Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade1, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents2. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases3 or as enzymes that directly attack ubiquitin4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes5. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.
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Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012)
Zinngrebe, J., Montinaro, A., Peltzer, N. & Walczak, H. Ubiquitin in the immune system. EMBO Rep. 15, 28–45 (2014)
Zhou, Y. & Zhu, Y. Diversity of bacterial manipulation of the host ubiquitin pathways. Cell. Microbiol. 17, 26–34 (2015)
Cui, J. et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010)
Xu, L. & Luo, Z. Q. Cell biology of infection by Legionella pneumophila . Microbes Infect. 15, 157–167 (2013)
Luo, Z. Q. & Isberg, R. R. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl Acad. Sci. USA 101, 841–846 (2004)
Huang, L. et al. The E Block motif is associated with Legionella pneumophila translocated substrates. Cell. Microbiol. 13, 227–245 (2011)
Lifshitz, Z. et al. Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal. Proc. Natl Acad. Sci. USA 110, E707–E715 (2013)
Fontana, M. F. et al. Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila . PLoS Pathog. 7, e1001289 (2011)
Choy, A. et al. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 (2012)
Simon, S., Wagner, M. A., Rothmeier, E., Muller-Taubenberger, A. & Hilbi, H. Icm/Dot-dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator. Cell. Microbiol. 16, 977–992 (2014)
Rolando, M. et al. Legionella pneumophila effector RomA uniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication. Cell Host Microbe 13, 395–405 (2013)
Hsu, F. et al. Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase. Proc. Natl Acad. Sci. USA 109, 13567–13572 (2012)
Zhu, W. & Luo, Z. Q. Cell biology and immunology lessons taught by Legionella pneumophila. Sci. China Life Sci. 59, 3–10 (2016)
Bardill, J. P., Miller, J. L. & Vogel, J. P. IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Mol. Microbiol. 56, 90–103 (2005)
Sakurai, J., Nagahama, M., Oda, M., Tsuge, H. & Kobayashi, K. Clostridium perfringens iota-toxin: structure and function. Toxins (Basel) 1, 208–228 (2009)
Wilde, C. & Aktories, K. The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases. Toxicon 39, 1647–1660 (2001)
Ganesan, A. K., Frank, D. W., Misra, R. P., Schmidt, G. & Barbieri, J. T. Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites. J. Biol. Chem. 273, 7332–7337 (1998)
Simon, N. C., Aktories, K. & Barbieri, J. T. Novel bacterial ADP-ribosylating toxins: structure and function. Nature Rev. Microbiol. 12, 599–611 (2014)
Havey, J. C. & Roy, C. R. Toxicity and SidJ-mediated suppression of toxicity require distinct regions in the SidE family of Legionella pneumophila effectors. Infect. Immun. 83, 3506–3514 (2015)
Jeong, K. C., Sexton, J. A. & Vogel, J. P. Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ. PLoS Pathog. 11, e1004695 (2015)
Tan, Y., Arnold, R. J. & Luo, Z. Q. Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination. Proc. Natl Acad. Sci. USA 108, 21212–21217 (2011)
Swanson, M. S. & Isberg, R. R. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63, 3609–3620 (1995)
Liu, Y. & Luo, Z. Q. The Legionella pneumophila effector SidJ is required for efficient recruitment of endoplasmic reticulum proteins to the bacterial phagosome. Infect. Immun. 75, 592–603 (2007)
Sherwood, R. K. & Roy, C. R. A. Rab-centric perspective of bacterial pathogen-occupied vacuoles. Cell Host Microbe 14, 256–268 (2013)
Ortiz Sandoval, C. & Simmen, T. Rab proteins of the endoplasmic reticulum: functions and interactors. Biochem. Soc. Trans. 40, 1426–1432 (2012)
Itoh, T. et al. Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation. Mol. Biol. Cell 19, 2916–2925 (2008)
Sheedlo, M. J. et al. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc. Natl Acad. Sci. USA 112, 15090–15095 (2015)
Herhaus, L. & Dikic, I. Expanding the ubiquitin code through post-translational modification. EMBO Rep. 16, 1071–1083 (2015)
Glowacki, G. et al. The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse. Protein Sci. 11, 1657–1670 (2002)
Berger, K. H. & Isberg, R. R. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila . Mol. Microbiol. 7, 7–19 (1993)
Xu, L. et al. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6, e1000822 (2010)
Tilney, L. G., Harb, O. S., Connelly, P. S., Robinson, C. G. & Roy, C. R. How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114, 4637–4650 (2001)
Li, Z., Solomon, J. M. & Isberg, R. R. Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole. Cell. Microbiol. 7, 431–442 (2005)
Pan, X., Luhrmann, A., Satoh, A., Laskowski-Arce, M. A. & Roy, C. R. Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors. Science 320, 1651–1654 (2008)
Fan, H. Y., Cheng, K. K. & Klein, H. L. Mutations in the RNA polymerase II transcription machinery suppress the hyperrecombination mutant Hpr1Δ of Saccharomyces cerevisiae . Genetics 142, 749–759 (1996)
Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360 (1995)
Fazzio, T. G. & Tsukiyama, T. Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism. Mol. Cell 12, 1333–1340 (2003)
Duménil, G. & Isberg, R. R. The Legionella pneumophila IcmR protein exhibits chaperone activity for IcmQ by preventing its participation in high-molecular-weight complexes. Mol. Microbiol. 40, 1113–1127 (2001)
Conover, G. M., Derre, I., Vogel, J. P. & Isberg, R. R. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol. Microbiol. 48, 305–321 (2003)
We thank P. Hollenbeck (Purdue University) for critical reading of the manuscript. J. Barbieri (Medical College of Wisconsin) for plasmids. This work was supported by National Institutes of Health grants R56AI103168, K02AI085403 and R21AI105714 (Z.-Q.L.), 2R01GM103401 (C.D.) and National Natural Science Foundation of China grants 21305006 and 21475005 (X.L.).
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Inhibition of the secretion of SEAP by SidE, SdeB and SdeC and the recruitment of an ER marker by the L. pneumophila mutant lacking the SidE family.
a, GFP fusions of the indicated proteins were co-expressed with SEAP in 293T cells for 24 h. The SEAP index was determined by measuring alkaline phosphatase activity in culture supernatant or in cells. Similar results were obtained in three independent experiments, and data shown are from one representative experiment done in triplicate. Note that mutations in the putative mART motif abolished the inhibitory effects. Error bars represent s.e.m. (n = 3). b, Quantitation of the vacuoles positive for GFP–HDEL. The indicated bacterial strains were used to infect a line of D. discoideum stably expressing GFP fusion to the ER retention signal HDEL and the recruitment of the GFP–HDEL signal to the phagosome was evaluated 10 h after infection. At least 150 phagosomes were scored in each sample done in triplicate. Results shown are from one representative experiment done in triplicate and similar results were obtained from three independent experiments. Error bars represent s.e.m. (n = 3). c, Representative images of L. pneumophila phagosomes associated with GFP–HDEL. Images are from one representative of three independent experiments with similar results. Scale bar, 5 μm.
Extended Data Figure 2 SdeA does not ADP-ribosylate mammalian proteins, the modification of Rab33b by other members of the SidE family and SdeA-mediated post-translational modification of Rab1 during bacterial infection.
a, SdeA, SdeAE/A or ExoS and 5 μCi 32P-NAD were added to 100 μg total protein of 293T cells. After incubation at 22 °C for 1 h, samples were separated by SDS–PAGE. Gels were stained with Coomassie brilliant blue (left panel) and then by autoradiography for the indicated time duration (middle and right panels). In samples receiving SdeA, no ADP-ribosylation signal was detected in many experiments performed in various reaction conditions. Lane 1: 32P-α-NAD + SdeA + 293T lysates; lane 2: 32P-α-NAD + SdeAE/A + 293T lysates; lane 3: no sample; lane 4: 32P-α-NAD + ExoS78-453 + FAS + 293T lysates. b, Flag-tagged Rab33b was co-expressed with GFP-tagged testing proteins in 293T cells for 24 h. Cell lysates were subjected to immunoprecipitation with Flag beads and the precipitated products were probed with the Flag antibody (right panel). 5% of each lysate was probed for the expression of Rab33b (left panel) or for GFP fusions (middle panel). Proteins used: 1, GFP; 2, GFP–SdeB1-1751; 3, GFP–SdeC; 4, GFP–SidE. c, 293T cells transfected to express Flag–Rab1 were infected with the indicated L. pneumophila strains for 2 h and the Rab1 enriched by immunoprecipitation was probed by immunoblotting. For all panels, similar results were obtained from three experiments. a–c, Uncropped blots and autoradiograph images are shown in Supplementary Fig. 1.
Extended Data Figure 3 The extracted ion chromatograms of ubiquitin tryptic fragments detected by mass spectrometry, expression of Rab33b and its mutants in COS1 cells, and in vitro ubiquitination of Rab33b by SdeA with E1 and a series of E2 proteins.
a, Proteins in bands corresponding to normal (upper panel) or shifted (lower panel) Rab33b were digested with trypsin and the resulting protein fragments were identified by mass spectrometry. Note that the ubiquitin tryptic fragments are present only in the shifted band of higher molecular mass. b, COS1 cells were transfected with GFP or GFP fusion of Rab33b or its mutants for 14 h. Total cell lysates resolved by SDS–PAGE were probed with a GFP-specific antibody. Tubulin was detected as a loading control. c, Reactions containing E1 and the indicated E2 proteins were allowed to proceed at 37 °C for 2 h. Proteins in the reactions were resolved by SDS–PAGE followed by immunoblotting to detect ubiquitinated proteins with higher molecular mass (left panel). SdeA in the reaction was detected with specific antibodies by using 10% of the reactions (lower panel). Control reactions with wild-type Legionella E3 ligase SidC1-542 and its enzymatically inactive mutant SidC1-542C46A with E1 and the E2 UbcH7 were established to monitor the activity of E1 (right panel). Note the robust self-ubiquitination of SidC1-542 (second lane right panel). Results in a are representative of three experiments with similar results; b and c are a representative of two and five independent experiments, respectively. b, c, Uncropped blots are shown in Supplementary Fig. 1.
a, SdeA or SdeAE/A dialysed against a buffer containing 10 mM EDTA was used for in vitro ubiquitination of Rab33b. Reactions were allowed to proceed for 2 h at 37 °C. Samples resolved by SDS–PAGE were detected by Coomassie staining (upper panel), by immunoblotting with antibodies specific for ubiquitin (middle panel) or for the Flag tag (lower panel). Note that the addition of exogenous NAD is sufficient to allow SdeA-mediated ubiquitination of Rab33b (lane 2). b, In vitro ubiquitination of Rabs by SdeA. Reactions containing indicated proteins and NAD were allowed to proceed for 2 h at 37 °C. After SDS–PAGE, ubiquitinated proteins were detected by staining 50% of the reactions resolved by SDS–PAGE with Coomassie (upper panel) or by immunoblotting with antibodies specific for ubiquitin (lower panel). Similar results were obtained from two experiments. c, In vitro ubiquitination of Rab33b by SidE, SdeB1-1751 and SdeC. Indicated testing proteins were incubated with NAD, ubiquitin and Flag–Rab33b for 2 h at 37 °C. Proteins resolved by SDS–PAGE were detected by antibodies specific for Flag (upper panel) or for ubiquitin (middle panel). His6-tagged SdeA, SdeB1-1751 and SdeC and SdeAE/A used in the reactions were probed 10% of the proteins with an antibody against His (lower panel). Similar results were obtained from two independent experiments. a–c, Uncropped blots are shown in Supplementary Fig. 1.
Extended Data Figure 5 SdeA does not detectably ADP-ribosylate Rab33b or Rab1 and the deubiquitinase (DUB) activity of SdeA does not interfere with its ubiquitin-conjugation activity.
a, 5 μg of SdeA or SdeAE/A were incubated with 5 μg of GST–Rab1, 4×Flag–Rab33b and 5 μCi of 32P-α-NAD. A reaction containing 200 ng of ExoS78-453, 2 μg of FAS and 5 μg Rab5 was established as a positive control. All reactions were allowed to proceed for 1 h at 22 °C before being terminated by adding 5 × SDS loading buffer. Samples resolved by SDS–PAGE were detected by Coomassie staining (upper panel) and then by autoradiography (middle and lower panels). Lane 1: 32P-α-NAD + SdeA + GST–Rab1; lane 2: 32P-α-NAD + SdeAE/A + GST–Rab1; lane 3: 32P-α-NAD + SdeA + 4×Flag–Rab33b; lane 4: 32P-α-NAD + SdeAE/A + 4×Flag–Rab33b; lane 5: no sample; lane 6: 32P-α-NAD + ExoS78-453 + FAS + Rab5. Note the strong ADP-ribosylation signals in the reaction with ExoS78-453 (lane 6). b, SdeA, its mutants SdeAC118A or SdeAC118AE/A was used for in vitro NAD-dependent ubiquitination of Rab33b. Reactions containing the indicated components were allowed to proceed for 2 h at 37 °C before being terminated with SDS sample buffer. Samples resolved by SDS–PAGE were probed by Coomassie staining (upper panel) or by immunoblotting with antibody specific for ubiquitin (middle panel) or for the Flag tag (lower panel). c, Reactions containing GST–ubiquitin were similarly established to detect self-ubiquitination by SdeA. Note that SdeA and SdeAC118A exhibited similar activity in these reactions. Data in all panels are one representative of two independent experiments with similar results. a–c, Uncropped blots and autoradiograph images are shown in Supplementary Fig. 1.
a, Arg42 in ubiquitin is important for SdeA-mediated ubiquitination. Ubiquitin or ubiquitinR42A was included in reactions catalysed by SdeA or the bacterial E3 ubiquitin ligase SidC (E1 and the E2 UbcH7 were added in the latter category of reactions). After allowing the reaction to proceed for 2 h at 37 °C. Samples separated by SDS–PAGE were probed with antibody against the Flag tag (on Rab33b) (middle panel) or ubiquitin (right panel). Note that ubiquitinR42A can be used by ubiquitination catalysed by SidC but not SdeA. b, GST–ubiquitinR42A cannot be used for self-ubiquitination by SdeA. GST–ubiquitin or GST–ubiquitinR42A was used in reactions with SdeA or SdeAE/A. Self-modification was detected by the shift of SdeA detected by Coomassie staining (left panel) or by immunoblotting with a GST-specific antibody (right panel). c, The lysine residues or the carboxyl terminus of ubiquitin is not important for SdeA-catalysed Rab33b ubiquitination. Reactions containing SdeA or SdeAE/A, NAD, Flag-Rab33b and the indicated ubiquitin mutants were allowed to proceed for 2 h at 37 °C. Proteins were detected by Coomassie staining (upper panel) or probed by immunoblotting with antibody against ubiquitin. d, Utilization of the ubiquitin di-glycine mutant by different ligases. Reactions with indicated components were allowed to proceed for 2 h at 37 °C. Proteins resolved by SDS–PAGE were detected by staining (upper panel) or by immunoblotting with antibodies specific to ubiquitin (lower panel). Note that the wild type but not the di-glycine ubiquitin mutant (AA) can be conjugated to proteins in a reaction containing E1 and E2 and the bacterial E3 ligase SidC (Lanes 6 and 7). This di-glycine mutant (AA) can still be attached to Rab33b by SdeA (Lane 4). e, Addition of 6 histidine residues to the carboxyl end of ubiquitin did not affect SdeA-mediated ubiquitination. Reactions containing the indicated components were established and allowed to proceed for 2 h at 37 °C. SDS–PAGE resolved samples were probed by Coomassie staining (left panel) or by immunoblotting with a GST-specific antibody (right panel). The data in all panels are one representative of three independent experiments with similar results. a–e, Uncropped blots are shown in Supplementary Fig. 1.
Extended Data Figure 7 Ubiquitination catalysed by SdeA is insensitive to the cysteine modifying agent maleimide.
a, Ubiquitination reactions by SdeA or SidC together with E1 and E2 were established; maleimide was added to 50 μM to a subset of these reactions. After incubation at 37 °C for 2 h, ubiquitination was detected by Coomassie staining (left panel) or by immunoblotting with the Flag- (middle panel) or ubiquitin-specific (right) antibody. Note that maleimide completely inhibits ubiquitination in the reaction catalysed by SidC, E1 and its cognate and E2 (lane 6) but does not affect the activity of SdeA (lane 4). b, Maleimide does not affect self-ubiquitination of SdeA. Reactions containing the indicated components were established and the modification of SdeA was probed by Coomassie staining (left panel) or by immunoblotting with the GST-specific antibody (right panel). For all panels, similar results were obtained from four independent experiments. a, b, Uncropped blots are shown in Supplementary Fig. 1.
Extended Data Figure 8 SdeA-mediated ubiquitination affects the activity but not stability of Rab33b and SdeA ubiquitinates Rab33b independently of its nucleotide binding status.
a, Evaluation of the ubiquitinated Rab33b. 4×Flag–Rab33b was loaded with unlabelled GDP (5 mM) before ubiquitination reaction. GDP-loaded Rab33b was subjected to ubiquitination by SdeA or SdeAE/A for 2 h at 37 °C; 20% of the samples were withdrawn to determine the extent of ubiquitination by Coomassie staining. b, Ubiquitination affected the GTP loading activity of Rab33b. Ubiquitinated or non-ubiquitinated 4×Flag–Rab33b was incubated in 50 μl nucleotide exchange buffer containing 5 μCi 35SγGTP at 22 °C. Aliquots of reactions were withdrawn at indicated time points and passed through nitrocellulose membrane filters. Membranes were washed for three times using exchange buffer before being transferred into scintillation vials containing scintillation fluid to detect incorporated 35SγGTP with a scintillation counter. c, Ubiquitination affected the GTPase activity of Rab33b. Samples withdrawn from Ub~Rab33b or Rab33b loaded with 32PγGTP were measured for the associated radioactivity to set as the starting point. Equal volumes of samples were withdrawn at the indicated time points to monitor intrinsic GTP hydrolysis. The GTP hydrolysis index was calculated by dividing the readings obtained in later time points by the values of the starting point. Similar results (a–c) were obtained in three independent experiments and the data shown were from one representative experiment. d, SdeA-mediated ubiquitination does not lead to degradation of Rab33b. GFP fusion of SdeA or SdeAE/A was co-transfected with Rab33b for 14 h. The proteasome inhibitor MG132 (10 μM) was added to one of the SdeA samples. The levels of Rab33b were detected by immunoblotting following immunoprecipitation with the Flag-specific antibody. Note that the addition of MG132 does not affect the level of modified Rab33b in samples co-transfected with SdeA. Similar results were obtained from two independent experiments. e, The nucleotide binding status of Rab33b does not affect its suitability as substrate in SdeA-mediated ubiquitination. Equal amounts of Rab33b, its dominant negative mutant Rab33b(T47N), or the dominant positive mutant Rab33b(Q92L) was incubated with SdeA. Samples withdrawn at the indicated time points were detected for ubiquitination by Coomassie staining (upper panel); 293T cells transfected to express these mutants were infected the indicated L. pneumophila strains and ubiquitinated Rab33b or its mutants were probed by molecular mass shift in Rab33b obtained by immunoprecipitation (lower panel). Data in this panel are one representative of two independent experiments with similar results. a, d, e, Uncropped blots and gel images are shown in Supplementary Fig. 1.
a, Controls were analysed by HPLC of NAD alone and in the presence of SdeA, Ub, and SdeA and Ub. In these reactions, AMP and NAD were identified with retention times of 3.6 and 6.8 min, respectively. b, Both AMP (left) and NAD (right) were additionally identified by ESI mass spectrometry. Both NAD and a product in which the nicotinamide group has been lost were observed in these experiments. c, To determine whether other fragments are generated in this reaction, retention time for nicotinamide mononucleotide (NMN, left) and nicotinamide (Nic, right) was determined by HPLC to be 5.6 and 2.6 min respectively. d, To identify additional components, a reaction was set up and the individual components were identified by HPLC. In the reaction mixture, AMP (3.5 min), nicotinamide (Nic 5.5 min), and NAD (6.5 min) were observed. An additional component to the reaction mixture (labelled X) was observed (6.1 min), but could not be further identified by mass spectrometry. Data in all panels are one representative from three independent experiments with similar results.
a, Full-length SdeA cannot produce 32P-labelled product in reactions using 32P-α-NAD. Reaction samples resolved by SDS–PAGE were detected by Coomassie staining (left panel) and then by autoradiography (right panel). Note the 32P-α-AMP-GST-ubiquitin complex can be detected in the reaction containing E1 but not SdeA. b, c, SdeA519-1100 is defective in auto-ubiquitination. Reactions containing the indicated components were allowed to proceed for the indicated time duration and the production of ubiquitinated Rab33b (b) or SdeA519-1100 was detected by immunoblotting. d, SdeA519-1100 induces the production of nicotinamide from NAD and ubiquitin. Retention time for nicotinamide and NAD was first determined by HPLC and nicotinamide can only be detected in the reaction containing SdeA519-1100, NAD and ubiquitin. e, SdeA519-1100 induces the production of 32P-ADPR-labelled ubiquitin. GST-ubiquitin or GST–ubiquitinR42A was incubated with 32P-α-NAD and SdeA519-1100 for 0036 h. Classical E1 incubated with GST–ubiquitin was included as a control. Samples resolved by SDS–PAGE before autoradiography (20 min) (right panel). Note that GST–ubiquitinR42A cannot be labelled by 32P. Data in panels a–e are one representative from two independent experiments with similar results. f, The detection of a peptide with m/z 737.33 corresponding to the tryptic peptide E34GIPPDQQRLIFAGK48 containing one ADP-ribosylation site was detected only after ubiquitin was incubated with SdeA519-1100. As a loading control, another unmodified ubiquitin peptide T55LSDYNIQK63 was detected in both control and treated samples. g, Tandem mass analysis revealed that ADP-ribosylation occurred on Arg42 evidenced by the extensive fragmentation of the ADP-ribosylation into adenine, adenosine, AMP and ADP ions. Although not as extensive, the fragmentation of the peptide backbone helps confirm the peptide sequence. Data shown in all panels are one representative from two independent experiments with similar results. a–c, e, Uncropped blots and autoradiograph images are shown in Supplementary Fig. 1.
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Qiu, J., Sheedlo, M., Yu, K. et al. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature 533, 120–124 (2016). https://doi.org/10.1038/nature17657
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