact with the Ub degron or simply support the structural integrity of the binding surface, this mutation analysis further demonstrated the involvement of repeats 2 and 3 in interaction with Ub-GST. To examine if the ARM repeats of Ufd4 also bind free Ub or Ub-chains, we conducted two assays. First, we measured if free Ub could inhibit the binding of haUfd4202–437 to Ub-GST in a competition assay. As shown in Fig. 3a, free Ub even in excess of Ub-GST was unable to interfere with the interaction between Ub-GST and haUfd4202–437. Second, we constructed

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GST, respectively. In line with the interaction between haUfd4202–437 and Ub-GST (Fig. 2), Ub-DHFRhis6 was readily pulled down by GST-Ufd4(202–437) but not GST (Fig. 3c, lanes 2 and 3). Interestingly, GST-Ufd4(202–437) retained UbDHFRhis6 more efficiently than GST-Rad23 (Fig. 3c, compare lanes 3 and 4), even though the latter had a much higher affinity for free Ub and Ub-chains. We conclude that the ARM repeats of Ufd4 preferentially, if not exclusively, interact with the Ub degron of the UFD substrates. The results from the pulldown assays prompted us to measure the effect of disruption of the ARM repeats in Ufd4 on the degradation of UbV76-V-bgal, a well-known UFD model

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a GST fusion with residues 202–437 of Ufd4 fused to the C-terminus of GST. The GST-Ufd4(202–437) fusion protein was applied in a pulldown assay with a mix of free Ub and K48linked Ub-chains. A GST fusion with Ub-binding protein Rad23 (GST-Rad23) was used as a positive control, whereas GST alone served as a negative control. As expected, GSTRad23 preferentially bound Ub-chains of 4 or more Ub moieties (Fig. 3b, left panel, lane 4). Binding of Ub monomer to GST-Rad23 was also observed in experiments with more input of free Ub (data not shown). By contrast, GST-Ufd4(202–437) was unable to interact with free Ub or Ub-chains (Fig. 3b, left panel, lane 3, and data not shown). To validate the pulldown assays, we incubated another model UFD substrate, UbDHFRhis6, with GST-Ufd4(202–437), GST-Rad23 and

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Fig. 3. The Ufd4 ARM repeats specifically bind UFD substrates. (a) The binding of Ufd4 ARM repeats to Ub-GST is not inhibited by free Ub. Increasing amounts of Ub (0, 0.5, 2.5 and 12.5 lg in lanes 3–6) were added in the pulldown assays measuring the binding of haUfd4202–437 to Ub-GST as in Fig. 2b. (b) The Ufd4 ARM repeats do not bind free Ub or Ub-chains. A mix of free Ub and K48-linked Ub chains was used in pulldown assays with GST-Ufd4(202–437), GST-Rad23 and GST. The retained proteins were resolved by SDS– PAGE, followed by immunoblotting with anti-Ub antibody (left panel). 2% of the input Ub and Ub-chains mix was included in the immunoblotting as control (lane 1). The amounts of GST-Ufd4(202– 437), GST-Rad23 and GST used in the pulldown assays were shown by Coomassie blue staining in a separate gel (right panel). (c) The Ufd4 ARM repeats recognize Ub-DHFRhis6. Purified Ub-DHFRhis6 was used in pulldown assays with GST-Ufd4(202–437), GST-Rad23 and GST, following the same procedure in (b).

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Fig. 4. The Ufd4 ARM repeats are essential for Ub-chain synthesis. (a) Pulse-chase analysis was performed to compare the stability of UbV76-V-bgal in ufd4D co-expressing ha-tagged Ufd4 (lanes 5–8) or Ufd4D263–335 (lanes 9–12) or a void vector (1–4). The monoubiquitylated (Ub1), diubiquitylated (Ub2) and multiubiquitylated [Ub(n)] species of UbV76-V-bgal are indicated. (b) Quantitation of the results of (a) by PhosphorImager to show the decay curves of UbV76-V-bgal in the presence of haUfd4 (s), haUfd4D263–335 (d), or void vector (h). The 35S in the bands of UbV76-V-bgal (plus the first six of its ubiquitylated species) at each time point was plotted. (c) The expression levels of haUfd4 (lane 2) and haUfd4D263–335 (lane 3) were comparable in the cells used in (a) as shown by immunoprecipitation analysis with anti-ha antibody. Lane1 represents the void vector control. (d) Deletion of residues 263–335 does not affect the Ufd4-Rpt6 interaction. Extracts prepared from yeast cells expressing haUfd4 or haUfd4D263–335 were applied to pulldown assay with GST-Rpt6, followed by immunoblotting with anti-ha antibody.

D. Ju et al. / FEBS Letters 581 (2007) 265–270

substrate [2]. ufd4D cells expressing UbV76-V-bgal were transformed with a plasmid encoding N-terminally ha-tagged Ufd4 (haUfd4) or Ufd4D263–335 (haUfd4D263–335), or a void vector. haUfd4D263–335 lacks repeat 3 and helices H2-2 and H2-3. The stability of UbV76-V-bgal in these transformants was measured by pulse-chase analysis. The normally short-lived UbV76-V-bgal was stabilized in ufd4D, with ubiquitylated UbV76-V-bgal being confined to a monoubiquitylated species (Fig. 4a, lanes 1–4, 4b). As expected, UbV76-V-bgal was multiubiquitylated and rapidly degraded in the presence of haUfd4 (t1/2 < 10 min) (Fig. 4a, lanes 5–8, 4b). Similar to previous observations [14], 30–40% of the pulse-labeled UbV76-V-bgal was degraded before the chase, i.e. during the pulse period in the presence of haUfd4, as compared to the same test protein in the absence of haUfd4 (Fig. 4a, compare lanes 5 and 1). This so-called ‘‘zero-point’’ effect stems from the activity of the UFD pathway. By contrast, haUfd4D263–335, expressed at a comparable level as haUfd4 (Fig. 4c), was unable to rescue multiubiquitylation and degradation of UbV76-V-bgal in ufd4D (Fig. 4a, lanes 9–12, 4b). Consistently, haUfd4D263–335 did not cause ‘‘zero-point’’ degradation of UbV76-V-bgal (Fig. 4a, compare lanes 9 and 1). Note that haUfd4D263–335 was pulled down by GST-Rpt6, a GST fusion with the Rpt6 proteasome subunit that has been shown to interact with Ufd4 [14], as efficiently as haUfd4 (Fig. 4d), suggesting that deletion of residues 263–335 of Ufd4 does not drastically affect the protein folding. We noticed that a diubiquitylated derivative of UbV76-V-bgal was formed in ufd4D expressing haUfd4D263–335 in addition to the monoubiquitylated species (Fig. 4a, lanes 9–12). Interestingly, this diubiquitylated species of UbV76-V-bgal had a different mobility as compared to that synthesized by haUfd4 (Fig. 4a, compare lanes 9–12 and 5–8). It remains to be determined if the mobility difference reflects different Ub–Ub linkage. Together, these results indicate that the ARM repeats of Ufd4 are essential for multi-Ub chain assembly on the UFD substrates.

4. Discussion We demonstrated in this study that the ARM repeats of Ufd4 interact with the N-terminal Ub degron of the UFD substrates. Interestingly, the Ufd4 ARM repeats appear not to bind (or bind with very low affinity) free Ub or K48-linked Ub-chains, suggesting that the Ub moiety in the UFD substrates may have a unique surface recognized by the Ufd4 ARM repeats, and such a surface does not exist in free Ub or K48-linked Ub-chains. Recent studies have shown that different families of Ub-binding domains target different surfaces on Ub and Ub-chains, not limited to the well-characterized hydrophobic pocket around Ile-44 [15,16]. Our data further suggest that Ub attached to protein substrates may display different interfaces for protein–protein interaction, perhaps partially depending on the context of the conjugated substrates. This may provide yet another layer of specificity for recognition of ubiquitylated proteins by a variety of Ub receptors in the cell. The Ufd4 ARM repeats represent a novel Ub-binding domain that selectively interacts with the N-terminal Ub moiety of UFD substrates. It will be of interest to examine if other ARM proteins also serve as Ub receptors and what types of ubiquitylated substrates (Ub interfaces) they would bind. Deciphering the atomic structure of the Ub-degron of the UFD

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substrates will provide a clearer understanding of how the Ufd4 ARM repeats recognize the Ub degron. Although the detailed mechanism remains to be investigated, the Ufd4 ARM repeats are essential for multiubiquitylation of the UFD substrates. We speculate that the ARM repeats of Ufd4 may have a dual role. On the one hand, the ARM repeats recognize the N-terminal Ub degron and therefore serve as a substrate-docking site. On the other hand, via the noncovalent binding, the ARM repeats may position the acceptor Ub to the donor Ub, which is covalently linked, via a thioester bond, to the catalytic active Cys of the HECT domain in Ufd4. This positioning allows the transfer of the donor Ub to a specific Lys residue on the acceptor Ub, first the N-terminal Ub degron and then the growing Ub-chain, producing a substrate-linked multi-Ub chain with defined Ub–Ub linkage. The involvement of two Ub-binding sites in Ub-chain assembly, while not reported for E3 before, has been shown for several E2s [17, and references therein]. For instance, the yeast Ubc13/Mms2 E2 heterodimer (and related human Ubc13/ Uve1A) bears 2 potential Ub-binding sites. One is the active site of Ubc13 for the formation of thioester with the donor Ub. The second site noncovalently binds the acceptor Ub and positions its K63 to the donor Ub, leading to the synthesis of K63-linked Ub-chain. Our current work provides the first example of E3 that uses two types of Ub-binding activities in Ub-chain synthesis. The involvement of ARM repeats in protein ubiquitylation is unlikely limited to Ufd4 since large-scale analyses of the Arabidopsis genome have revealed the existence of ARM repeats in at least 41 U-box E3s and two F-box proteins in addition to the Ufd4 homologs, UPL3 and UPL4 (Fig. 1a; Refs. [18,19]). Further biochemical analysis of these ARM-containing E3s will provide new insight into the role of ARM repeats in protein ubiquitylation. Acknowledgements: This study was supported by grants to Y.X. from the Barbara Ann Karmanos Cancer Institute, and the American Cancer Society (RGS-0506401-GMC). Xiaogang Wang is a visiting scholar from Fudan University, Shanghai, China.

References [1] Pickart, C.M. and Eddins, M.J. (2004) Ubiquitin: structure, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72. [2] Johnson, E.S., Ma, P.C., Ota, I.M. and Varshavsky, A. (1995) A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456. [3] Dantuma, N.P., Lindsten, R., Glas, M., Jellne, M. and Masucci, M.G. (2000) Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotech. 18, 538–543. [4] Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D., Mayer, T.U. and Jentsch, S. (1999) A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644. [5] Davydov, I.V., Patra, D. and Varshavsky, A. (1998) The N-end rule pathway in Xenopus egg extracts. Arch. Biochem. Biophys. 357, 317–325. [6] Wang, L., Mao, X., Ju, D. and Xie, Y. (2004) Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase. J. Biol. Chem. 279, 55218–55223. [7] Riggleman, B., Wieschaus, E. and Schedl, P. (1989) Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene. Genes Dev. 3, 96–113. [8] Hatzfeld, M. (1999) The armadillo family of structural proteins. Int. Rev. Cytol. 186, 179–224.

270 [9] Huber, A.H., Nelson, W.J. and Weis, W.I. (1997) Three-dimensional structure of the armadillo repeat region of beta-catenin. Cell 90, 871–882. [10] Kang, R.S., Daniels, C.M., Francis, S.A., Shih, S.C., Salerno, W.J., Hicke, L. and Radhakrishnan, I. (2003) Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding. Cell 113, 621–630. [11] Ohno, A., Jee, J., Fujiwara, K., Tenno, T., Goda, N., Tochio, H., Kobayashi, H., Hiroaki, H. and Shirakawa, M. (2005) Structure of the UBA domain of Dsk2p in complex with ubiquitin: molecular determinants for ubiquitin recognition. Structure 13, 521–532. [12] Dieckmann, T., Wither-Ward, E.S., Jarosinski, M.A., Liu, C.F., Chen, I.S. and Feigon, J. (1998) Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr. Nat. Struct. Biol. 5, 1042–1047. [13] Mueller, T.D. and Feigon, J. (2002) Solution structures of UBA domains reveal a conserved hydrophobic surface for protein– protein interactions. J. Mol. Biol. 319, 1243–1255.

D. Ju et al. / FEBS Letters 581 (2007) 265–270 [14] Xie, Y. and Varshavsky, A. (2002) UFD4 lacking the proteasomebinding region catalyses ubiquitination but is impaired in proteolysis. Nat. Cell Biol. 4, 1003–1007. [15] Hicke, L., Schubert, H.L. and Hill, C.P. (2005) Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6, 610–621. [16] Harper, J.W. and Schulman, B.A. (2006) Structural complexity in ubiquitin recognition. Cell 124, 1133–1136. [17] Hochstrasser, M. (2006) Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34. [18] Downes, B.P., Stupar, R.M., Gingerich, D.J. and Vierstra, R.D. (2003) The HECT ubiquitin-protein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development. Plant J. 35, 729–742. [19] Mudgil, Y., Shiu, S.H., Stone, S.L., Salt, J.N. and Goring, D.R. (2004) A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family. Plant Physiol. 134, 59–66. [20] Rost, B., Yachdav, G. and Liu, J. (2004) The PredictProtein server. Nucleic Acids Res. 32 (Web Server issue), W321–W326.