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.

HeLa-FcγRII cells transiently expressing RFP-Rab10 were infected with the indicated L. pneumophila strains at an MOI of 2 for 7h. (a) Representative images of infected cells. Fixed cells were stained for FLAG-SdcB (green) and DNA (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. (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.

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 ΔsidC ΔsdcA ΔsdcB 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 ΔsidC ΔsdcA ΔsdcB 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 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, retaining Rab10 on the vacuole (right). However, MavC-mediated crosslinking between Ub and SdcB disrupts the catalytic activity of SdcB, resulting in releasing Rab10 from the LCV (left).

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.

Ub, E1 enzyme, His-UbcH6, purified His-SdcB and His-MavC 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 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-.

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.