The SidE- and SidC-family proteins differentially contribute towards ubiquitination of Rab10.

HEK293T-FcγRII cells transiently expressing 3xFLAG-Rab10 and HA-Ub were infected with the indicated L. pneumophila strains for 1 h at an MOI of 20. Rab10 was isolated from cell lysate by immunoprecipitation using anti-FLAG magnetic beads and was probed with anti-FLAG and with anti-HA antibodies. Triple-HA-Ub (3xHA-Ub) or Ub in which the C-terminal GG were replaced with AA (3xHA-UbAA) was expressed instead of HA-Ub in (b). Bacterial lysates were probed with anti-Myc antibody in (b).

The SidE- and SidC-family proteins differentially contribute towards recruitment of Rab10 to the LCV.

HeLa-FcγRII cells transiently expressing RFP-Rab10 were infected with the indicated L. pneumophila strains at an MOI of 5 for 4 h (a) and for the indicated time (b). (a) Representative images of infected cells. Fixed cells were stained for L. pneumophila (green) and DNA (blue) and visualized with RFP-Rab10 (red). Magnified images in the white squares are shown in merged and in each channel. Arrows indicate the Rab10-positive LCVs. Scale bars, 10 μm. (b) Quantitation of Rab10-positive LCVs (%). Infections were performed in triplicate and each value represents scoring from 200 LCVs. Significance was determined using Student’s t-test.

SdcB associates with the LCV and plays a major role in Ub recruitment to the LCV at late stages of infection.

HeLa-FcγRII cells were infected with the indicated L. pneumophila strains at an MOI of 2 for 1 h (a, b) and for 7 h (c, d). (a, c) Representative images of infected cells. Fixed cells were stained for FLAG-SdcB or Ub (green), L. pneumophila (red) and DNA (blue). Magnified images in the white squares are shown in the lower panels. Scale bars, 10 μm. (b, d) Quantitation of SdcB-positive (left) and of Ub-positive (right) LCVs (%). Infections were performed in triplicate and each value represents scoring from 200 LCVs. Significance was determined using Student’s t-test.

The catalytic activity of SdcB enhances retention of Rab10 on the LCV.

(a) HEK293T-FcγRII cells transiently expressing 3xFLAG-Rab10 Q68L (QL) or Rab10 T23N (TN) with HA-Ub were infected with the indicated L. pneumophila strains for 1 h at an MOI of 50. Rab10 was isolated from cell lysate by immunoprecipitation using anti-FLAG magnetic beads and was probed with anti-FLAG antibody. (b) HeLa-FcγRII cells transiently expressing RFP-Rab10QL were infected with the indicated L. pneumophila strains at an MOI of 10 for 1 h (see Figure 4-figure supplement 2). Rab10-positive LCVs (%) were quantified. Infections were performed in triplicate and each value represents scoring from 50 LCVs. Significance was determined using Student’s t-test. (c) HEK293T-FcγRII cells transiently expressing 3xFLAG-Rab10QL and HA-Ub were infected with the L. pneumophila strains expressing Myc-tagged SdcB or its catalytic mutant for 7 h at an MOI of 20. Rab10 was isolated from cell lysate by immunoprecipitation using anti-FLAG magnetic beads and was probed with anti-FLAG and with anti-HA antibodies. For detection of translocated SdcB, it was isolated from cell lysate by immunoprecipitation using anti-Myc magnetic beads and was probed with anti-Myc antibody. Note that apparent reduction of the wild-type SdcB was caused by its auto-ubiquitination leading to the molecular weight shift (see text). (d, e) HeLa-FcγRII cells transiently expressing RFP-Rab10 were infected with the indicated L. pneumophila strains at an MOI of 2 for 7h. (d) Representative images of infected cells. Fixed cells were stained for FLAG-SdcB (green) and L. pneumophila (blue) and visualized with RFP-Rab10 (red). Magnified images in the white squares are shown in each channel. White arrows indicate the position of a bacterium. The red arrow indicates a Rab10 signal surrounding an LCV. Scale bars, 10 μm. (e) Quantitation of Rab10-positive LCVs (%) out of SdcB-positive ones. Infections were performed in triplicate and each value represents scoring from 200 SdcB-positive LCVs. Significance was determined using Student’s t-test.

The transglutaminase activity of MavC can mediate a unique Ub conjugation to SdcB.

(a) 3xFLAG-SdcB, HA-Ub and GFP-MavC were coexpressed in HEK293T-FcγRII cells. SdcB was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of SdcB. (b) 3xFLAG-SdcB and GFP-MavC were coexpressed in HEK293T-FcγRII cells. SdcB was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of SdcB. (c) In vitro transglutaminase assay was performed using purified proteins. The samples were analyzed by SDS-PAGE followed by silver staining (top) or by immunoblotting using the indicated antibodies (middle and bottom). The asterisks indicate the Ub-conjugated form of SdcB. (d) 3xFLAG-SdcA or SidC, GFP-MavC and HA-Ub were coexpressed in HEK293T-FcγRII cells. SdcA or SidC was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of SdcA. (e) 3xFLAG-SdcB, GFP-MavC and HA-Ub or Ub without any Lys residues (Ub No K) were coexpressed in HEK293T-FcγRII cells. SdcB was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of SdcB. (f) 3xFLAG-SdcB, GFP-MavC and HA-Ub or Ub in which the C-terminal GG were replaced with AA (Ub AA) were coexpressed in HEK293T-FcγRII cells. SdcB was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of SdcB.

Identification of residues on Ub and SdcB between which MavC can crosslink.

(a) MavC catalyzes the formation of an isopeptide bond between the Gln41 of Ub and the Lys518 of SdcB. The indicated proteins were expressed in HEK293T-FcγRII cells and SdcB was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads. The samples were resolved by SDS-PAGE. The Ub-conjugated SdcB was detected by immunoblotting and by CBB staining. The gel slices of areas of the bands shown with the red squares were subjected to mass spectrometric analysis. Product ion spectrum was shown for Ub peptide –AKIQDKEGIPPDQQR-crosslinked with SdcB peptide – VLLDKEVNDEGIAEAVASK-. (b, c) The indicated proteins were coexpressed in HEK293T-FcγRII cells. SdcB was isolated from cell lysates by immunoprecipitation using anti-FLAG magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of SdcB.

Catalytic activity of MavC negatively regulates the Rab10 localization to the LCV.

(a, b) HeLa-FcγRII cells transiently expressing RFP-Rab10 and HA-MavC or its catalytic mutant were infected with the Lp01 ΔsidCΔsdcAΔsdcB strain complemented with the plasmid expressing 3xFLAG-SdcB or its catalytic mutant at an MOI of 2 for 4 h. (a) Representative images of cells infected with the Lp01 ΔsidCΔsdcAΔsdcB strain complemented with the plasmid expressing 3xFLAG-SdcB. Fixed cells were stained for FLAG-SdcB (green) and DNA (blue), and visualized with RFP-Rab10 (red). Magnified images in the white squares are shown in each channel. White arrows indicate the position of a bacterium. The red arrow indicates a Rab10 signal surrounding an LCV. Scale bars, 10 μm. (b) Quantitation of Rab10-positive LCVs (%) out of SdcB-positive ones. Infections were performed in triplicate and each value represents scoring from 200 SdcB-positive LCVs. Significance was determined using Student’s t-test. (c) HeLa-FcγRII cells transiently expressing RFP-Rab10 were infected with the indicated Lp01 strains at an MOI of 2 for 9 h, and Rab10-positive LCVs (%) were quantified. Infections were performed in triplicate and each value represents scoring from 200 LCVs. Significance was determined using Student’s t-test. (d) The schematic of roles of SidE and SidC family ligases in Rab10 localization to the LCV and of negative regulation of SdcB-dependent Rab10 retention by the transglutaminase activity of MavC. Red arrows indicate canonical Ub conjugation by SidC, SdcA and SdcB. Purple arrows indicate the noncanonical Ub conjugation. In the early stage of infection, Rab10 is recruited and retained to the LCV. This event is linked to its PR-ubiquitination catalyzed by the SidE effectors. The PR-ubiquitination of Rab10 provides a platform of its polyubiquitination in a manner depending on SidC and SdcA. In later stages, SdcB contributes towards sustained Ub accumulation on the LCV, enabling the LCV to maintain Rab10 on the vacuole. MavC-mediated crosslinking between Ub and SdcB disrupts the catalytic activity of SdcB, eventually releasing Rab10 from the LCV.

Mutation of Lys102, Lys136, and Lys154 on Rab10 did not eliminate ubiquitination of Rab10 upon infection.

HEK293T-FcγRII cells transiently expressing 3xFLAG-Rab10 or 3xFLAG-Rab10 K102A K136A L154A (KKK) (a) with HA-Ub were infected with the indicated L. pneumophila strains for 1 h (a) or 7 h (b) at an MOI of 20. Rab10 was isolated from cell lysate by immunoprecipitation using anti-FLAG magnetic beads and was probed with anti-FLAG and with anti-HA antibodies.

Active Rab10 is preferentially targeted for infection-induced ubiquitination.

HEK293T-FcγRII cells transiently expressing 3xFLAG-Rab10 Q68L (QL) or Rab10 T23N (TN) with HA-Ub were infected with the wild-type L. pneumophila strain or treated with media containing anti-Legionella antiserum (mock infection) for 1 h at an MOI of 20. Rab10 was isolated from cell lysate by immunoprecipitation using anti-FLAG magnetic beads and was probed with anti-FLAG and with anti-HA antibodies.

The SidE- and SidC-family proteins contribute towards retaining active Rab10 to the LCV.

HeLa-FcγRII cells transiently expressing RFP-Rab10QL were infected with the indicated L. pneumophila strains at an MOI of 10 for 1 h. Cells were fixed and stained with anti-Legionella antiserum (green) before permeabilization of cells for detection of extracellular bacteria. Permeabilized cells were stained with DAPI (blue) for detection of intracellular bacteria (and nuclei) and with RFP-Rab10 (red). Representative images of infected cells were shown. Magnified merged images in the white squares are shown as enlarged images. Scale bars, 5 μm.

SdcB has a catalytic activity of self-ubiquitination with preference of various E2 enzymes.

Ub, E1 enzyme, indicated E2 enzymes and purified His-SdcB or His-SidC were mixed in the reaction buffer in the presence of ATP and incubated at 30°C for 120 min. The samples were analyzed by SDS-PAGE followed by silver staining (top) or by immunoblotting using the anti-Ub antibody (bottom).

The catalytic activity of MavC negatively impacts on auto-ubiquitination of SdcB.

(a) 3xFLAG-SdcB, HA-Ub and GFP-MavC were coexpressed in HEK293T-FcγRII cells. After 24 h transfection, cell media was replaced with media containing 10 μM of MG132 or equivalent amount of DMSO, and incubation of cells was resumed for additional 6 h. The cell lysates were probed with the indicated antibodies. (b) The in vitro reaction was performed with recombinant Ub, E1 enzyme, His-UbcH6, purified His-SdcB and His-MavC in the reaction buffer in the presence of ATP by incubation at 30°C for 120 min. The samples were analyzed by SDS-PAGE followed by immunoblotting using the anti-Ub antibody.

Mass spectrometry analysis identified additional residues forming a covalent linkage between Ub and SdcB.

Product ion spectrum was shown for Ub peptide –IQDKEGIPPDQQR-crosslinked with SdcB peptide –GYVGVFFSGKENIK-.

Mutations on Lys518 and Lys891 of SdcB did not affect ubiquitination of Rab10.

HEK293T-FcγRII cells transiently expressing 3xFLAG-Rab10QL and HA-Ub were infected with the L. pneumophila ΔsidCΔsdcAΔsdcB strain expressing Myc-tagged SdcB, its catalytic mutant (SdcB C57A) or SdcB K518R K891R for 7 h at an MOI of 20. Rab10 was isolated from cell lysate by immunoprecipitation using anti-FLAG magnetic beads and was probed with anti-FLAG and with anti-HA antibodies. For detection of translocated SdcB, it was isolated from cell lysate by immunoprecipitation using anti-Myc magnetic beads and was probed with anti-Myc antibody. Note that apparent loss of SdcB was caused by its auto-ubiquitination leading to the molecular weight shift.

Bacterially delivered MavC mediates elimination of Rab10 from the LCV.

Representative images of cells infected with the indicated Lp01 strains at an MOI of 2 for 9 h (Rab10-positive LCVs (%) were quantified in Figure 7c). Fixed cells were stained for L. pneumophila (green) and DNA (blue), and visualized with RFP-Rab10 (red). Magnified images in the white squares are shown in each channel. Red arrows indicate the Rab10 signal surrounding an LCV. Scale bars, 10 μm.

Ectopic expression of SdcB does not proceed Rab10 polyubiquitination even in the presence of SdeA.

Indicated proteins were coexpressed in HEK293T-FcγRII cells. Rab10 was isolated from cell lysates by immunoprecipitation using anti-RFP magnetic beads and was probed with the indicated antibodies. The asterisks indicate the Ub-conjugated form of Rab10.