The acetylase activity of Cdu1 regulates bacterial exit from infected cells by protecting Chlamydia effectors from degradation
- Department of Integrative Immunobiology, Duke University, United States
- Department of Molecular Genetics and Microbiology, Duke University, United States
- Deparment of Molecular Biology, Umeå University, Sweden
- The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Sweden
- Umeå Centre for Microbial Research (UCMR), Umeå University, Sweden
- Department of Molecular Microbiology & Immunology, Oregon Health & Science University, United States
Figures
Figure 1 with 4 supplements
The C. trachomatis inclusion membrane proteins InaC, IpaM, and CTL0480 are ubiquitinated in the absence of Cdu1.
(A–C) Volcano plots (pairwise comparisons) of the relative abundance of human and Ct ubiquitinated peptides.(A) Mock infected HeLa cells versus HeLa cells infected with WT Ct (L2 434 Bu pBOMB) (24 …
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Figure 1—source data 1
Excel file containing MaxQuant intensity values used to generate volcano plots in Figure 1A–C.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-data1-v2.zip
Figure 1—figure supplement 1
TargeTron mediated disruption of the L2 cdu1 ORF.
(A) Depiction of the cdu1 ORFs in WT L2 and cdu1::GII strains with corresponding diagnostic PCR amplicons. (B) PCR analysis of the disrupted cdu1 ORF in the cdu1::GII strain. *Amplification of cdu1 …
Figure 1—figure supplement 2
Generation of a cdu1 null strain in C. trachomatis (L2).
(A) Western blot analysis of protein lysates derived from HeLa cells infected with a Ct L2 434 Bu parental strain (L2) and an L2 cdu1::GII aadA (cdu1::GII) strain for 24 hr. Blots were probed with …
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Figure 1—figure supplement 2—source data 1
Original files used for western blot analysis shown in Figure 1—figure supplement 2A (anti-Cdu1, anti-RpoB, and anti-α-tubulin).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-figsupp2-data1-v2.zip
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Figure 1—figure supplement 2—source data 2
Files containing original scans of western blot analysis shown in Figure 1—figure supplement 2A (anti-Cdu1, anti-RpoB, and anti-α-Tubulin) with sample labels and highlighted bands.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-figsupp2-data2-v2.zip
Figure 1—figure supplement 3
Proteins co-precipitating (non ubiquitinated) with human and Ct proteins enriched by TUBE1 pulldowns.
(A) Volcano plots (pairwise comparisons) of human proteins (non-ubiquitinated) that co-precipitate with TUBE1-bound proteins in mock infected HeLa cells and HeLa cells infected with L2 or cdu1 null …
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Figure 1—figure supplement 3—source data 1
Excel file containing MaxQuant intensity values used to generate volcano plots in panels A and B.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-figsupp3-data1-v2.zip
Figure 1—figure supplement 4
Pathway enrichment analysis of human and Ct proteins that co-precipitate (non ubiquitinated) with TUBE1-bound proteins.
(A) Metascape functional enrichment analysis of human co-precipitating proteins in mock infected HeLa cells. (B) Metascape functional enrichment analysis of human co-precipitating proteins in cdu1 …
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Figure 1—figure supplement 4—source data 1
Excel file with Metascape compiled GO clusters and associated p-values for GO analysis of human co-precipitating proteins in mock-infected Hela cells (panel A).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-figsupp4-data1-v2.zip
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Figure 1—figure supplement 4—source data 2
Excel file with Metascape compiled GO clusters and associated p-values for GO analysis of human co-precipitating proteins in cdu1 null infected HeLa cells (panel B).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-figsupp4-data2-v2.zip
Figure 2
Cdu1 associates with InaC, IpaM, and CTL0480.
(A) Co-localization of Cdu1(magenta) with endogenous InaC (green), IpaM (green), and ectopically expressed CTL0480-Flag (green) at the Ct (L2) inclusion membrane of HeLa cells infected for 24 hr. …
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Figure 2—source data 1
Excel files with pixel intensity values for generating line scan profiles in Figure 2B.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data1-v2.zip
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Figure 2—source data 2
Original western blot scans used to generate Figure 2D (anti-GFP [input], anti-Flag [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-Flag [IP:GFP]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data2-v2.zip
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Figure 2—source data 3
Original western blot scans with labeled samples and highlighted bands for Figure 2D.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data3-v2.zip
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Figure 2—source data 4
Original western blot scans used to generate Figure 2E (anti-GFP [input], anti-V5 [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-V5 [IP:GFP]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data4-v2.zip
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Figure 2—source data 5
Original western blot scans with labeled samples and highlighted bands for Figure 2E.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data5-v2.zip
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Figure 2—source data 6
Original western blot scans used to generate Figure 2F (anti-GFP [input], anti-V5 [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-V5 [IP:GFP]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data6-v2.zip
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Figure 2—source data 7
Original western blot scans with labeled samples and highlighted bands for Figure 2F.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data7-v2.zip
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Figure 2—source data 8
Original western blot scans used to generate Figure 2G (anti-GFP [input], anti-V5 [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-V5 [IP:GFP]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data8-v2.zip
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Figure 2—source data 9
Original western blot scans with labeled samples and highlighted bands for Figure 2G.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data9-v2.zip
Figure 3 with 1 supplement
Cdu1 stabilizes InaC, IpaM, and CTL0480.
(A) Western blot analysis of endogenous InaC in HeLa cells infected for 24, 36, and 48 hr with Wt Ct (L2 pBOMB), cdu1 null (cdu1::GII pBOMB), and inaC null (M407) strains. Ct Slc1 and human alpha …
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Figure 3—source data 1
Original western blot scans used to generate Figure 3A (anti-InaC, anti-Slc1, and anti-α-tubulin).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data1-v2.zip
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Figure 3—source data 2
Original western blot scans with labeled samples and highlighted bands for Figure 3A.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data2-v2.zip
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Figure 3—source data 3
Excel file containing densitometry data for Figure 3B.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data3-v2.zip
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Figure 3—source data 4
Original western blot scans used to generate Figure 3C (anti-IpaM, anti-Slc1, and anti-α-tubulin).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data4-v2.zip
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Figure 3—source data 5
Original western blot scans with labeled samples and highlighted bands for Figure 3C.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data5-v2.zip
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Figure 3—source data 6
Excel file containing densitometry data for Figure 3D.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data6-v2.zip
Figure 3—figure supplement 1
Quantification of CTL0480 at Ct inclusion membranes.
Quantification of CTL0480 localization at Ct inclusion membranes as shown in Figure 3E. CTL0480 fluorescent signal was used to determine the percent of inclusions displaying CTL0480 at the inclusion …
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Figure 3—figure supplement 1—source data 1
Excel files with numerical data for quantifying CTL0480 localization to WT and cdu1::GII inclusions.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-figsupp1-data1-v2.zip
Figure 4
The acetylase activity of Cdu1 is required to stabilize Cdu1, InaC, and IpaM.
(A) Western blot analysis of endogenous InaC and Cdu1-Flag catalytic variants expressed from a plasmid (pBOMB). HeLa cells were infected for 36 hr with WT Ct (L2 434 Bu pBOMB), a cdu1 null strain (cd…
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Figure 4—source data 1
Original western blot scans used to generate Figure 4A (anti-InaC, anti-Flag, anti-Slc1, and anti-α-tubulin).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data1-v2.zip
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Figure 4—source data 2
Original western blot scans with labeled samples and highlighted bands for Figure 4A.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data2-v2.zip
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Figure 4—source data 3
Original western blot scans used to generate Figure 4B (anti-IpaM, anti-Flag, anti-Slc1, and anti-α-tubulin).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data3-v2.zip
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Figure 4—source data 4
Original western blot scans with labeled samples and highlighted bands for Figure 4B.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data4-v2.zip
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Figure 4—source data 5
Original western blot scans used to generate Figure 4C (anti-Flag, anti-Slc1, and anti-α-tubulin).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data5-v2.zip
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Figure 4—source data 6
Original western blot scans with labeled samples and highlighted bands for Figure 4C.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data6-v2.zip
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Figure 4—source data 7
Original western blot scans used to generate Figure 4D (anti-Flag [input], anti-Slc1 [input], anti-α-tubulin [input], anti-Flag [IP:Flag], and anti-lys48 pUb [IP:Flag]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data7-v2.zip
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Figure 4—source data 8
Original western blot scans with labeled samples and highlighted bands for Figure 4D.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data8-v2.zip
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Figure 4—source data 9
Original western blot scans used to generate Figure 4E (anti-IncA [input], anti-InaC [input], anti-IpaM [input], anti-InaC [IP:Acetylated lysines], anti-IpaM [IP:Acetylated lysines], and anti-IncA [IP:Acetylated lysines]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data9-v2.zip
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Figure 4—source data 10
Original western blot scans with labeled samples and highlighted bands for Figure 4E.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data10-v2.zip
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Figure 4—source data 11
Original western blot scans used to generate Figure 4F (anti-IncA [input], anti-Flag [input], and anti-Flag [IP:Acetylated lysines]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data11-v2.zip
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Figure 4—source data 12
Original western blot scans with labeled samples and highlighted bands for Figure 4F.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data12-v2.zip
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Figure 4—source data 13
Original western blot scans used to generate Figure 4G (anti-IncA [input], anti-Flag [input], and anti-Flag [IP:Acetylated lysines]).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data13-v2.zip
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Figure 4—source data 14
Original western blot scans with labeled samples and highlighted bands for Figure 4G.
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data14-v2.zip
Figure 5 with 1 supplement
The DUB activity of Cdu1 is not required for blocking the ubiquitination of inclusion membranes and Cdu1 is not required for protection against IFNγ mediated cellular immunity.
(A) Representative images of Ct inclusions decorated with ubiquitin during infection of HeLa cells with a cdu1::GII strain for 24 hr. Representative images of infected HeLa cells used for …
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Figure 5—source data 1
Excel file with numerical data for quantifying ubiquitinated inclusions in HeLa cells infected with WT CT and cdu1::GII strains, and cdu1::GII strains expressing Cdu1 C345A, I225A, and K268E variants (Figure 5B).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data1-v2.zip
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Figure 5—source data 2
Excel files with numerical data for quantifying the number of ubiquitinated inclusions in HeLa cells infected with cdu1::GII and incA null strains, and cdu1::GII strains expressing Cdu1 variants that are ubiquitinated at inclusion junctions in co-infected HeLa cells (Figure 5D).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data2-v2.zip
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Figure 5—source data 3
Excel file containing numerical data for quantifying the number of inclusions in IFN-γ-primed A549 cells infected with WT CT, garD::GII, and cdu1::GII strains (Figure 5E).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data3-v2.zip
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Figure 5—source data 4
Excel file containing numerical data for quantifying the number of RNF213-positive inclusions in IFN-γ-primed A549 cells infected with WT CT, garD::GII, and cdu1::GII strains (Figure 5G).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data4-v2.zip
Figure 5—figure supplement 1
The Acetylase activity of Cdu1 is the predominant activity of Cdu1 responsible for protecting Ct inclusions from ubiquitination.
(A) Representative images of Ct inclusions (green, Cap1) decorated with Ubiquitin (magenta, FK2 antibody) in HeLa cells infected for 24 hr. Strains used: WT Ct (L2 434 Bu), cdu1::GII pBOMB MCI, cdu1:…
Figure 6 with 2 supplements
Cdu1 is required for assembly of F-actin, Golgi ministack repositioning, and MYPT1 recruitment to the inclusion.
(A) Examples of representative images of F-actin (arrowheads) (green, Alexa Fluor Phalloidin) assembled around the Ct inclusion (magenta, anti Cdu1 and Cap1 staining) in HeLa cells infected for 40 …
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Figure 6—source data 1
Excel file containing numerical data for quantifying the number of inclusions surrounded by filamentous actin in HeLa cells infected with WT CT, inaC null, inaC null complemented with InaC, cdu1::GII, and cdu1::GII strains expressing Cdu1, Cdu1 C345A, I225A, and K268E variants (Figure 6B).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig6-data1-v2.zip
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Figure 6—source data 2
Excel files containing numerical data for quantifying the number of inclusions surrounded by dispersed Golgi mini stacks in HeLa cells infected with WT CT, inaC null, inaC null complemented with InaC, cdu1::GII, and cdu1::GII strains expressing Cdu1, Cdu1 C345A, I225A, and K268E variants (Figure 6D).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig6-data2-v2.zip
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Figure 6—source data 3
Excel files with numerical data for quantifying the localization of MYPT1 to WT and cdu1::GII inclusions (Figure 6F).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig6-data3-v2.zip
Figure 6—figure supplement 1
The DUB activity of Cdu1 is not required for assembly of F-actin around the Ct inclusion.
Representative images of F-actin (arrowheads) (green, Alexa Fluor Phalloidin) assembled around the Ct inclusion (magenta, antiCdu1 and Cap1 staining) in HeLa cells infected for 40 hr. Strains used: …
Figure 6—figure supplement 2
The DUB activity of Cdu1 is not required for Golgi ministack repositioning around the Ct inclusion.
Representative images of Golgi (anti-GM130 staining, magenta) around Ct inclusions in HeLa cells infected for 24 hr. Strains used: WT L2 (Rif-R 434 Bu, parent of M407), M407 (inaC null strain) …
Figure 7 with 1 supplement
Cdu1, InaC, and IpaM are required for optimal extrusion of Ct inclusions from HeLa cells.
(A) Representative images of extrusions isolated from HeLa cell monolayers infected with Ct strains for 52 hr. Scale bar: 200 μm (B) Quantification of the number of extruded inclusions produced by …
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Figure 7—source data 1
Excel files containing numerical data for quantifying the number of extrusions produced and the size of extrusions in WT CT, cdu1::GII strains, and cdu1::GII strains expressing Cdu1, I225A, and K268E variants from infected HeLa cells (Figure 7B and C).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig7-data1-v2.zip
Figure 7—figure supplement 1
The number of inclusions and the size of inclusions in cdu1 null, inaC null, ipaM null, and ctl0480 null strains are similar across each strain.
(A) Representative images of inclusions in infected HeLa cell monolayers at 48 hpi. Scale bar: 200 μm (B) Quantification of the number of inclusions in infected HeLa cell monolayers. To determine …
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Figure 7—figure supplement 1—source data 1
Excel files with numerical data for quantifying the number of inclusions produced and inclusion size in WT CT, cdu1::GII strains, and cdu1::GII strains expressing Cdu1, I225A, and K268E variants in infected HeLa cells (panels B and C).
- https://cdn.elifesciences.org/articles/87386/elife-87386-fig7-figsupp1-data1-v2.zip
Figure 8
A model for acetylation mediated protection of the Inc proteins InaC, IpaM, and Ctl0480 from degradation.
The cellular Ub machinery targets C. trachomatis effectors, including the Inc proteins InaC, IpaM, and CTL0480 for ubiquitination and subsequent protein degradation. C. trachomatis counters such …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Chlamydia trachomatis L2 434 Bu) | cdu1 | BV-BRC (Bacterial and Viral Bioinformatics resource center); Refseq (NCBI Reference Sequence Database) | fig|471472.4.peg.264 (BV-BRC) CTL0247 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | inaC | BV-BRC; Refseq | fig|471472.4.peg.199 (BV-BRC) CTL0184 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | ipaM | BV-BRC; Refseq | fig|471472.4.peg.511 (BV-BRC) CTL0476 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | CTL0480 | BV-BRC; Refseq | fig|471472.4.peg.516 (BV-BRC) CTL0480 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | cpoS | BV-BRC; Refseq | fig|471472.4.peg.517 (BV-BRC) CTL0481 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | incA | BV-BRC; Refseq | fig|471472.4.peg.406 (BV-BRC) CTL0374 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | slc1 | BV-BRC; Refseq | fig|471472.4.peg.323 (BV-BRC) CTL0299 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | rpoB | BV-BRC; Refseq | fig|471472.4.peg.608 (BV-BRC) CTL0567 (Refseq) | |
| Gene (Chlamydia trachomatis L2 434 Bu) | garD | BV-BRC; Refseq | fig|471472.4.peg.421 (BV-BRC) CTL0390 (Refseq) | |
| Gene (Homo sapiens) | MYPT1 | NCBI | PPP1R12A | |
| Cell line (Homo sapiens) | Hela | ATCC | Cat# CCL-2 RRID:CVCL_0030 | |
| Cell line (Cercopithecus aethiops) | Vero | ATCC | Cat# CCL-81 RRID:CVCL_0059 | |
| Cell line (Homo sapiens) | HEK 293T | ATCC | Cat# CRL-3216 RRID:CVCL_0063 | |
| Cell line (Homo sapiens) | A549 | ATCC | Cat# CCL-185 RRID:CVCL_0023 | |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pOPIN-GFP_Cdu1 FL (aa 1–401) | Jonathan Pruneda (Oregon Health and Science University) | N/A | Used for Cdu1 co-immunoprecipitations in transfected cells |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pOPIN-GFP_Cdu1 TMD- (aa 71–401) | Jonathan Pruneda (Oregon Health and Science University) | N/A | Used for Cdu1 co-immunoprecipitations in transfected cells |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pOPIN-GFP_Cdu1 CD- (aa 1–130) | Jonathan Pruneda (Oregon Health and Science University) | N/A | Used for Cdu1 co-immunoprecipitations in transfected cells |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pCDNA-DEST53 (w/o GFP)_InaC (CT813)–3XFLAG | This paper | N/A | Construct for showing Cdu1-InaC co-IP in transfected cells |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pcDNA3.1/nV5-DEST_IpaM | This paper | N/A | Construct for showing Cdu1-IpaM co-IP in transfected cells |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pcDNA3.1/nV5-DEST_CTL0480 | This paper | N/A | Construct for showing Cdu1-CTL0480 co-IP in transfected cells |
| Transfected construct (Chlamydia trachomatis L2 43 Bu) | pcDNA3.1/nV5-DEST_CpoS | This paper | N/A | Construct for showing lack of Cdu1-Cpos co-IP in transfected cells |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | Chlamydia trachomatis LGV biovar L2 434 Bu (L2) | Richard Stephens (UC Berkeley) | N/A | Reference Chlamydia trachomatis (L2) strain |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 pBOMB-MCI (Parent: LGV L2 434 Bu) | This paper | N/A | L2 strain transformed with empty expression plasmid |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 pBOMB-MCI_CTL0480-3XFlag (Parent: LGV L2 434 Bu) | This paper | N/A | L2 strain transformed with plasmid expressing CTL0480-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 pBOMB-MCI_IpaM-3XFlag (Parent: LGV L2 434 Bu) | This paper | N/A | L2 strain transformed with plasmid expressing IpaM-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 pBOMB-MCI_Cdu1-3XFlag (Parent: LGV L2 434 Bu) | This paper | N/A | L2 strain transformed with plasmid expressing Cdu1-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 pBOMB-MCI_Cdu1C345A-3XFlag (Parent: LGV L2 434 Bu) | This paper | N/A | L2 strain transformed with plasmid expressing Cdu1C345A –3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 pBOMB-MCI_Cdu1K268E-3XFlag (Parent: LGV L2 434 Bu) | This paper | N/A | L2 strain transformed with plasmid expressing Cdu1K268E –3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 cdu1::GII aadA (Parent: LGV L2 434 Bu) | This paper | N/A | L2 cdu1 null strain (SpecR) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 cdu1::GII aadA pBOMB-MCI (Parent: L2 cdu1::GII aadA) | This paper | N/A | L2 cdu1 null strain transformed with empty expression plasmid |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 cdu1::GII aadA pBOMB-MCI_Cdu1-3XFlag (Parent: L2 cdu1::GII aadA) | This paper | N/A | L2 cdu1 null strain transformed with plasmid expressing WT Cdu1-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 cdu1::GII aadA pBOMB-MCI_Cdu1C345A-3XFlag (Parent: L2 cdu1::GII aadA) | This paper | N/A | L2 cdu1 null strain transformed with plasmid expressing WT Cdu1C345A-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 cdu1::GII aadA pBOMB-MCI_Cdu1I225A-3XFlag (Parent: L2 cdu1::GII aadA) | This paper | N/A | L2 cdu1 null strain transformed with plasmid expressing WT Cdu1I225A-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 cdu1::GII aadA pBOMB-MCI_Cdu1K268E-3XFlag (Parent: L2 cdu1::GII aadA) | This paper | N/A | L2 cdu1 null strain transformed with plasmid expressing WT Cdu1K268E-3XFlag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 Rif-R (Parent: L2 434 Bu) | PMID:22232666 | N/A | Rifampin resistant L2 strain (L2 434 Bu) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | M407 (inaC C307T, InaC Q103*) (Parent: L2 Rif-R) | PMID:25920978 | N/A | L2 strain bearing a nonsense mutation in inaC (inaC null srain) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | M407 p2TK2 (Parent: M407) | PMID:25920978 | N/A | L2 inaC null strain transformed with empty expression plasmid |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | M407 p2TK2_InaC (Parent: M407) | PMID:25920978 | N/A | L2 inaC null strain transformed with plasmid expressing WT InaC (untagged) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | M407 p2TK2_InaC-3X Flag (Parent: M407) | This paper | N/A | L2 inaC null strain transformed with plasmid expressing WT InaC-3X Flag |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | inaC::GII bla (Parent: L2 434 Bu) | PMID:28465429 | N/A | L2 inaC null strain (PenicillinR) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | ipaM::GII cat (Parent: L2 434 Bu) | PMID:37530528 | N/A | L2 ipaM null strain (ChloramphenicolR) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | ctl0480::GII aadA (Parent: L2 434 Bu) | PMID:30555802 | N/A | L2 ctl0480 null strain (SpecR) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | M923 (incA C589T, IncA R197*) (Parent: L2 Rif-R) | PMID:25920978 | N/A | L2 strain bearing a nonsense mutation in incA (incA null srain) |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | M923 pBOMB-MCl (Parent: L2 M923) | PMID:28041929 | N/A | L2 incA null strain transformed with empty expression plasmid |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 434 Bu (Parent of L2 garD::GII) | PMID:36084633 | N/A | Wild type L2 parent of garD null strain |
| Biological sample (Chlamydia trachomatis L2 434 Bu) | L2 ctl0390::GII aadA (garD::GII) | PMID:36084633 | N/A | L2 garD null strain (SpecR) |
| Antibody | CT L2 Cdu1 (a.a 71–401) Rabbit polyclonal | This paper | N/A | Dilution IF: 1:100 Dilution WB 1:500 Antibody raised against L2 Cdu1 |
| Antibody | CT Cap1 Rabbit polyclonal | PMID:26981769 | N/A | Dilution IF: 1:250 |
| Antibody | CT CT813 (InaC) (D/UW-3/CX) Mouse monoclonal | PMID:16861671 | N/A | Dilution IF: 1:100 |
| Antibody | CT CTL0480 Rabbit polyclonal | PMID:23727243 | N/A | Dilution IF: 1:100 |
| Antibody | CT IncA Mouse monoclonal | Dan Rockey Oregon State Univ. Corvallis | N/A | Dilution IF: 1:100 |
| Antibody | CT IpaM Mouse monoclonal 12ES IgG1 | PMID:11207561 | N/A | Dilution IF: 1:100 Dilution WB 1:200 See Supplementary file 9 |
| Antibody | CT RpoB Rabbit polyclonal | Ming Tang (UC Irvine) | N/A | Dilution WB 1:100 See Supplementary file 9 |
| Antibody | CT Slc1 Rabbit polyclonal | PMID:24586162 | N/A | Dilution WB 1:400 See Supplementary file 9 |
| Antibody | Hs Acetylated lysine Rabbit polyclonal | Cell signalling | Cat# 9441 RRID:AB_331805 | Dilution WB 1:4000 See Supplementary file 9 |
| Antibody | Hs Alpha tubulin clone B-5-1-2 Mouse monoclonal | Sigma-Aldrich | Cat# T5168 RRID:AB_477579 | Dilution WB 1:500 See Supplementary file 9 |
| Antibody | Flag epitope Mouse monoclonal | Sigma-Aldrich | Cat# F3165 RRID:AB_259529 | Dilution WB 1:1000 See Supplementary file 9 |
| Antibody | Flag epitope Mouse monoclonal | Sigma-Aldrich | Cat# F1804 RRID:AB_262044 | Dilution IF: 1:250 |
| Antibody | Hs GM130 Mouse monoclonal | BD Biosciences | Cat# 610822 RRID:AB_398141 | Dilution IF: 1:1000 |
| Antibody | Lys48-linkage specific polyubiquitin (D9D5) Rabbit monoclonal | Cell signalling | Cat# 8081 RRID:AB_10859893 | Dilution WB 1:1000 See Supplementary file 9 |
| Antibody | Hs MYPT1 Rabbit polyclonal | US Biological | Cat# M9925-01C RRID:AB_2927397 | Dilution IF: 1:100 |
| Antibody | Hs Ubiquitin (P4D1) Mouse monoclonal | Cell signalling | Cat# 3936 RRID:AB_331292 | Dilution IF: 1:50 |
| Antibody | V5 epitope Mouse monoclonal | Abcam | Cat# ab27671 RRID:AB_471093 | Dilution WB 1:5000 See Supplementary file 9 |
| Antibody | Hs RNF213 Rabbit Polyclonal | Sigma | Cat# HPA003347 RRID:AB_1079204 | Dilution IF: 1:1000 |
| Antibody | Rabbit IgG-HRP Goat polyclonal | ThermoFisher | Cat# 31460 RRID:AB_228341 | Dilution WB 1:1000 See Supplementary file 9 |
| Antibody | Mouse IgG-HRP Goat polyclonal | ThermoFisher | Cat# 31430 RRID:AB_228307 | Dilution WB 1:1000 See Supplementary file 9 |
| Antibody | Rabbit IgG-A488 Goat polyclonal | ThermoFisher | Cat# A-11008 RRID:AB_143165 | Dilution IF: 1:1000 |
| Antibody | Rabbit IgG-A647 Goat polyclonal | ThermoFisher | Cat# A-21244 RRID:AB_2535812 | Dilution IF: 1:1000 |
| Antibody | Mouse IgG-A488 Goat polyclonal | ThermoFisher | Cat# A-11001 RRID:AB_2534069 | Dilution IF: 1:1000 |
| Antibody | Mouse IgG-A647 Goat polyclonal | ThermoFisher | Cat# A-21235 RRID:AB_2535804 | Dilution IF: 1:1000 |
| Sequence-based reagent | See Supplementary file 10 | This paper | Primers used in this study | |
| Peptide, recombinant protein | 3xFLAG peptide | APExBIO | Cat# A6001 | Peptide for eluting Flag immunoprecipitated proteins |
| Peptide, recombinant protein | Recombinant human interferon gamma (IFNγ) | Millipore | Cat# IF005 | For inducing Type II interferon responses in cell cultures |
| Peptide, recombinant protein | LGV L2 434 Bu Cdu1 recombinant protein | Jonathan Pruneda (Oregon Health and Science University) | N/A | Recombinant Cdu1 protein used to generate Rabbit polyclonal antibodies against Cdu1 |
| Commercial assay or kit | TargeTron gene knockout system | Sigma-Aldrich | Cat# TA0100 | Kit for generating cdu1::GII null strain |
| Chemical compound, drug | PR-619 (DUB inhibitor) | Sigma-Aldrich | Cat# SML0430 | Used during TUBE affinity enrichment |
| Chemical compound, drug | MG132 | Sigma-Aldrich | Cat# 474791 | Proteasome inhibitor |
| Chemical compound, drug | TUBE-1 | LifeSensors | Cat# UM401M | Pan-polyubiquitination affinity capture reagent |
| Chemical compound, drug | Acti-stain 488 (Phalloidin 488) | Cytoskeleton Inc. | Cat# PHDG1 | Reagent for visualizing Actin by IF |
| Chemical compound, drug | ChromoTek GFP-Trap Agarose | Proteintech | Cat# gta | Agarose for immunoprecipitating GFP tagged proteins |
| Chemical compound, drug | Protein A/G PLUS-Agarose | Santa Cruz Biotechnology | Cat# sc-2203 | Agarose for immunoprecipitations |
| Software | Targetronics | Targetronics, LLC; https://www.targetrons.com/ | Algorithm for generating TargeTron homing sequences | |
| Software | Proteome Discoverer 2.3 | Thermo Fisher; https://www.thermofisher.com/in/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html | Proteomics software | |
| Software | Mascot software | Matrix Science; https://www.matrixscience.com | Proteomics software | |
| Software | MaxQuant 1.6.2.3 | https://www.maxquant.org | Proteomics software | |
| Software | Scaffold PTM | Proteome Software; https://www.proteomesoftware.com/products/scaffold-ptm | Software for viewing proteomics data | |
| Software | VolcaNoseR | PMID:33239692; https://huygens.science.uva.nl/VolcaNoseR/ | Online software for generating volcano plots | |
| Software | Metascape | PMID:30944313; https://metascape.org/gp/index.htm#/main/step1 | Online program for Gene Ontology clustering | |
| Software | DAVID Bioinformatic Resources | PMID:19033363; PMID:19131956 https://david.ncifcrf.gov/tools.jsp | Online program for Gene Ontology clustering | |
| Software | Image J | PMID:22930834; https://imagej.nih.gov/ij/ | Software for image viewing and processing | |
| Software | NEBaseChanger | New England Biolabs; https://nebasechanger.neb.com | Cloning website | |
| Software | Prism 9 | GraphPad; https://www.graphpad.com/updates/prism-900-release-notes | Software for statistical analysis | |
| Software | HCS Studio Cell Analysis Software | Thermo Fischer Scientific | Cat# CX51110 | Software for high content image analysis |
Additional files
-
Supplementary file 1
TUBE1 co-precipitating proteins mass spectrometry data for all biological replicates.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp1-v2.xlsx
-
Supplementary file 2
Numerical data used to generate all volcano plots.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp2-v2.xlsx
-
Supplementary file 3
Numerical mass spectrometry data used to identify high confidence TUBE1 human co-precipitating proteins.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp3-v2.xlsx
-
Supplementary file 4
Numerical data for Gene Ontology (GO) classification of TUBE1 human co-precipitating proteins.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp4-v2.xlsx
-
Supplementary file 5
Numerical mass spectrometry data used to identify high confidence TUBE1 Chlamydia co-precipitating proteins.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp5-v2.xlsx
-
Supplementary file 6
Human ubiquitinated peptides mass spectrometry data.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp6-v2.xlsx
-
Supplementary file 7
Chlamydia ubiquitinated peptides mass spectrometry data.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp7-v2.xlsx
-
Supplementary file 8
Numerical mass spectrometry data used tp identify high confidence ubiquitinated proteins.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp8-v2.xlsx
-
Supplementary file 9
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp9-v2.docx
-
Supplementary file 10
Primer sequences used in this study.
- https://cdn.elifesciences.org/articles/87386/elife-87386-supp10-v2.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/87386/elife-87386-mdarchecklist1-v2.pdf
