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New Classes of Mind Bomb-Interacting Proteins Identified from Yeast Two-Hybrid Screens

Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.


Introduction
Notch signaling pathway is an evolutionarily conserved signal transduction cascade in flies, worms and vertebrates. It is a shortrange cell communication. Through the Notch signaling pathway, signal-sending cells transfer a lateral inhibitory signal to the adjacent signal-receiving cells to control cell fate decision during development. It also plays roles in cell proliferation, cell death and self-renewal of adult stem cells [1]. The multiple functions of the Notch signaling pathway explain why improper Notch signaling causes human disorders such as Alagille syndrome, spondylocostal dysostosis and cancers [2][3][4].
Notch signaling is activated by the interaction of DSL ligands (Delta and Serrate for Drosophila and Lag-2 for C. elegans) on the surface of signal-sending cells with the Notch receptor on signalreceiving cells. Accompanying the interaction, ligands are internalized into cells that provide a pulling force on the Notch receptors leading to the exposure of the second cleavage site cut by ADAM metalloproteases. Then, the third cleavage of the Notch receptor by c-secretase releases the Notch intracellular domain (NICD) into the nucleus. NICD forms a complex with CSL transcription factors (CBF1 for human; Suppressor of Hairless for Drosophila and Lag-1 for C. elegans) to regulate the expression of downstream genes [5][6][7][8].
The endocytosis of DSL ligands is required for the activation of the Notch signaling pathway. The process is triggered by the ubiquitylation of ligands by Neuralized and Mind bomb (Mib). Mind bomb is an E3 ubiquitin ligase with two mib/herc2 domains, zz zinc finger domain, two mib repeats, eight ankyrin repeat domains (AND) and three RING finger domains ( [9], Figure 1A). Mib in zebrafish can ubiquitylate and/or internalize ligands DeltaB, DeltaC, DeltaD, Jagged 1a, Jagged 1b and Jagged 2a [9][10][11][12]. This activity is essential for the Notch activation. Zebrafish mib mutants and Mib-deficient mice exhibit developmental defects in somites, neurons, pronephric duct, angiogenesis and heart due to a blockage in the Notch signaling pathway in early embryonic development [9,[12][13][14]. A paralog called Mind bomb 2 (Mib2) in zebrafish with similar protein structure to Mib but with only two RING finger domains ( Figure 1A) also has the ability to ubiquitylate the ligand DeltaC [10]. Both Mib and Mib2 are able to auto-ubiquitylate themselves and form homodimer or heterodimer with each other. Overexpression of mib2 in the zebrafish mib mutant can rescue the phenotypes, indicating a redundant role of Mib2 [15].
Ubiquitylation is a posttranslational modification with ubiquitin by the sequential action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligase (E3). Ubiquitin can be conjugated to substrates at many different sites and in many distinct topological configurations, which behaves as complex signals to regulate protein activity, localization and degradation [16][17]. This process is reversible and ubiquitin chain can be removed and edited by deubiquitylases (DUBs, also known as deubiquitinases, deubiquitylating enzymes or deubiquitinating enzymes). Therefore, the two-sided cooperation of ubiquitylation and deubiquitylation allows a precisely and immediately cellular response and control.
Studies have shown that both HIF activation and deregulated Notch signaling were linked to tumorigenesis and development [18][19][20]. Recent studies further emphasize the crosstalk between hypoxia and Notch signaling in cell differentiation [21][22]. Hypoxia blocks cell differentiation in a Notch-dependent manner through HIF-1a interaction with Notch intracellular domain and synergize to activate Notch downstream target genes [21].
The most well-known function of Mind bomb is its role in the Notch signaling pathway. However, it is not the only function of Mib. Mib has been reported to be involved in the apoptosis by interaction with death-associated protein kinase (DAPK) and cellular Fas-associated death domain (FADD)-like IL-1b converting enzyme (FLICE)-like inhibitory proteins (cFLIP) [23][24]. Mib also regulates the Wnt signaling pathway through the interaction with receptor-like tyrosine kinase (RYK) [25].
In this study, to further explore the function of Mib and Mib2, yeast two-hybrid screens were used to identify Mib/Mib2-interacting proteins. By this approach we isolated 81 putative Mib/Mib2-binding proteins and classified them into six groups: ubiquitin proteasome system, cytoskeleton, trafficking, replication/ transcription/translation, cell signaling and others. The potential roles of Mib and Notch activity in these pathways require further investigation. Additional analysis was carried out on several interesting candidates. Hif1an/Fih-1 (hypoxia-inducible factor 1, alpha subunit inhibitor/factor inhibiting HIF 1) and Trabid/ Zranb1 (tumor-necrosis-factor-receptor-associated factor-binding domain/zinc finger, RAN-binding domain containing 1) were shown to interact with and be ubiquitylated by Mib/Mib2 in COS7 cells. Usp1 and Usp9 can be co-immunoprecipitated by Mib/Mib2. These data suggest that Mib and Mib2 may be required for hypoxia-regulated differentiation, tumorigenesis and NF-kB pathway in addition to their prominent function in Notch signaling.

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].  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 inframe 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).
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 (b), 14-3-3 zeta (f) and 14-3-3 theta (h) 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.

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][30][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).

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 RFdependent 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.

Discussion
Yeast two-hybrid screen is a feasible approach to discover binding/interacting proteins and unexplored functions of Mib/ Mib2. 81 putative interacting proteins were identified in our yeast two-hybrid screens. Most of them are novel. The interaction of Usp1, Usp9, Trabid, Fih-1 and Mib/Mib2 were confirmed by immunoprecipitation. Notably, the interacting proteins identified from Mib were also co-immunoprecipitated with Mib2, and vice versa (Figures 2 and 3). We also demonstrated that Trabid and Fih-1 are substrates of Mib and Mib2. Our screens showed that Mib and Mib2 are likely to interact with proteins involved in different biological processes, including ubiquitin proteasome system, cytoskeleton, trafficking, replication/transcription/translation, cell signaling and others. It reveals the roles of Mib and Mib2 are versatile and not just limited to the Notch signaling pathway.
In these two screens, putative interacting proteins of Mib/Mib2 are classified according to their biological functions, but there is no identical clone detected from both screens. The reason is likely due to their different screen strategies. While in the first screen, the interaction of bait and prey occurred in the nucleus by reconstitution of binding and activating domains of transcription factors; in the second, it took place close to plasma membrane by restoration of a functional Ras signaling pathway. In addition, the baits for the first screen were Mib-C-RF123a and Mib2-C-RF12, which are not major binding domains [10]; and much more interacting proteins were identified by baits Mib-M-Ank and Mib-ZnF+Ank than Mib-C-RF123b in the second screen. Furthermore, the screening cDNA libraries are different. Last but not least, different degree of saturation in screen is another possibility  that cannot be ruled out. All of these parameters can affect the outcomes of these two yeast two-hybrid screens.

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 autoubiquitylat-ing 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)-a and interleukin (IL)-1 are proinflammatory cytokines that initiate signaling pathways in endothelial cells leading to activation of nuclear factor kB (NF-kB) and plays an important role in the regulation of immune and inflammatory responses [38][39]. NF-kB 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-kB activation.
Recently, Fih-1 was found that it can associate with and hydroxylate proteins containing AND, such as Notch receptor 1, 2 and 3 [43], IkBa [44], and Tankyrase-2, Rabankyrin-5 and RNase L [45]. At hypoxia, NICD enhances recruitment of HIF-1a to its target promoters and derepresses HIF-1a function by sequestering HIF-1a 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.
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-kB pathway through the interaction with B-cell CLL/lymphoma 10 (BCL10), inhibitor of nuclear factor kappa-B kinase subunit gamma (IKKc) and transforming growth factor b-activated kinase 1 (TAK1) [67]. In the past, the roles of Mib in the neurogenesis, gliogenesis, thyroid morphogenesis, T helper cell differentiation and pancreatic b-cell formation were focused on the Notchdependent pathway [71][72][73][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.

Materials and Methods
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 59 and 39 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 (CytoTrapH 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 (25uC), but prevents growth at 37uC. 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 37uC and 25uC. 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 37uC. 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 59 primer (59-ACTACTAGCAGCTGTAATAC-39) 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 PCRamplified fragments into the pCS2+FLAG vector [10]. Full-length fih-1 and partial trabid C (295-716 aa) cDNA fragments were PCRamplified 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. Cterminal usp1 C (349-772 aa) cDNA fragments were PCRamplified 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-HAubiquitin 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).
In vivo substrate ubiquitylation assay  harvested at 48 h and lysed in IONIC buffer and 10 mM MG132. The cell lysate was clarified by centrifugation and incubated with rabbit anti-Myc antibody (Santa Cruz) for 2 h at 4uC, and then incubated with protein A Sepharose beads for 1 h at 4uC. The eluted proteins were electrophoresed on a SDS-PAGE followed by Western blotting with mouse anti-HA (Roche) or mouse anti-Myc (Roche) antibody.