Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

Abstract

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 cascade, 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 agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. 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.

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 standard error of the mean (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 standard error of the mean (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. 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 posttranslational modification of Rab1 during bacterial infection

a, SdeA, SdeA_E/A or ExoS and 5 μCi ³²P-NAD were added to 100 μg total proteins 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+SdeA_E/A+293T lysates; Lane 3: no sample; Lane 4: ³²P-α-NAD+ExoS_78-453 +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-SdeB_1-1751; 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, b, 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 E2s

a, Proteins in bands corresponding to normal (upper panel) or shifted Rab33b (lower panel) 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 weight. 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 weight (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 SidC_1-542 and its enzymatically inactive mutant SidC_1-542C46A with E1 and the E2 UbcH7 were established to monitor the activity of E1 (right panel). Note the robust self-ubiquitination of SidC_1-542 (2^nd 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.

Extended Data Figure 4. The activity of EDTA-dialyzed SdeA and other members of the SidE family

a, SdeA or SdeA_E/A dialyzed 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, SdeB_1-1751 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, SdeB_1-1751 and SdeC and SdeA_E/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, b, 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 SdeA_E/A were incubated with 5 μg of GST-Rab1, 4xFlag-Rab33b and 5 μCi of ³²P-α-NAD. A reaction containing 200 ng of ExoS_78-453, 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 5xSDS 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+SdeA_E/A+GST-Rab1; Lane 3: ³²P-α-NAD+SdeA+4F-Rab33b; Lane 4: ³²P-α-NAD+SdeA_E/A+4F-Rab33b; Lane 5: no sample; Lane 6: ³²P-α-NAD+ExoS_78-453+FAS+Rab5. Note the strong ADP-ribosylation signals in the reaction with ExoS_78-453 (lane 6). b, SdeA, its mutants SdeA_C118A or SdeA_C118AE/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 SdeA_C118A exhibited similar activity in these reactions. Data in all panels are one representative of two independent experiments with similar results. a, b, c, Uncropped blots and autoradiograph images are shown in Supplementary Fig. 1.

Extended Data Figure 6. The reactivity of ubiquitin mutants in SdeA-mediated ubiquitination

a, Arg₄₂ in ubiquitin is important for SdeA-mediated ubiquitination. Ubiquitin or ubiquitin_R42A was included in reactions catalyzed 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 ubiquitin_R42A can be used by ubiquitination catalyzed by SidC but not SdeA. b, GST-ubiquitin_R42A cannot be use for self-ubiquitination by SdeA. GST-ubiquitin or GST-ubiquitin_R42A was used in reactions with SdeA or SdeA_E/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-catalyzed Rab33b ubiquitination. Reactions containing SdeA or SdeA_E/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 Ub 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, b, c, d, e, Uncropped blots are shown in Supplementary Fig. 1.

Extended Data Figure 7. Ubiquitination catalyzed 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 catalyzed 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. 4xFlag-Rab33b was loaded with unlabeled GDP (5 mM) before ubiquitination reaction. GDP loaded Rab33b was subjected to ubiquitination by SdeA or SdeA_E/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 4xFlag-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 SdeA_E/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 Rab33bT47N, or the dominant positive mutant Rab33bQ92L 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 weight 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.

Extended Data Figure 9. Detection of the reaction intermediates in SdeA-catalyzed ubiquitination

a, Controls were analyzed 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 (labeled 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.

Extended Data Figure 10. Detection of the ubiquitination intermediate by using SdeA_519-1100

a, Full-length SdeA cannot produce ³²P-labeled 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, SdeA_519-1100 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 SdeA_519-1100 was detected by immunoblotting. d, SdeA_519-1100 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 SdeA_519-1100, NAD and ubiquitin. e, SdeA_519-1100 induces the production of ³²P-ADPR-labeled ubiquitin. GST-ubiquitin or GST-ubiquitin_R42A was incubated with ³²P-α-NAD and SdeA_519-1100 for 6 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-ubiquitin_R42A cannot be labeled by ³²P. Data in panels a to 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 SdeA_519-1100. 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, b, c, e, Uncropped blots and autoradiograph images are shown in Supplementary Fig. 1.

Figure 1. A putative mono ADP-ribosyltransferase (mART) motif is important for yeast toxicity of SdeA

a. Alignment of the central region of the SidE family members and several toxins with mART activity. Proteins identified by PSI-BLAST were manually aligned. Shown mART toxins are IotA from Clostridium perfringens , the C3 exoenzyme from Clostridium botulinum and ExoS from Pseudomonas aeruginosa . Residues important for the mART motif were highlighted in red. b-c. The mART is essential for yeast toxicity and for secretion inhibition by SdeA. Yeast cells were spotted on the indicated medium for 3 d before image acquisition. The secretion of SEAP was examined in 293T cells transfected to express SEAP and GFP-tagged testing proteins; the strong SEAP inhibitor AnkX was used as a control. Error bars represent standard error of the mean (s.e.m.) (n=3). The expression of the proteins (the lower panel in b for yeast and the right panel in c for mammalian cells) was probed with indicated antibodies. The PGK (3-phosphoglyceric phosphokinase) and tubulin were probed as a loading control, respectively. SdeA_E/A, SdeA with Glu₈₆₇ and Glu₈₆₉ mutated to Ala. IB, immunoblotting. The yeast toxicity results in b and protein levels in b and c are from one representative of three independent experiments. The SEAP results in c are one representative done in triplicate from three independent experiments. b, c, Uncropped blots are shown in Supplementary Fig. 1.

Figure 2. The predicted mART motif is essential for the role of SdeA in intracellular bacterial growth

a, The indicated bacterial strains were used to infect D. discoideum and the bacterial yields were monitored at 24-h intervals. Note that SdeA but not the SdeA_E/A mutant restored the defect exhibited by the ΔsidEs strain. b, Expression and Dot/Icm-mediated translocation of SdeA and SdeA_E/A. The bacteria used for infections were probed for protein expression; the metabolic enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control (upper panels). Saponin-soluble fractions of infected cells were probed for translocated SdeA with tubulin as a loading control (lower panel). c-d, L. pneumophila was used to infect a strain of D. discoideum stably expressing the ER retention fusion GFP-HDEL and the recruitment of the ER marker to the phagosome was evaluated 2 h after infection. IB, immunoblotting. Results in a and c are from one representative experiment done in triplicate from three independent experiments; error bars represent standard error of the mean (s.e.m.) (n=3). Results in b and d are one representative from three independent experiments. Bar, 5 μm. b, Uncropped blots are shown in Supplementary Fig. 1.

Figure 3. SdeA induces a posttranslational modification on multiple ER-associated Rab proteins

a, Lysates of 293T cells co-transfected to express SdeA and Flag-tagged small GTPases were subjected to immunoprecipitation with Flag beads and the products were probed with the Flag-specific antibody. Note the appearance of shifted bands for Flag-tagged ER-associated Rabs but not for Rab5 and Rac1. M, SdeA_E/A; W, SdeA. b, SdeA-dependent posttranslational modification of Rab33b during bacterial infection. Cells expressing Flag-Rab33b were infected with relevant L. pneumophila strains for 2 h and Flag-Rab33b purified from cell lysates was probed by immunoblotting. c-f, SdeA induces Rab33b ubiquitination. Flag-Rab33b purified from cells co-expressing SdeA (c) or infected with wild type L. pneumophila (e) was subjected to mass spectrometric analysis and tryptic ubiquitin fragments were identified in proteins of the shifted bands (d and f). g, Overexpression of Rab33b restricts intracellular bacterial growth. COS1 cells transfected with Rab33b and the indicated mutants were infected with L. pneumophila and the formation of replicative vacuoles was determined. IB, immunoblotting. Data shown are one representative experiment of three independent experiments (a-f); results in g are one representative done in triplicate from three independent experiments. Error bars represent standard error of the mean (s.e.m.) (n=3). a, b, c, e, Uncropped blots and gel images are shown in Supplementary Fig. 1.

Figure 4. SdeA catalyzes ubiquitination independent of E1 and E2

a, A heat stable molecule from cells is required for ubiquitination induced by SdeA. Reactions resolved by SDS-PAGE were probed with the indicated antibodies. Note the production of ubiquitinated Rab33b in reactions containing boiled mammalian cell lysates and E. coli lysates. TCL, total cell lysates. b, NAD is required for SdeA-catalyzed ubiquitination. Ubiquitinated Rab33b and SdeA were probed by Coomassie staining or by immunoblotting with antibodies specific for ubiquitin or Flag. c, Self-ubiquitination by SdeA. SdeA or SdeA_E/A was incubated with GST-ubiquitin and NAD; ubiquitination was detected by immunoblotting or by Coomassie staining. Note the formation of the high molecular weight self-ubiquitinated SdeA when GST-ubiquitin was included in the reactions. d, Ubiquitination catalyzed by the central domain of SdeA. SdeA_178-1000 or SdeA_178-1000E/A was used for ubiquitination of Rab33b and the products were probed by Coomassie staining or by immunoblotting. IB, immunoblotting. a-d, similar results were obtained from four experiments. a, b, c, d, Uncropped blots are shown in Supplementary Fig. 1.