Canonical and phosphoribosyl ubiquitination coordinate to stabilize a proteinaceous structure surrounding the Legionella-containing vacuole
- G.W. Hooper Foundation, University of California, San Francisco, United States
- Department of Microbiology and Immunology, University of California, San Francisco, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 1 supplement
Rab5A forms a ‘cloud’ around the WT LCV in a bacterial effector dependent manner.
(A) Endogenous Rab5A recruitment to the WT or dotA LCV in HeLa FcγR. (B) LCV-associated Rab5A area normalized to bacterial cell area for WT and dotA strains. N=5, 8–30 LCVs measured per strain per replicate. Welch’s two-sample t-test, p=0.0009. In this and all subsequent jitter plots, small points represent individual measurements, large points are mean values across a given biological replicate, and horizontal bars depict the sample mean across replicates. Color corresponds to biological replicate. (C) Endogenous Rab5A staining in cells overexpressing EGFP or indicated fusion constructs. (D) Mean endosome area per cell in samples overexpressing indicated construct. N=3, 20–40 cells scored per replicate. ANOVA, Tukey–Kramer post hoc test, ** p<0.005, ***p<0.0005. (E) Manual scoring of endogenous Rab5 recruitment to WT or dotA LCVs in cells overexpressing indicated construct. N=3, 50–130 LCVs scored per replicate. G test, Bonferroni adj. p-value = 0.0125, ***p<0.000125. (F) LCV-associated Rab5A area as in panel (B) in cells expressing indicated construct. N=3, 10–30 LCVs scored per replicate. ANOVA, Tukey–Kramer post hoc test, n.s. p>0.05. For all infection experiments, cells were fixed at 1 hpi.
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Figure 1—source data 1
CellProfiler output and processed data for plot in Figure 1B (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig1-data1-v1.csv
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Figure 1—source data 2
CellProfiler output and processed data for plot in Figure 1D (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig1-data2-v1.csv
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Figure 1—source data 3
Original counts for plot in Figure 1E (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig1-data3-v1.csv
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Figure 1—source data 4
CellProfiler output and processed data for plot in Figure 1F (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig1-data4-v1.csv
Figure 1—figure supplement 1
Rab5 nucleotide binding mutants are recruited to the WT LCV and ubiquitinated during infection.
(A) Representative image of mCherry Rab5A S34N recruitment to the WT LCV at 4 hpi in HeLa FcγR cells. Scale bar = 10 μm. (B) Quantification of recruitment of the indicated mCherry fusion Rab5 variant to the WT LCV at 4 hpi. Three biological replicates, N=3, 25–50 LCVs analyzed per condition per replicate. (C) Western blot analysis of whole cell lysates prepared from HEK293T FcγR cells transiently transfected with the indicated Flag-tagged Rab5A variant and infected with L.p. WT for 5 hours. Molecular weight indicated in kDa.
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Figure 1—figure supplement 1—source data 1
Tif file for original image of Western blot in Figure 1, Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file for original image of Western blot in Figure 1, Figure 1—figure supplement 1C with relevant bands labeled.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
The SidE family is required for Rab5 cloud formation at the LCV.
(A) Endogenous Rab5A recruitment to the LCV for the indicated strains in HeLa FcγR. (B) LCV-associated Rab5 area normalized to bacterial cell size for the indicated strains. N=3, 5–60 LCVs scored per strain per replicate. One-way ANOVA followed by Tukey–Kramer post hoc test, n.s. p>0.05, *p<0.05, **p<0.005. (C) Immunoprecipitation of Flag-Rab5A from cells expressing HA ubiquitin ΔGG and indicated myc-tagged SidE family effector, or vector control. (D) Immunoprecipitation of Flag-Rab5A from cells expressing HA ubiquitin ΔGG and infected with indicated L.p. strain. For (C) and (D), * indicates non-specific band, most likely light chain. For all infection experiments, cells were fixed or lysed at 1 hpi. For all blots, molecular weight is indicated in kDa.
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Figure 2—source data 1
CellProfiler output and processed data for plot in Figure 2B (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig2-data1-v1.csv
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Figure 2—source data 2
Tif files for original images of Western blots in Figure 2C, D.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig2-data2-v1.zip
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Figure 2—source data 3
PDF file with relevant bands for Western blots in Figure 2C and D with relevant bands labeled.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig2-data3-v1.zip
Figure 2—figure supplement 1
SdeB EE/AA is unable to catalyze PR-ubiquitination.
Western blot analysis of HA-Ub ΔGG conjugation in whole cell lysates prepared from HEK293T FcγR cells infected with the indicated L.p. strain for 1 hour. Molecular weights indicated in kDa.
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Figure 2—figure supplement 1—source data 1
Tif files for original image of Western blots in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF file for original images of Western blots in Figure 2—figure supplement 1 with relevant bands labeled.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
Rab5 is detergent resistant when associated with the WT LCV.
(A) Saponin vs SDS permeabilized uninfected cells stained for endogenous Rab5A. (B) Quantification of normalized total Rab5 fluorescence in cells permeabilized as in panel (A). N=3, 50 cells scored per condition per replicate. Welch’s two-sample t-test, p=0.00069.(C) Endogenous Rab5A immunofluorescence in infected cells after either saponin or SDS permeabilization. (D) Quantification of experiment described in panel (C). N=3, 80–250 LCVs scored per condition per replicate. Welch’s ANOVA followed by Games–Howell post hoc test, n.s. p>0.05, *p<0.05. For all infection experiments, cells were fixed at 1 hpi.
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Figure 3—source data 1
Fiji output and processed data for Figure 3B (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig3-data1-v1.csv
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Figure 3—source data 2
CellProfiler output and processed data for Figure 3D (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig3-data2-v1.csv
Figure 3—figure supplement 1
Validation of dual-stain method for detection of extracellular bacteria.
(A) Representative images of cells permeabilized either before or after Bap31 primary antibody and GAR-405 staining. (B) Pearson correlation of GAR-633 and GAR-405 signal in cells prepared as in flowchart shown in (A). N=2, 50 cells analyzed per replicate per condition. Welch’s two-sample t-test, p=0.006. (C) Representative images of cells infected with L.p. WT and processed using dual stain method for the L.p. opsonization antibody, as well as immunofluorescence analysis of endogenous Rab5. Double stained (extracellular) bacteria are indicated by blue arrows, whereas single stained (intracellular) bacteria are indicated by magenta arrows. (D) Quantification of normalized Rab5 intensity at either double (405+) or single (405-) stained bacteria. N=4,~30–90 bacteria scored per replicate per condition. Welch’s two-sample t-test, p=0.0127. (E) Measurement of normalized GAR405 intensity at bacterial cell bodies for same dataset described in (D). Welch’s t-test, p=0.0002. (F) Distribution of Rab5 intensity values for dataset described in (D). For each replicate, data was pooled, and the percentage of 405+versus 405- bacteria falling into each quartile was tabulated. (G) Normalized GAR405 intensity vs. normalized Rab5 intensity for dataset described in (D). Note that very few datapoints show strong signal for both Rab5 and GAR405. Color represents biological replicate (N=4), 418 total bacteria scored.
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Figure 3—figure supplement 1—source data 1
CellProfiler output and processed data for plot in Figure 3—figure supplement 1B (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig3-figsupp1-data1-v1.csv
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Figure 3—figure supplement 1—source data 2
CellProfiler output and processed data for plot in Figure 3—figure supplement 1D–G (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig3-figsupp1-data2-v1.csv
Figure 4
The SidE family of effectors regulates ubiquitin morphology at the LCV.
(A) Manual scoring immunofluorescence imaging of endogenous ubiquitin recruitment to the indicated L.p. strain. N=3, 60–120 LCVs scored per strain per replicate. G test, Bonferroni adj. p-value = 0.01. ***p<0.0001. (B) Representative images of endogenous ubiquitin recruitment to indicated strain in experiment described in panel (A). (C) Quantification of LCV-associated ubiquitin area normalized to bacterial cell area for indicated strain for experiment described in (A). N=4, 20–120 LCVs scored per strain per replicate. Welch’s ANOVA followed by Games–Howell post hoc test, n.s. p>0.05, *p<0.05.
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Figure 4—source data 1
Manual counts for plot in Figure 4A (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig4-data1-v1.csv
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Figure 4—source data 2
CellProfiler output and processed data for plot in Figure 4C.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig4-data2-v1.csv
Figure 5 with 1 supplement
Ubiquitin at the WT LCV resists detergent washout in a SidE family-dependent manner.
(A) Endogenous ubiquitin immunofluorescence in cells infected with the indicated strain and permeabilized with either saponin or SDS. (B) Quantification of normalized ubiquitin fluorescence at the LCV for experiment described in (A). N=3, 87–240 LCVs scored per strain per replicate. ANOVA, Tukey–Kramer post hoc test, n.s. p>0.05. (C) Background-normalized cytosolic ubiquitin intensity in infected or uninfected cells after SDS washout. 17–72 cells analyzed per condition per replicate. Welch’s two-sample t-test, p=0.09334. (D, E) Immunoblot analysis of total ubiquitin in the (D) soluble or (E) insoluble fraction of cells infected with the indicated L.p. strain from the ΔsidE/sdeABC panel, heat shocked, or left untreated, with total protein (StainFree, Bio-Rad) shown as the loading control. (F) Quantification of normalized high molecular weight ubiquitin in experiments described for (D) and (E). Total endogenous ubiquitin intensity was measured at 150 kDa and above and normalized to total protein and the fold change over the dotA infected control was calculated. N=3, ANOVA, Tukey–Kramer post hoc test when applicable, n.s. p>0.05, *p<0.05, **p<0.005. (G–I) Western blot analysis and quantification of soluble-insoluble fractionation carried out on cells infected with the L.p.ΔsidC/sdcA strain panel as described for (D–F). For all blots, molecular weight is indicated in kDa.
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Figure 5—source data 1
CellProfiler output and processed data for plot in Figure 5B (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig5-data1-v1.zip
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Figure 5—source data 2
CellProfiler output and processed data for plot in Figure 5C (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig5-data2-v1.csv
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Figure 5—source data 3
Tif files for original images of Western blots and StainFree images in Figure 5D, E, G and H.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig5-data3-v1.zip
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Figure 5—source data 4
PDF file with original images of Western blots and StainFree images in Figure 5D, E, G and H with relevant bands labeled.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig5-data4-v1.zip
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Figure 5—source data 5
Blot quantification for Figure 5F (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig5-data5-v1.csv
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Figure 5—source data 6
Blot quantification for Figure 5I (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig5-data6-v1.csv
Figure 5—figure supplement 1
Extended representative images of ubiquitin detergent resistance.
Endogenous ubiquitin staining in cells infected with the indicated strain and permeabilized with either saponin or SDS at 1 hpi.
Figure 6 with 1 supplement
LCV-associated ubiquitin stability decreases as infection progresses.
(A) Immunofluorescence analysis of endogenous ubiquitin localization to the WT LCV at the indicated timepoint post-infection after permeabilization with either saponin or SDS. (B) Normalized ubiquitin intensity quantified for the experiment described in (A). Bacteria per LCV were approximated by dividing the area of each LCV by the mean LCV area for the 1 hpi timepoint for each experiment and permeabilization condition. Intensity data was subjected to one-way ANOVA, followed by a Tukey–Kramer post hoc test, n.s p>0.05, ***p<0.0005. (C, D) Immunoblot analysis of endogenous ubiquitin in cells infected with either L.p. WT or dotA for the indicated length of time and fractionated by solubility. Total protein (StainFree) is shown as a loading control. (E) Quantification of normalized high molecular weight ubiquitin in experiments described for (C) and (E). Total endogenous ubiquitin intensity was measured at 150 kDa and above and normalized to total protein, and the fold change over the dotA infected control was calculated. N=3, ANOVA, Tukey–Kramer post hoc test, n.s. p>0.05, **p<0.005, ***p<0.0005. (F) Quantification of normalized ubiquitin intensity at the WT LCV at 1 hpi after saponin or SDS permeabilization in host cells expressing either EGFP or EGFP DupA. Points represent mean values for each biological replicate for a given condition, and replicates are indicated by color. (G) Analysis of the distribution of the normalized LCV-associated ubiquitin intensity values for the experiment described in panel (E). For all blots, molecular weight is indicated in kDa.
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Figure 6—source data 1
CellProfiler output and processed data for plot in Figure 6B (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-data1-v1.csv
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Figure 6—source data 2
Tif files for original images of Western blots and StainFree images in Figure 6C and D.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-data2-v1.zip
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Figure 6—source data 3
PDF file with original images of Western blots and StainFree images in Figure 6C and D with relevant bands labeled.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-data3-v1.zip
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Figure 6—source data 4
Blot quantification for Figure 6E (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-data4-v1.csv
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Figure 6—source data 5
CellProfiler output and processed data for plots in Figure 6F and G.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-data5-v1.csv
Figure 6—figure supplement 1
Ectopically expressed EGFP-DupA is active against PR-ubiquitin conjugates.
(A) Western blot analysis of lysates from cells transfected with HA-ubiquitin ΔGG and the indicated construct (LotC is a canonical ubiquitin ligase effector), and either left uninfected or infected with L.p. WT. Molecular weight is indicated in kDa. (B) Representative images of endogenous ubiquitin staining in cells transfected with either EGFP alone or EGFP-DupA, infected with L.p. WT, and permeabilized with either saponin or SDS. For all infection experiments, cells were fixed or lysed at 1 hpi.
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Figure 6—figure supplement 1—source data 1
Tif files for original images of Western blots in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
PDF file with images of Western blots in Figure 6—figure supplement 1A with relevant bands labeled.
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig6-figsupp1-data2-v1.zip
Figure 7 with 5 supplements
Ubiquitin expands outwards from the WT LCV during early infection and is co-recruited with Rab5.
(A) Insets from live imaging time courses (see movies S1 and S2) of cells expressing EGFP ubiquitin infected with indicated L.p. strain labeled using the HaloTag system. (B) LCV-associated ubiquitin area normalized to bacterial cell area for LCVs shown in panel (A). (C) Percentage of LCVs falling into ‘compact’ or ‘expansive category’. ‘Compact’ defined as normalized ubiquitin area at T30/T0≤1.5, ‘expansive’ indicates normalized ubiquitin area at T30/T0>1.5. Data was pooled across three biological replicates, 49 LCVs total for L.p. WT, 41 for L.p.ΔsidE/sdeABC. (D) Normalized LCV-associated ubiquitin area across imaging time course for indicated L.p. strains. Violin plots show distribution of pooled data, and dots indicate mean values for each biological replicate. N=3, 6–30 LCVs scored per replicate. ANOVA, Tukey–Kramer post hoc test, n.s. p>0.05, *p<0.05, **p<0.005. (E, F) Insets from timelapse imaging in infected cells expressing mCherry Rab5 and EGFP ubiquitin, infected with either L.p. WT or ΔsidE/sdeABC (see Movies S3 and S4). Graphs in F are intensity measurements for each FP tagged protein for these insets. (G) Visual scoring of ubiquitin and Rab5 recruitment patterns across all replicates. R5 is Rab5, Ub is ubiquitin. Percentages are calculated for all pooled data. 164 LCVs were scored in total for L.p. WT, and 175 for L.p.ΔsidE/sdeABC. (H) Manual approximation of Rab5 association time with either the WT or ΔsidE/sdeABC LCV across three biological replicates. Each point represents a single LCV. LCVs that transition from Rab5 positive to negative are labeled as +to -, whereas LCVs that remain Rab5 positive at the end of the time course are marked as +only. 26 LCVs were scored for L.p. WT, and 24 for L.p.ΔsidE/sdeABC. (I) Comparison of the timepoints at which the intensity maximum occurs for Rab5 and ubiquitin for each LCV across three biological replicates. 3–9 LCVs were scored per strain per replicate. Welch’s two-sample t-test, p=0.00473.
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Figure 7—source data 1
Fiji output and processed data for plots in Figure 7B–D (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig7-data1-v1.csv
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Figure 7—source data 2
Fiji output and processed data for plots in Figure 7F (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig7-data2-v1.csv
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Figure 7—source data 3
Manual positive/negative scoring for plot in Figure 7G (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig7-data3-v1.csv
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Figure 7—source data 4
Manual Rab5 dwell time scoring for plot in Figure 7H (.csv).
- https://cdn.elifesciences.org/articles/108254/elife-108254-fig7-data4-v1.csv
Figure 7—figure supplement 1
Uncommon LCV-associated ubiquitin morphology patterns observed during live imaging.
Example cases of (A) diffuse ubiquitin localization throughout imaging at WT LCV, (B) compact ubiquitin localization at WT LCV, and (C) highly dynamic compact-to-expansive ubiquitin morphology at the SidE family knockout strain LCV.
Figure 7—video 1
Timelapse imaging of GFP-ubiquitin dynamics at the WT LCV.
Scale bar length corresponds to 10 μm.
Figure 7—video 2
Timelapse imaging of GFP-ubiquitin dynamics at the ΔsidE/sdeABC LCV.
Scale bar length corresponds to 10 μm.
Figure 7—video 3
Timelapse imaging of GFP-ubiquitin and mCherry Rab5A dynamics at the WT LCV.
Scale bar length corresponds to 10 μm.
Figure 7—video 4
Timelapse imaging of GFP-ubiquitin and mCherry Rab5A dynamics at the ΔsidE/sdeABC LCV.
Scale bar length corresponds to 10 μm.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Legionella pneumophila) | Legionella pneumophila serogroup 1 strain Lp02 rpsL hsdR thyA (‘WT’) | Berger and Isberg, 1993 Gift from Dr. Craig Roy | LEG003 | |
| Strain, strain background (L. pneumophila) | Legionella pneumophila serogroup 1 strain Lp02 rpsL hsdR thyA dotA (‘dotA’) | Berger and Isberg, 1993 Gift from Dr. Craig Roy | LEG004 | |
| Strain, strain background (L. pneumophila) | Lp02 ΔsidE ΔsdeC ΔsdeBA (Δlpg0234, Δlpg2153 Δlpg2156-2157), annotated as ‘ΔsidE/sdeABC’ for brevity | Jeong et al., 2015 Gift from Dr. Ralph Isberg | LEG151/JV6113 | |
| Strain, strain background (L. pneumophila) | Lp02 ΔsidE/sdeABC pJB1806::SdeB | Steinbach et al., 2024 | LEG171 | |
| Strain, strain background (L. pneumophila) | Lp02 ΔsidE/sdeABC pJB1806::SdeB EE/AA | This study | LEG196 | L.p. ΔsidE/sdeABC transformed with plasmid encoded IPTG-inducible SdeB EE/AA (pCM003) |
| Strain, strain background (L. pneumophila) | Lp02 pON::Halotag | This study | LEG195 | L.p. WT transformed with plasmid encoded constitutively expressed HaloTag7 (pAS100) |
| Strain, strain background (L. pneumophila) | Lp02 ΔsidE/sdeABC pON::HaloTag | This study | LEG160 | L.p. ΔsidE/sdeABC transformed with plasmid encoded constitutively expressed HaloTag7 (pAS100) |
| Cell line (Homo sapiens) | HEK293T FcgR | This study | Derived from parental HEK39T from ATCC (CRL-3216) | |
| Cell line (H. sapiens) | HeLa FcgR | Gift from Dr. Craig Roy | ||
| Transfected construct (H. sapiens) | EGFP in pcDNA3.1 | This study | pAS079 | Transient expression of EGFP in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | EGFP hRabex5 in pcDNA3.1 | This study | pAS061 | Transient expression of EGFP-Rabex5 in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | EGFP hRabapatin5 in pcDNA3.1 | This study | pAS062 | Transient expression of EGFP-Rabaptin5 in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | Myc-SdeA in pMSCV | Gift from Dr. Ralph Isberg | L. pneumophila effector gene | |
| Transfected construct (H. sapiens) | Myc-SdeB in pMSCV | Gift from Dr. Ralph Isberg | L. pneumophila effector gene | |
| Transfected construct (H. sapiens) | Myc-SdeC in pMSCV | Gift from Dr. Ralph Isberg | L. pneumophila effector gene | |
| Transfected construct (H. sapiens) | Myc-SidE in pMSCV | Gift from Dr. Ralph Isberg | L. pneumophila effector gene | |
| Transfected construct (H. sapiens) | Myc-LotC in pMSCV | Gift from Dr. Ralph Isberg | L. pneumophila effector gene | |
| Transfected construct (H. sapiens) | pcDNA3.1 | Gift from Dr. Craig Roy | pSM022 | |
| Transfected construct (H. sapiens) | 3 X Flag hRab5A in pcDNA3.1 | Steinbach et al., 2024 | pAS034 | |
| Transfected construct (H. sapiens) | HA ubiquitin in pRK5 | Gift from Dr. Kohei Arasaki | pSM099 | |
| Transfected construct (H. sapiens) | HA ubiquitin ∆GG in pRK5 | This study | pAS117 | HA-tagged ubiquitin with C terminal GG truncation for transient expression in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | DupA in pEGFPc2 | This study | pCM004 | EGFP- DupA for transient expression in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | Ubiquitin in pEGFPc1 | Gift from Dr. Nico Dantuma | 11928 (Addgene) | |
| Transfected construct (H. sapiens) | 3 X Flag hRab5A Q79L in pcDNA3.1 | This study | pAS039 | Flag-Rab5a Q79L for transient expression in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | 3 X Flag hRab5A S34N in pcDNA3.1 | This study | pAS040 | Flag-Rab5a S34N for transient expression in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | 3 X Flag hRab5A 1–211 in pcDNA3.1 | Steinbach et al., 2024 | pAS041 | Flag-Rab5a 1–211 for transient expression in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | mCherry hRab5A in pcDNA3.1 | Steinbach et al., 2024 | pAS042 | |
| Transfected construct (H. sapiens) | mCherry Rab5A S34N in pcDNA3.1 | This study | pAS048 | mCherry-Rab5a S34N for transient expression in mammalian cells under CMV promoter |
| Transfected construct (H. sapiens) | mCherry Rab5A Q79L in pcDNA3.1 | This study | pAS047 | mCherry-Rab5a Q79L for transient expression in mammalian cells under CMV promoter |
| Antibody | Rab5A (E6N8S), mouse monoclonal | Cell Signaling Technology | 46449, RRID:AB_2799303 | IF (1:100) |
| Antibody | Flag (M2), mouse monoclonal | Sigma Aldrich | F1804, RRID:AB_262044 | WB (1:2500), IP (1:50) |
| Antibody | HA, HRP conjugate, mouse monoclonal | Thermo Fisher | 26183-HRP, RRID:AB_2533056 | WB (1:1000) |
| Antibody | L. pneumophila, rabbit polyclonal | Thermo Fisher | PA1-7227, RRID:AB_559903 | Opsonization (1:2000) |
| Antibody | Myc, mouse monoclonal | Proteintech | 60003–2-Ig, RRID:AB_2734122 | WB (1:1000) |
| Antibody | GFP, mouse monoclonal | Roche | 11814460001, RRID:AB_390913 | WB (1:1000) |
| Antibody | Ubiquitin (P4D1), mouse monoclonal | Santa Cruz | sc-8017, RRID:AB_628423 | WB (1:500) |
| Antibody | Ubiquitin conjugates (FK2) | Millipore | ST1200, RRID:AB_2043482 | IF (1:200) |
| Recombinant DNA reagent | SdeB in pJB1806 (plasmid) | Steinbach et al., 2024 | pAS083 | Transformed into L. pneumophila |
| Recombinant DNA reagent | SdeB EE/AA in pJB1806 (plasmid) | This study | pCM003 | Transformed into L. pneumophila |
| Recombinant DNA reagent | HaloTag in pON (plasmid) | This study | pAS100 | Transformed into L. pneumophila |
| Software, algorithm | Fiji | Schindelin et al., 2012 | https://fiji.sc/ | |
| Software, algorithm | CellProfiler | Stirling et al., 2021 | https://cellprofiler.org/releases | |
| Software, algorithm | ggplot2 | https://ggplot2.tidyverse.org/ | ||
| Software, algorithm | dplyr | https://dplyr.tidyverse.org/ | ||
| Software, algorithm | userfriendlyscience | https://rdrr.io/cran/userfriendlyscience/man/userfriendlyscience-package.html | ||
| Software, algorithm | ImageLab | Bio-Rad | ||
| Other | JF646 | Gift from Dr. Luke Lavis | Far red HaloTag ligand |
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Canonical and phosphoribosyl ubiquitination coordinate to stabilize a proteinaceous structure surrounding the Legionella-containing vacuole
eLife 14:RP108254.
https://doi.org/10.7554/eLife.108254.3
