81 putative zebrafish Mib/Mib2-binding proteins were identified

To further investigate Mib function and identify putative Mib/Mib2-binding proteins, two yeast two-hybrid screens with different interaction strategies were performed using different regions of Mib and Mib2 as baits (Figure 1B) to screen the zebrafish whole embryo cDNA libraries. In the first screen, where the reconstitution of the functional transcription factor of LexA (DNA binding domain)-bait fusion protein and prey-Gal4 (activation domain) fusion protein activates the HIS3 reporter gene, 33 positive clones of 13 genes were identified by Mib-C-RF123a (783–1029 aa) from 60 million colonies, and 232 positive clones of 26 genes were identified by Mib2-C-RF12 (781–999 aa) from 52 million colonies (Table 1). Notably, there are much more positive clones identified by Mib2-C-RF12 (232 clones) than by Mib-C-RF123a (33 clones). With the bait Mib2-C-RF12, Mib was one of the positive clones, which is consistent with the finding that Mib2 is an interacting protein of Mib [10].

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Table 1. Zebrafish Mib/Mib2-binding proteins isolated in the first yeast two-hybrid screen.

https://doi.org/10.1371/journal.pone.0093394.t001

In the second screen with CytoTrap system, the bait and prey proteins are expressed and retained in the cytoplasm, where, unlike in the nucleus, they may undergo post-translational modifications. In addition, transcriptional activators and inhibitors may be used as baits to screen for protein-protein interactions. In this screen, we picked up a total of 95 positive clones that have in-frame cDNA sequence with the myristylation sequence, in which 52 clones of 28 genes by Mib-M-Ank (454–785 aa), 6 clones of 4 genes by Mib-C-RF123b (789–1030 aa) and 37 clones of 20 genes by Mib-ZnF+Ank (74–785 aa) (Table 2). Mib-M-Ank was screened for about 0.4 million colonies; Mib-C-RF123b and Mib-ZnF+Ank baits were individually screened for around 0.5 million colonies. Conspicuously, there are much more positive clones identified by Mib-M-Ank (52 clones) and Mib-ZnF+Ank (37 clones) than by Mib-C-RF123b (6 clones), which is consistent with the previous report that N-terminus and middle ankyrin repeats, but not C-terminal RF123, are the major binding domains of Mib protein to itself or other interacting proteins [10]. The interacting candidates through Mib-M-Ank and Mib-ZnF+Ank include both unique (19 genes and 11 genes, respectively) and common clones (9 genes) (Table 2).

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Table 2. Zebrafish Mib-binding proteins isolated in the second yeast two-hybrid screen.

https://doi.org/10.1371/journal.pone.0093394.t002

In these two screens, the baits Mib-N (1–462 aa), Mib-N+Ank (1–782 aa) and Mib2-N+Ank (54–780 aa) showed autoactivation and were not used for screens to avoid false positive clones. Interestingly, all of these autoactivated baits contains two mib/herc2 domains, whose configuration and/or conformation may have unexpected higher affinity with or non-specific binding to the reporter constructs or non-binding activation of Ras signal. In the comparison of these two screens, many positive clones are functionally related and can be classified into six groups: ubiquitin proteasome system, cytoskeleton, trafficking, replication/transcription/translation, cell signaling and others (Tables 1 and 2). Although no identical gene was found from both screens, interestingly, the isoforms of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3): 14-3-3 beta (β), 14-3-3 zeta (ζ) and 14-3-3 theta (θ) appeared in these two screens. 14-3-3 zeta was picked up for fifteen times by the bait Mib2-C-RF12 in the first screen. In addition, the family of actinin and paired box genes were also identified with different baits. In these two yeast two-hybrid screens, totally, 81 putative interacting proteins were isolated, implying that Mib and Mib2 may be involved in many cellular events that are intriguing to be investigated further.

Some genes are frequently picked-up. Those that have more than 5 clones identified include ribosomal protein S27a (rps27a)/ubiquitin A-80-residue ribosomal protein fusion product (UBA80) (46), ubiquitin C (ubc) (45), ubiquitin B (ubb) (31), ubiquitin specific protease 5 (usp5) (31), ubiquitin A-52 residue ribosomal protein fusion product (uba52) (30), myosin, light chain, phosphorylatable, fast skeletal muscle a (mylpfa) (21), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide b (ywhaz) (14-3-3 zeta) (15), actinin alpha 4 (actn4) (7), insulin-degrading enzyme (ide) (7), nascent polypeptide-associated complex alpha polypeptide (naca) (7), DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (ddx5) (6). They belong to either proteasome/ubiquitin system or structural housekeeping genes, except 14-3-3 zeta, ide, naca and ddx5. The higher number of clones may reflect the abundance of these genes.

Mib and Mib2 interact with deubiquitylases Usp1, Usp9 and Trabid

In our screens, the ubiquitin proteins: ubiquitin B, ubiquitin C, ubiquitin A-52 and ubiquitin A80 appeared with a high frequency. Proteasome subunits including proteasome 26S subunit 1 (psmd1), proteasome subunit 5 (psma5) and proteasome 26S subunit 4 (psmd4) were also identified from both screens, reflecting a role of protein degradation and the E3 ligase activity of Mib and Mib2. Other intriguing interacting proteins were deubiquitylases (DUBs): ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 5 (Usp5), ubiquitin specific protease 9 (Usp9) and tumor-necrosis-factor-receptor-associated factor-binding domain (Trabid) in the ubiquitin proteasome system group (Tables 1 and 2).

Ubiquitylation is a reversible reaction that ubiquitin can be removed by deubiquitylases. DSL ligands can be ubiquitylated by Neuralized and Mib. However, deubiquitylation of ligands is fully unknown. Usp1, Usp5, Usp9 and Trabid picked out from our screens might be potential candidates to regulate the Notch signaling pathway. To further verify the physical interactions of Mib with these deubiquitylases, co-immunoprecipitation was performed in COS7 cells. Cells were coexpressed with different FLAG-tagged mib or -mib2 and Myc- or HA- tagged DUBs. The whole cell lysate was immunoprecipitated (IP) by anti-FLAG antibody and then subjected to Western blotting (IB) by anti-Myc antibody or anti-HA antibody to detect the interacting proteins. The results indicated that Usp1 C (from 349 to 772 amino acids), Usp9 and Trabid C (from 295 to 716 amino acids) were co-immunoprecipitated with Mib (Figure 2A, lane 2; Figure 2B, lane 2; Figure 2C, lane 2), but Usp5 was not (data not shown). Because Mib and Mib2 are E3 ubiquitin ligase in Notch activation through Delta ubiquitylation and endocytosis [9]–[10], [14], [26]–[28] and zebrafish Mib and Mib2 have both common and specific Delta substrate [10], the interaction of Mib2 and these DUBs was also examined by immunoprecipitation. The results showed that Usp1 C, Usp9 and Trabid C were also co-immunoprecipitated with Mib2 (Figure 2A, lane 3; Figure 2B, lane 3; Figure 2C, lane 4), but Usp5 was not (data not shown). Mib^ta52b and Mib2RF12m (E3 ligase-inactivated form, point mutation in the C-terminal-most RF [10] also bound to Trabid C (Figure 2C, lanes 3 and 5), reflecting that E3 enzymatic activity is not required for binding.

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Figure 2. Interaction of Mib and Mib2 with Usp1, Usp9 and Trabid.

Immunoprecipitation of (A) Usp1 C (349–772 aa), (B) Usp9 (full length) and (C) Trabid C (295–716 aa) by Mib and Mib2. COS7 cells were co-transfected with the expressing plasmids for HA- or Myc-tagged DUBs and FLAG-tagged Mib/Mib2. Immunoprecipitation (IP) were performed with anti-FLAG M2-Agarose and detected by Western blotting (IB) with anti-FLAG, anti-Myc or anti-HA antibody. Star indicates non-specific signals.

https://doi.org/10.1371/journal.pone.0093394.g002

Mib and Mib2 interact with Fih-1 that is involved in the hypoxia signaling pathway

In addition to DUBs, interacting candidates: atonal homolog-1 (Zath-1), heat shock cognate (Hsc70) and factor inhibiting HIF-1 (Fih-1) were investigated to verify their binding activities with Mib and Mib2 for the reasons that they have been shown to interact with Notch signaling genetically and/or biochemically [21], [29]–[31]. Results of immunoprecipitation done in COS7 cells indicated that Fih-1 is able to interact with Mib and Mib2 physically (Figure 3, lanes 2 and 4), but neither Zath-1 nor Hsc70 was pulled down by Mib or Mib2 (data not shown). Similar to Trabid, Mib^ta52b and Mib2RF12m also bound to Fih-1 (Figure 3, lanes 3 and 5).

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Figure 3. Interaction of Mib and Mib2 with Fih-1.

IP of Fih-1 by various Mibs and Mib2s. COS7 cells were co-transfected with pCS2-FLAG tag and pCS2-MT tag constructs. The cell lysate was incubated with anti-FLAG M2-Agarose and then subjected to IB with anti-Myc (top panel) or anti-FLAG antibody (bottom panel).

https://doi.org/10.1371/journal.pone.0093394.g003

Trabid and Fih-1 are substrates of Mib and Mib2

Zebrafish Mib and Mib2 are E3 ubiquitin ligase and can mediate ubiquitylation of their substrates in a most C-terminal RF-dependent manner [10]. Since both Mib and Mib2 can bind to Fih-1 and Trabid, we next asked whether Mib and Mib2 could promote the most C-terminal RF-dependent substrate ubiquitylation. COS7 cells were cotransfected with pcDNA3.1-HA-ubiquitin, different pCS2-FLAG-mib or -mib2 and pCS2-MT-fih-1 or -trabid C (295–716 aa). The whole cell lysate was immunoprecipitated with anti-Myc antibody and then subjected to immunoblotting with anti-Myc antibody as a control or anti-HA to detect the ubiquitylated proteins. Both Mib and Mib2 facilitated Fih-1 and Trabid C ubiquitylation (Figure 4A and 4B, lanes 2, 4), while the E3 ligase-inactivated Mib^ta52b or Mib2RF12m failed to do so (Figure 4A and 4B, lanes 3, 5). Trabid FL (full length) was also ubiquitylated by Mib (Figure 4C). These data imply that in addition to DeltaC, zebrafish Mib and Mib2 have two novel common substrates: Fih-1 and Trabid.

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Figure 4. Both Mib and Mib2 promote Fih-1and Trabid ubiquitylation.

(A) Ubiquitylation of Fih-1, (B) Trabid C (295–716 aa) and (C) Trabid FL (full-length) by Mib or Mib2. COS7 cells were co-transfected with pcDNA3.1-HA-ubiquitin, pCS2-FLAG tag and pCS2-MT tag constructs. The cell lysate was incubated with anti-Myc antibody and then subjected to IB with anti-HA (top panel) or anti-Myc antibody (bottom panel).

https://doi.org/10.1371/journal.pone.0093394.g004

E3 ligases and deubiquitylases (DUBs)

Ubiquitin and proteasome subunits are binding proteins of Mib and Mib2 identified in our screens. It reflects a role of Mib/Mib2 in proteasome-dependent degradation of their substrates such as DAPK and RYK [23], [25]. In this study, we demonstrated that Usp1, Usp9 and Trabid are interacting proteins of Mib and Mib2 by yeast two-hybrid and immunoprecipitation. DUB/E3 interaction has been reported that it can fine-tune protein degradation on their common substrates and/or stabilize the autoubiquitylated E3 ligase. For example, USP7 (HAUSP) interacts with MDM2 to regulate the turnover of p53 together, and it is also able to increase the stability of MDM2 [32]. USP9X (FAM) deubiquitylates the autoubiquitylated E3 ligases Itch and SMURF1 and, thereby, increases their stability [33]–[34]. Mib/Mib2 with autoubiquitylating activity stimulates its turnover through proteasome, and so deubiquitylation is required for the maintenance of Mib. Therefore, Usp1, Usp9 and Trabid are potential candidates in regulating the stability of Mib/Mib2. However, whether the interaction between Mib/Mib2 and these DUBs is related to Notch signaling remains unclear. Of notice, another two DUBs, eIF3f and Bap1, have been shown in regulating Notch activation [35] and implicated in participating in Notch signaling [36]–[37], respectively.

Tumor necrosis factor (TNF)-α and interleukin (IL)-1 are pro-inflammatory cytokines that initiate signaling pathways in endothelial cells leading to activation of nuclear factor κB (NF-κB) and plays an important role in the regulation of immune and inflammatory responses [38]–[39]. NF-κB activation through IL-1 requires several adaptors, including tumor-necrosis-factor-receptor-associated factor 6 (TRAF6) [40]. Human TRAF-binding domain (TRABID) was able to interact with TRAF6 [39], suggesting a role of Trabid in regulating NF-κB activation.

Notch and hypoxia signaling pathways

The regulation of hypoxia signaling pathway is conducted by O₂ sensing enzymes, such as HIF-α-specific prolyl hydroxylase domain proteins (PHDs) and Hif1an/Fih-1, an asparaginyl hydroxylase. FIH-1 interacts with and mediates hydroxylation of HIF-1α and, therefore, inhibits HIF-1 transcriptional activity [41]–[42].

Recently, Fih-1 was found that it can associate with and hydroxylate proteins containing AND, such as Notch receptor 1, 2 and 3 [43], IκBα [44], and Tankyrase-2, Rabankyrin-5 and RNase L [45]. At hypoxia, NICD enhances recruitment of HIF-1α to its target promoters and derepresses HIF-1α function by sequestering HIF-1α through its higher affinity to FIH-1 [30]. Moreover, the crosstalks between hypoxia and Notch signaling pathways were also found to be critical in stem cell maintenance [21], [46], arterial cell fate decision [47], tumor invasion [48], blood cell survival [49] and angiogenesis [50]. Although these lines of evidence indicate a close cooperation between Notch and hypoxia molecules, the interaction of Mib, a critical Notch signaling molecule, and Fih-1, an important hypoxia sensor, was first documented here.

Despite the fact that Mib was not identified in a previous proteomics-based screen for FIH substrates [45], we found that Mib and Mib2 were associated with Fih-1 in both yeast-two hybrid and co-immunoprecipitation experiments. This suggests that Mib/Mib2 and Fih-1 may regulate hypoxia signaling pathway and Notch signaling pathway through each other. The function of Fih-1 on Mib is probably to stabilize the folding structure of Mib, as Fih-1 was found to stabilize the folding of AND through hydroxylation [51], and, thereby, further enhance Notch activation. Mib may facilitate Fih-1 ubiquitylation and degradation as Siah-1, also an E3 ligase, does [52]–[53].

Mib and Mib2 in cellular trafficking

Enodcytosis of ligands is required for the activation of Notch signaling [54]. Ligands ubiquitylated by E3 ligase Neuralized or Mib are recognized as cargos by the endocytic adaptor Epsin that initiates the Clathrin/Dynamin dependent endocytosis. So far, it is believed that the major role of Mib/Mib2 in Notch signaling is to ubiquitylate ligands. However, fewer studies work on the possible role of Mib/Mib2 in endocytosis. In fact, substrates of Mib/Mib2, such as Delta and RYK, were translocated to the intracellular vesicles with Mib/Mib2 [10], [25]. Results from our screens showed that trafficking molecules like EPS15 and Epsin-2-like are possible binding proteins of Mib2. What are their roles? One possibility is that the interaction of EPS15 and Epsin-2-like with Mib2 is an advantage for forming endocytotic structure immediately after ligands are ubiquitylated by Mib2. Another possibility is that Mib2 ubiquitylates EPS15 and Epsin-2-like to regulate the endocytosis directly. Monoubiquitylation of EPS15 and Epsin is known as a negative regulation to inhibit their binding activity with ubiquitylated cargo on the cell membrane [55]–[56]. The study in fly Drosophila indicates that Fat facets (Usp9 ortholog) promotes endocytosis of Delta by deubiquitylating Liquid facets (Epsin ortholog) [57]–[58]. In addition, we have demonstrated Usp9 is an interacting protein of Mib2 by the yeast two-hybrid screen and a binding protein of Mib and Mib2 by immunoprecipitation, which is consistent with previous study by affinity purification/tandem mass spectrometry - Fat facets in Mouse (FAM) or mUSP9x interacts with Mib1 [59]. Therefore, we postulate that Mib and Mib2 may cooperate with Usp9 in regulating endocytosis of Delta through the ubiquitylation/deubiquitylation of Epsin.

Mib may also participate in other trafficking processes. Trafficking molecules like exocyst complex component 1, exocyst complex component 3, and vesicle docking protein p115 were found in our screens. Similarly, other trafficking components have also been identified by other group, such as Importin alpha and beta, Kinesin heavy chain member 2, Nipsnap1, Rba11-F1p2, Rab11-FIP5 and early endosome antigen 1 [59]. These findings imply that trafficking functions of Mib/Mib2 are worthy of further exploration.

Status quo and perspectives

It has been reported that yeast protein interactomes obtained from different research groups by large-scale two-hybrid screens fail to overlap to a great extent [60]–[61]. Reasons ranging from methodology, plasmid constructs, strategy, stringency, saturation and stochastic activation of reporter genes have been discussed. Similarly, these reasons can explain why our screens did not pick up the genes identified by other groups (e.g., DSL ligands, listed in Table 3). The ubiquitylation of Mib-interacting proteins affects their localization (e.g., Jagged 1, Jagged 2, DeltaC, DeltaD, CDK5, Snx5 and RYK), protein stability (e.g., DAPK, RYK and SMN) or activity (e.g., TBK1 and NMDAR) [9]–[12], [23]–[25], [59], [62]–[65]. Similar to Mib, Mib2 can regulate the localization (e.g., DeltaC and Jagged 2) and activity (e.g., TAK1) of its interacting proteins [10], [66]–[67]. Additionally, another Mib-interacting protein PAR-1 can phosphorylate Mib. This results in Mib degradation and, therefore, suppresses Notch signaling and stimulates neuronal differentiation [68]. Most importantly, our screens complement previous studies and add novel interacting proteins to an expanding list (Table 3) for further exploration of Mib/Mib2 functions.

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Table 3. The confirmed interacting proteins of Mind bomb and Mind bomb 2.

https://doi.org/10.1371/journal.pone.0093394.t003

In our screens, several proteins involved in the cytoskeleton, including actinin alpha 2, actinin alpha 3, actinin alpha 4, myosin light chain and cofilin 1, were identified. Cytoskeleton member like dystrobrevin and dystrophin-associated protein A1 were also identified as Mib-interacting proteins by affinity purification and mass spectrometry [59]. Whether Mib participates in the muscle development or maintenance is unclear. Studies in Drosophila indicated that Mib2 expressed in founder myoblasts regulates myoblast fusion and muscle stability through a Notch-independent way [69]–[70]. Although no evidence supports the involvement of Mib in Drosophila myogenesis, the putative Mib-interacting proteins related to cytoskeleton do implicate such a role in vertebrates.

Proteins involved in cell signaling also appear in our screens, such as polo-like-kinase 4, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteins (14-3-3), TGF-beta activated kinase 1/MAP3K7 binding protein 3 (TAK1-binding protein 3), creatine kinase, and casein kinase 2 beta (ck2b), suggesting that Mib/Mib2 are potentially involved in several signaling pathways in addition to the Notch signaling pathway. Indeed, Mib plays a positive role in the Wnt signaling pathway by regulating the stability and localization of RYK [25]. Mib2 activates NF-κB pathway through the interaction with B-cell CLL/lymphoma 10 (BCL10), inhibitor of nuclear factor kappa-B kinase subunit gamma (IKKγ) and transforming growth factor β-activated kinase 1 (TAK1) [67]. In the past, the roles of Mib in the neurogenesis, gliogenesis, thyroid morphogenesis, T helper cell differentiation and pancreatic β-cell formation were focused on the Notch-dependent pathway [71]–[74]. In the future, it is rational to take other potential cell signaling pathways into consideration for a more deep understanding of the roles of Mib and Mib2 in various developmental processes.

Yeast two-hybrid screen

For the first yeast two-hybrid screen, a conventional LexA and Gal4 system relying on transcriptional activation of reporter genes in the nucleus to detect interactions was used. Bait fragments used for this screen were Mib-N+Ank (1–782 aa), Mib-C-RF123a (783–1029 aa), Mib2-N+Ank (54–780 aa) and Mib2-C-RF12 (781–999 aa) (Figure 1B). Yeast two-hybrid screening was performed by Hybrigenics (Paris, France). The corresponding fragments of mib and mib2 [15] was PCR-amplified and cloned into pB27 plasmid for N-terminal LexA DNA binding domain fusion proteins (N-LexA-Mib/Mib2-C). The constructs were checked by sequencing and used as baits to screen a random-primed Danio rerio embryo (stages 18–20 hpf) RP1 cDNA library (Hybrigenics) constructed into pP6 plasmid for C-terminal Gal4 transcription activating domain fusion proteins. Mib-N+Ank (1–782 aa) and Mib2-N+Ank (54–780 aa) have autoactivation effect and, therefore, are not used for further screen. 60 million colonies for Mib-C-RF123a (783–1029 aa) and 52 million colonies for Mib2-C-RF12 (781–999 aa) were respectively screened. cDNA fragments corresponding to positive “prey” clones were amplified by PCR and sequenced at their 5′ and 3′ junctions. The resulting sequences were searched against GenBank using a fully automated procedure, assigned a quality score (PBS, for Predicted Biological Score) indicative of the confidence of interaction [75] and listed in Table 1.

For the second yeast two-hybrid screen, zebrafish Mib-N (1–462 aa), Mib-M-Ank (454–785 aa), Mib-C-RF123b (789–1030 aa) and Mib-ZnF+Ank (74–785 aa) (Figure 1B) were subcloned into pSos vector (Stratagene) as baits by PCR amplification of mib cDNA sequences (using BamHI and XhoI as cloning sites). These baits were then used to screen the prey, pMyr-cDNA library (CytoTrap® XR Zebrafish Embryo Plasmid cDNA Library, Stratagene; normal, whole, zebrafish embryos, average insert size: 1.0 kb) within the temperature-sensitive yeast mutant strain cdc25H according to the manufacturer's instructions (Stratagene). cdc25 is the yeast homolog of the human Sos (hSos) gene, which encodes a guanyl nucleotide exchange factor that activates Ras signal transduction pathway. The cdc25 mutation present in the cdc25H strain allows normal growth at the permissive temperature (25°C), but prevents growth at 37°C. pSos bait vector generates a fusion protein of hSos and the bait protein. pMyr-cDNA is expressed as a fusion protein with a myristylation sequence that anchors the fusion protein to the plasma membrane. The physical interaction of the bait and prey proteins results in the localization of hSos to the plasma membrane and reconstitutes Ras-signaling pathway activity, which allows the yeast growth at both 37°C and 25°C. pSos-Mib-N (1–462 aa) was unsuitable for detecting protein-protein interactions in the CytoTrap system, because the bait plasmid cotransformed with the pMyr empty vector autoactivated Ras signal and induced cdc25H yeast growth at 37°C. The screen then focused on the other three baits. Around 0.4, 0.5 and 0.5 million colonies were screened for Mib-M-Ank (454–785 aa), Mib-C-RF123b (789–1030 aa) and Mib-ZnF+Ank (74–785 aa), respectively. Plasmid DNA was isolated from the putative positive pMyr-cDNA clones. The two-hybrid interaction was confirmed by retransformation of the yeast strain cdc25H with the bait vector and purified prey plasmid. All positive prey plasmids were sent for DNA sequencing by the 5′ primer (5′-ACTACTAGCAGCTGTAATAC-3′) in the pMyr vector and blasted again NCBI database. Clones that have in-frame cDNA sequence with the myristylation sequence were finally selected and listed in Table 2.

Plasmids

pCS2-FLAG-mib and -mib2 were made by subcloning the PCR-amplified fragments into the pCS2+FLAG vector [10]. Full-length fih-1 and partial trabid C (295–716 aa) cDNA fragments were PCR-amplified and subcloned into pCS2-MT vector at EcoRI and XhoI sites. Full-length trabid cDNA fragments were PCR-amplified and subcloned into pCS2-MT vector at XhoI and XbaI sites. C-terminal usp1 C (349–772 aa) cDNA fragments were PCR-amplified and subcloned into pCS2-MT vector at EcoRI and XhoI sites. Full-length usp9 cDNA fragment was PCR-amplified and subcloned into pCMV-HA vector at SfiI and NotI sites. All PCR products were verified by DNA sequencing. pcDNA3.1-HA-ubiquitin is a gift from Dr. Margaret A. Shipp. Usp1, Usp9, Trabid and Fih-1 protein sequences are shown in Supporting Information with comparison to those of other species (Figures S1–S4).

Cell culture and co-immunoprecipitation assay

COS7 cells [10], [15] were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin. Cells were transiently transfected using Lipofectamine 2000 transfection reagent (Invitrogen). 4 µg of pCS2-MT-fih-1 was cotransfected with 4 µg of pCS2-FLAG-mib or -mib2 into COS7 cells. 2 µg of pCS2-MT-trabid C (295–716 aa), pCS2-MT-usp1 (349–772 aa) or pCMV-HA-usp9 was cotransfected with 2 µg of pCS2-FLAG-mib or -mib2 into COS7 cells. Cells were harvested and lysed 24 h after transfection in IONIC buffer (50 mM Tris HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, containing a protease inhibitor cocktail, Calbiochem). The cell lysate was clarified by centrifugation and incubated with anti-FLAG M2 affinity gel (Sigma) for 2 h at 4°C. The gel was boiled in SDS gel loading buffer and the eluted proteins were electrophoresed on a SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). Blots were incubated with primary antibody: anti-HA (Protech), anti-Myc (Santa Cruz) or anti-FLAG (Sigma) for 2 hr. The signal was visualized using a secondary antibody: HRP-conjugated anti-mouse IgG antibody (GeneTex) or HRP-conjugated anti-rabbit IgG antibody (GeneTex), with a chemiluminescence detection system (PerkinElmer Inc).

In vivo substrate ubiquitylation assay

COS7 cells were transiently transfected with 2 µg of pcDNA3.1-HA-ubiquitin, 2 µg of pCS2-MT-fih-1 [or pCS2-MT-trabid C (295–716 aa)] as substrate and 4 µg of pCS2-FLAG-mib or -mib2 as putative E3 ubiquitin ligases. 2 µM proteasome inhibitor MG132 was added to the cell culture medium after 24 h. Cells were harvested at 48 h and lysed in IONIC buffer and 10 µM MG132. The cell lysate was clarified by centrifugation and incubated with rabbit anti-Myc antibody (Santa Cruz) for 2 h at 4°C, and then incubated with protein A Sepharose beads for 1 h at 4°C. The eluted proteins were electrophoresed on a SDS-PAGE followed by Western blotting with mouse anti-HA (Roche) or mouse anti-Myc (Roche) antibody.