Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria
2011; Springer Nature; Volume: 31; Issue: 2 Linguagem: Inglês
10.1038/emboj.2011.397
ISSN1460-2075
AutoresTal Keren‐Kaplan, Ilan Attali, Khatereh Motamedchaboki, Brian A. Davis, Neta Tanner, Yael Reshef, Einat Laudon, Mikhail Kolot, Olga Levin‐Kravets, Oded Kleifeld, Michael H. Glickman, Bruce Horazdovsky, Dieter A Wolf, Gali Prag,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle11 November 2011free access Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria Tal Keren-Kaplan Tal Keren-Kaplan Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ilan Attali Ilan Attali Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Khatereh Motamedchaboki Khatereh Motamedchaboki Signal Transduction Program and NCI Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Brian A Davis Brian A Davis Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Neta Tanner Neta Tanner Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Yael Reshef Yael Reshef Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Einat Laudon Einat Laudon Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Mikhail Kolot Mikhail Kolot Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Olga Levin-Kravets Olga Levin-Kravets Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Oded Kleifeld Oded Kleifeld Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Michael Glickman Michael Glickman Department of Biology, Technion Israel Institute of Technology, Haifa, Israel Search for more papers by this author Bruce F Horazdovsky Bruce F Horazdovsky Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Dieter A Wolf Dieter A Wolf Signal Transduction Program and NCI Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Gali Prag Corresponding Author Gali Prag Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Tal Keren-Kaplan Tal Keren-Kaplan Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ilan Attali Ilan Attali Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Khatereh Motamedchaboki Khatereh Motamedchaboki Signal Transduction Program and NCI Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Brian A Davis Brian A Davis Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Neta Tanner Neta Tanner Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Yael Reshef Yael Reshef Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Einat Laudon Einat Laudon Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Mikhail Kolot Mikhail Kolot Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Olga Levin-Kravets Olga Levin-Kravets Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Oded Kleifeld Oded Kleifeld Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Michael Glickman Michael Glickman Department of Biology, Technion Israel Institute of Technology, Haifa, Israel Search for more papers by this author Bruce F Horazdovsky Bruce F Horazdovsky Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Dieter A Wolf Dieter A Wolf Signal Transduction Program and NCI Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Gali Prag Corresponding Author Gali Prag Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Author Information Tal Keren-Kaplan1, Ilan Attali1, Khatereh Motamedchaboki2, Brian A Davis3, Neta Tanner1, Yael Reshef1, Einat Laudon1, Mikhail Kolot1, Olga Levin-Kravets1, Oded Kleifeld4, Michael Glickman5, Bruce F Horazdovsky3, Dieter A Wolf2 and Gali Prag 1 1Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 2Signal Transduction Program and NCI Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA 3Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA 4Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia 5Department of Biology, Technion Israel Institute of Technology, Haifa, Israel *Corresponding author. Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: +972 3 640 9828; Fax: +972 3 640 6834; E-mail: [email protected] The EMBO Journal (2012)31:378-390https://doi.org/10.1038/emboj.2011.397 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings. Introduction Ubiquitin (Ub) is a key post-translational modifier in all eukaryotes. The process of ubiquitylation, in which Ub attaches to a lysine residue of a substrate protein, regulates the function of thousands of proteins and is involved in numerous cellular processes such as protein degradation, DNA remodelling and repair, and protein trafficking. Malfunctions in the Ub system underlie various human disorders, including cancer and neurodegenerative, metabolic and infectious diseases (Ciechanover and Brundin, 2003; Nalepa et al, 2006; Mizushima et al, 2008). Ubiquitylation involves the concerted action of Ub-activating, Ub-conjugating, and Ub-ligase enzymes (E1, E2 and E3, respectively); the latter conclude the process by promoting the covalent attachment of Ub to a lysine residue of a specific target protein (Hershko et al, 1980; Wilkinson et al, 1980; Bachmair et al, 1986; Li et al, 2008; Deshaies and Joazeiro, 2009). Some forms of ubiquitylation serve as canonical signals for rapid degradation by proteasomes and lysosomes, while others perform various signalling functions. Ubiquitylated proteins are recognized by a large set of cognate Ub receptors (Hicke et al, 2005; Hurley et al, 2006). These Ub receptors decrypt the Ub signal by tethering a Ub-binding domain (UBD) to a functional domain, thus linking the ubiquitylated target to a specific function in trans. Many Ub receptors undergo coupled monoubiquitylation themselves, presumably in order to impose a closed (cis) conformation that prevents binding of ubiquitylated targets (Di Fiore et al, 2003; Prag et al, 2003; Shih et al, 2003; Hoeller et al, 2006). Deubiquitylating enzymes (DUBs) are able to rapidly reverse this auto-regulatory signal by opening the cis conformation. Hence, the current models suggest that Ub receptors may exist in three states: (i) in the free, unmodified apo form, (ii) in the ubiquitylated and closed cis form, which is inactive due to intramolecular Ub–UBD interactions and (iii) in the trans form, which binds ubiquitylated target proteins through the UBD. The rapid dynamics of ubiquitylation/deubiquitylation has impeded the structure–function analysis of Ub receptors. Indeed, of the >100 UBD structures in the Protein Data Bank (PDB; January 2011), none is of a ubiquitylated cis form. To bypass this obstacle, we reconstructed the entire eukaryotic ubiquitylation system in Escherichia coli. The system is built modularly, facilitating the straightforward production of ubiquitylated proteins. Two affinity tags, one on Ub and the other on the substrate, enable the ubiquitylated proteins to be purified in quantities and homogeneity suitable for biochemical, biophysical and crystallographic analyses. This system will facilitate the determination of the structures of ubiquitylated proteins, and will enable high-resolution insight into cellular mechanisms for interpreting Ub signals. Moreover, permutations of the system can be devised to screen for targets of E3 enzymes and UBDs or to study in detail the ubiquitylation of human disease proteins, including the facile identification of their modified residues. In this work, we demonstrate the utility of this expression system by reporting several new biological findings. First, we demonstrate E2-specific auto-ubiquitylation of the E3 enzyme, Mind bomb. Next, we show that Epsin proteins undergo E3-independent ubiquitylation. Finally, we focus on the ubiquitylation of the yeast Ub receptor Vps9, in which a UBD is tethered to a guanine nucleotide exchanging factor (GEF) domain. We characterize in vitro the GEF activity and Ub-binding functions of this Ub receptor in its apo, trans and cis forms. A cycle of Ub binding and monoubiquitylation was postulated to regulates the association of the human orthologue of Vps9, Rabex-5, with endosomes (Mattera and Bonifacino, 2008). Our study provides a direct explanation for the regulatory function of Ub in controlling the cellular localization of Vps9. Results Construction of an integrated recombinant ubiquitylation system in bacteria We developed a ubiquitylation system in bacteria that permits the purification of stably modified ubiquitylated proteins. The system consists of two compatible expression vectors. One is a generic plasmid (pGEN) that harbours His6–Ub, E1-activating enzyme and E2-conjugating enzyme (Figure 1). We cloned these genes into a modified pHis6-parallel2 vector in which the β-lactamase gene was replaced with a kanamycin resistance cassette. A second plasmid (pCOG) encodes a selected substrate for ubiquitylation and its cognate E3 ligase. In this vector, the substrate is typically fused to glutathione S-transferase (GST) or to maltose-binding protein (MBP) affinity tags. We constructed each type of vector such that it expressed its corresponding genes from a single promoter (pT7 or pTac), giving rise to a polycistronic mRNA. We produced a large collection of pGEN and pCOG vectors expressing different E2s, E3s, substrates, and their mutants (Supplementary Table SIV). Co-expression of the proteins encoded by these vectors yields large quantities of ubiquitylated substrates (0.5–1.0 mg of purified ubiquitylated Vps9 or Rpn10 per 1 l culture). Affinity tags such as His6 fused to Ub, and MBP or GST fused to the substrate facilitate separation of the ubiquitylated from the unmodified substrate (as demonstrated for Rpn10 and Vps9, respectively). Moreover, tobacco etch virus (TEV) protease or rhinovirus protease recognition sites located between the tags and the ubiquitylated substrate enable tag removal, which, upon further affinity chromatography, facilitates the separation of the ubiquitylated product from the non-modified form. Using this method, the ubiquitylated protein is highly purified and ready for biochemical and biophysical studies. Figure 1.Bacterial system for expression and purification of ubiquitylated proteins. A scheme describing the ubiquitylation system and the steps for purifying ubiquitylated proteins from E. coli (see also Supplementary Table SIV). Download figure Download PowerPoint To assess the functionality of the system, we first produced pGEN vectors co-expressing Ub and wheat Uba1 with either human UbcH5b or yeast Ubc5, the most promiscuous E2s with regards to association with E3s. Since most E3 ligases undergo auto-ubiquitylation in the absence of a specific substrate (Lorick et al, 1999), we simplified the system by first testing whether E3 ligases would undergo auto-ubiquitylation when expressed in bacteria from pCOG. E3 ligases fall into two large groups, U-Box/RING (really interesting new gene) and HECT (homologous to E6-AP carboxyl terminus) domain enzymes (Lorick et al, 1999; Hatakeyama et al, 2001; Hagglund and Roizman, 2002). To assess the general applicability of the reconstituted system, we first asked whether RING- and HECT-domain E3 ligases undergo auto-ubiquitylation in bacteria (Figure 2). Figure 2.Reconstituted E3 auto-ubiquitylation activity in bacteria. Auto-ubiquitylation of the RING and HECT E3-ligase protein families is shown. (A–C) Bacterial lysates co-expressing of His6–Ub (or the indicated mutants), Uba1, UbcH5b (pGEN1), and GST–Mib (pCOG9) were purified on Ni+2 and/or GSH beads as indicated. (A) On the left, a Coomassie blue-stained SDS–PAGE of auto-ubiquitylated D. rerio (zebrafish) Mib, a representative RING-containing E3s ligase is shown. Wild-type Ub (lane 1 from the left), Ub K48R, K63R double mutant (lane 2), Ub K0 (lane 3) or without pGEN (lane 4). On the right, anti-His tag western blot of GSH purified Mib is shown, without or with pGEN1 (lanes 1 and 2, respectively). (B, C) Ubiquitylated Mib was purified on GSH (uncut), then GST tag was cleaved and the sample was bound to GSH beads. Flow through (FT) and elution (Elution) were analysed using western blot with anti-GST tag (B) or with anti-His tag (C). (D) Scheme of Mib ubiquitylation sites detected by mass spectrometry (see Supplementary Figure S1 and Supplementary Table SI). (E–G) Auto-ubiquitylation of yeast Rsp5, a representative HECT-containing E3s. Bacterial lysates co-expressing of His6–Ub, Uba1, Ubc5 (pGEN5), and MBP–Rsp5 WT (pCOG3) or C777A mutant (pCOG7) were purified on amylose beads. (E) Input expression levels are shown. (F) Coomassie blue-stained SDS–PAGE of bound fraction is shown. (G) Western blot using anti-His tag of bound fraction is shown. Download figure Download PowerPoint The RING-containing E3 Mind bomb (Mib) of Danio rerio (zebrafish), which is known to promote endocytosis in the Delta/Notch pathway (Itoh et al, 2003), was chosen as a representative protein. A GST–Mib construct containing its three RING domains (a.a. 738–1030) was co-expressed with His6–Ub, E1 and UbcH5b, sequentially purified on reduced glutathione- and Ni+2-affinity matrices and resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified protein displayed a laddering pattern that entirely depended on the co-expression of Ub, indicating that Mib underwent multi-monoubiquitylation or polyubiquitylation (Figure 2A). A similar pattern was observed by western blot analysis with an anti-His antibody recognizing the His6–Ub fusion (Figure 2A). The ubiquitylation pattern of Mib observed here was consistent with the pattern previously found to occur in vitro (Itoh et al, 2003), confirming the reliability of the system. We next tested whether the ubiquitylation signal on Mib is K48- or K63-polyUb, the most abundant polyUb modifications (Peng et al, 2003). We found that replacing the wild-type Ub with a point mutant in which lysines 48 and 63 were replaced by arginine residues (K48R, K63R double mutant) did not affect the ubiquitylation pattern, suggesting that Mib undergoes either multi-monoubiquitylation or polyubiquitylation through lysines other than the K48 or K63. We then used Ub K0 (in which all Lys residues were mutated to Arg). The ladder pattern was retained, demonstrating that Mib is multi-monoubiquitylated in the bacterial system. Using mass spectrometry analysis, we identified five distinct lysine residues that underwent ubiquitylation (Figure 2D; Supplementary Table SI; Supplementary Figure S1). Since Mib is expressed as GST fusion, we tested whether ubiquitylation takes place on Mib or on the GST moiety. The linker between the fusion partners was cleaved and the proteins were separated by chromatography on a GSH-affinity matrix. As shown in Figure 2B and C, the flow through (FT) contained heavily ubiquitylated Mib. Using anti-GST antibody, we found no evidence for ubiquitylation of GST (Figure 2B, Elution). However, blotting with anti-His antibody detected a faint band at ∼39 kDa which may represent a minor fraction of monoubiquitylated GST (see Elution in Figure 2C). Thus, the majority of the Ub modification was attached to Mib not to GST. To determine whether HECT-containing E3 ligases are also active in the reconstituted ubiquitylation system, we cloned a representative HECT protein, yeast Rsp5, as MBP fusion, into pCOG. We found that, like Mib, Rsp5 underwent auto-ubiquitylation when co-expressed with its cognate E2s yeast Ubc4 or Ubc5 (Figure 2E–G). HECT proteins contain a catalytic cysteine that transfers the Ub to the substrate. When we substituted the predicted catalytic cysteine by alanine (C777A), the ligase failed to charge with Ub (Figure 2G), although the expression levels of ubiquitylation components were equal in both bacterial lysates (Figure 2E; Supplementary Figure S4). Interestingly, the level of Ub-charged E2 was very low apparently because Ub is rapidly transferred to the E3 ligase (Figure 2G). However, in the presence of catalytically inactive Rsp5, accumulation of Ub-charged E2 was apparent (compare WT and C777A lanes in Figure 2F). Nevertheless, Ub-charged E3 did not accumulate when a substrate such as Rpn10 was added to the reconstituted system, ostensibly because of rapid Ub transfer to the substrate (Supplementary Figure S4), since Ub was transferred to it. Taken together, this series of experiments demonstrates our successful reconstitution of the entire ubiquitylation cascade for RING and HECT E3 ligases in E. coli. The reconstituted system faithfully recapitulates Rpn10 ubiquitylation It has recently been shown that Rpn10 (regulatory proteasomal non-ATPase subunit) is ubiquitylated by Rsp5 in vivo at a single lysine residue (K84) (Isasa et al, 2010). However, in-vitro assays performed by the same team and others showed that Rpn10 and its human orthologue S5a undergo multi-monoubiquitylation or polyubiquitylation (Lu et al, 2008; Uchiki et al, 2009; Isasa et al, 2010). To test whether, unlike an in-vitro ubiquitylation assay, the reconstituted bacterial system would faithfully recapitulate Rpn10 ubiquitylation observed in S. cerevisiae in vivo, we studied the dependence of Rpn10 ubiquitylation on the concerted action of E1, E2 and E3 enzymes. As shown in Figure 3A, co-expression of His6–Ub, Uba1, Ubc5 and Rsp5 was necessary and sufficient for Rpn10 monoubiquitylation. To assess the requirement of individual components, we systematically omitted each of the enzymes and the substrate (Figure 3A and B; for typical expression levels of E1, E2 and E3, see Supplementary Figure S4). As expected, Rpn10 monoubiquitylation was only observed when all components were present. Applying the scheme depicted in Figure 1, we purified ubiquitylated Rpn10. Sequential purification via the two affinity tags, MBP on Rpn10 and His6 on Ub, efficiently isolated an apparently homogenous species corresponding to monoubiquitylated Rpn10 (Figure 3C). Size-exclusion chromatography upon enzymatic removal of the MBP and the His6 tags resulted in a highly symmetric peak at the elution volume expected for correctly folded Ub-Rpn10 (Figure 3C). The yield of pure Ub-Rpn10 obtained from 1 L of bacterial culture was ∼1.0 mg. Figure 3.Purification of K84-monoubiquitylated Rpn10. (A, B) Rpn10 ubiquitylation in bacteria is shown. Bacterial lysates co-expressing of His6–Ub, Uba1, yeast Ubc5 (expressed from pGEN5) and GST–Rpn10 and MBP–Rsp5 (expressed from pCOG5) were purified on GSH beads. Systematic deletions of the ubiquitylation enzymes (−E1, −E2 and −E3) show that all are required for ubiquitylation. Only when all components are co-expressed, ubiquitylation is observed (All). (A) Coomassie blue-stained SDS–PAGE of GSH beads bound fraction is shown. Input expression levels of His6—Ub, MBP–Rsp5 and GST–Rpn10 are shown below. Typical expression levels of E1 and E2 are shown in Supplementary Figure S4. (B) Western blot analysis of same samples shown in (A) using anti-His tag antibody is shown. (C) Coomassie blue-stained SDS–PAGE of the purification steps of ubiquitylated Rpn10 is shown. Bacterial lysates co-expressing pGEN24, Rsp5 and MBP–Rpn10 expressed from pCOG30 and pCOG31, respectively, were purified on amylose-affinity matrix (amylose), followed by Ni+2 beads (Ni+2). The apo Rpn10 was removed by wash (FT-Ni+2), Rpn10 was eluted (elution); His6 and MBP tags were cleaved and removed (cleaved). The UV280 absorbance chromatogram output of the size exclusion column (SEC) is shown (right of the gel). (D) A scheme of Rpn10 ubiquitylation site detected by mass spectrometry (see Supplementary Figure S2 and Supplementary Table SII) is shown. Download figure Download PowerPoint Using an in-vitro ubiquitylation assay followed by mass spectroscopy analysis, Crosas and co-workers identified four lysine residues in Rpn10 that were modified by Ub (K71, K99, K84 and K268), although K84 was found as the major site (52%). Using tandem mass spectrometry, we only identified K84 as ubiquitylated in Ub-Rpn10 produced in the bacterial system (Figure 3D; Supplementary Figure S2; Supplementary Table SII). Although the mass spectrometry on an LTQ-Orbitrap instrument employed here is exquisitely sensitive, we cannot entirely exclude the possibility of minor modifications of other lysine residues in Rpn10. Rpn10 contains a Ub interaction motif (UIM), a domain that in some cases can mediate the ubiquitylation of a fusion partner such as GST (Oldham et al, 2002). Our mass spectrometry data revealed a similar phenomenon as we identified ubiquitylation of MBP within the context of the MBP–Rpn10 fusion protein (Supplementary Table SII). This finding corroborates the suggestion that UIMs can function to direct protein ubiquitylation. Specificity and fidelity of the bacterial reconstituted ubiquitylation system In the ubiquitylation process, particular protein–protein interactions govern several levels of specificity, including the interactions of E1:Ub-like protein (UbL), E1:E2, E2:E3 and E3:substrate. For example, the E1 Ub-activating enzyme (Uba1) associates with Ub but not with the UbL SUMO; whereas the E1-like enzyme of the autophagy system, ATG7 cooperates with two different UbLs, ATG12 and LC3 (Schulman and Harper, 2009). E. coli possesses at least two UbLs that serve as sulphur donors in the thiamine (vitamin B1) and the molybdenum cofactor biosynthesis pathways (Gutzke et al, 2001; Lake et al, 2001; Wang et al, 2001). We, therefore, asked whether bacterial E1-like enzymes interact with the eukaryotic factors in the context of the reconstituted system. The lack of Rpn10 ubiquitylation in the absence of Uba1 (Figure 3A) suggests that, assuming their constitutive expression under our culture conditions, E. coli E1-like proteins do not participate in reactions observed with the reconstituted ubiquitylation system. We demonstrated the E2:E3 specificity of the system by showing that Mib auto-ubiquitylation activity occurred in conjunction with UbcH5b, a cognate E2 (Itoh et al, 2003), but not in conjunction with Cdc34, a non-cognate E2, known to be involved in cell-cycle control (Goebl et al, 1988; Figure 4A and B). The functionality of Cdc34 is supported by the formation of free polyUb chains (Gazdoiu et al, 2005). Similar E2 specificity was found for the Rsp5-dependent ubiquitylation of Rpn10, which was clearly observed with the cognate E2 Ubc4 (Huibregtse et al, 1995), but barely with Cdc34 (Figure 4C and D). This indicates that Rps5 can associate with Cdc34, but with a significant lower efficiency compared with its cognate E2, Ubc4. Figure 4.Specificity and fidelity of the ubiquitylation system in the bacteria. Specificity to cognate E2s by RING-containing (A and B) and by HECT-containing (C and D) E3 ligases is shown. (A, B) Bacterial lysates co-expressing GST–Mib (pCOG9) and His6–Ub, Uba1, and UbcH5b or Cdc34 (pGEN1 or pGEN8, respectively), were purified on Ni+2 and/or GSH beads as indicated. The purified proteins were resolved on SDS–PAGE and subjected to western blot analysis with anti-His tag or anti-GST antibodies as indicated. (A) Shows that Mib is ubiquitylated by UbcH5b as indicated in both purifications (Ni2+ and GSH). Free polyUb chains are shown in the Cdc34 lane that was purified on Ni2+, but not seen in the GSH purification. (B) The ubiquitylation of Mib by UbcH5b; Cdc34 does not ubiquitylates Mib (only apo Mib was detected) in the GSH purification is shown. (C, D) Bacterial lysates co-expressing MBP–Rsp5 and GST–Rpn10 (pCOG5) and His6–Ub, Uba1, and Ubc4 or Cdc34 (pGEN4 or pGEN8, respectively), were purified on Ni+2 beads. The purified proteins were resolved on SDS–PAGE and subjected to western blot with anti-His tag or anti-GST antibodies as indicated. (C) Shows that Rpn10 is ubiquitylated by Rsp5 in the presence of Ubc4. Free polyUb chains are shown in the Cdc34 lane blotted with anti-His. (D) Rsp5-dependent ubiquitylation of Rpn10 is clearly evident in the Ubc4 lane. In the Cdc34 lane, a faint band of ubiquitylated Rpn10 is seen. (E) E3:substrate specificity is shown. Rsp5-dependent ubiquitylation of Cps1 (N-terminal residues PVEKAPR) fused to GST (lane 1) versus GST alone (lane 2). (F–H) Fidelity of the polyUb linkage is shown in free polyUb chains. Bacterial lysates co-expressing Ub, Uba1 and the specified E2s were subjected to western blot with the indicated antibodies. (F, G) Formation of K48-polyUb by Cdc34 or E2-25K, respectively and (H) the formation of K63-polyUb by UBC13/UEV1a are shown. (I, J) Coomassie blue-stained SDS–PAGE of fractions derived from size-exclusion chromatography of purified K48 and K63 diUb produced in the bacterial system is shown; proteins were purified using the Pickart's protocol (Pickart and Raasi, 2005) followed by gel filtration. Download figure Download PowerPoint To examine E3:substrate specificity, we used the yeast biosynthetic multi-vesicular body (MVB) cargo, Carboxypeptidase-S (Cps1p), as the substrate. Cps1p is a type II transmembrane protein in which a short N-terminal tail is facing the cytosol. Monoubiquitylation of the cytosolic tail was demonstrated to serve as crucial signal for its MVB sorting (Katzmann et al, 2
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