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. 2022 Jan 27;11:e73611. doi: 10.7554/eLife.73611

Figure 5. Ceg3 inhibits ADP/ATP exchange in mitochondria.

(A) Ceg3 interferes with the mitochondrial membrane potential (MMP). HEK293T cells transfected to express Ceg3 or its inactive mutant Ceg3E/A were used to determine MMP by the JC-10 dye. Samples treated with 20 μM CCCP for 1 hr were included as a positive control for loss of MMP integrity. Quantitation shown was from three independent experiments done in triplicate. Error bars: standard error of the mean (SEM). Statistical analysis was determined by two-tailed t-test. ns, not significant; *, p<0.05; **, p<0.01. (B) The workflow for measuring ADP/ATP exchange rates. (C) Ceg3 interferes with the ADP/ATP exchange by mitochondria. Mitochondria isolated from HEK293T cells expressing the indicated proteins were suspended in a reaction buffer containing 10 mM HEPES (pH 7.4), 250 mM sucrose, and 10 mM KCl. 2 mM ADP was added to initiate the ADP/ATP exchange process. After 5 min incubation, the concentrations of ATP transported from mitochondria were determined to calculate the ADP/ATP exchange rates. Quantitation shown was from three independent experiments. Error bars: standard error of the mean (SEM). Statistical analysis was determined by two-tailed t-test. ns, not significant; *, p<0.05. (D) Ceg3 perturbs ADP/ATP exchange in cells infected with Legionella pneumophila. Opsonized bacteria of the indicated L. pneumophila strains were used to infect HEK293T cells expressing the FcγII receptor at an MOI of 100 for 2 hr. Mitochondria isolated from the infected cells were used to determine ADP/ATP exchange rates. Results shown were from three independent experiments. Error bars: standard error of the mean (SEM). Statistical analysis was determined by two-tailed t-test. ns, not significant; *, p<0.05; **, p<0.01. (E) A diagram depicting the inhibition of mitochondrial ADP/ATP exchange by Ceg3. Ceg3-induced modification of ANTs by ADPR in the inner membrane blocks the ADP/ATP transport activity of the translocases. MOI, multiplicity of infection; NI, no infection.

Figure 5.

Figure 5—figure supplement 1. ADP-ribosylation of ANTs by Ceg3 does not affect mitophagy induction.

Figure 5—figure supplement 1.

(A) CCCP induces mitophagy in COS-1 cells. Lysates of the indicated cell lines that have been treated with 20 μM CCCP for 24 hr were resolved by SDS-PAGE and then probed for PDHA1 (mitochondrial matrix protein) and ATPB (mitochondrial inner membrane protein). Tubulin was probed as a loading control. (B) Ceg3 expressed in COS-1 cells by lentiviral transduction induced ADP-ribosylation of ADP/ATP translocase. Lysates of COS-1 cells transduced with a lentivirus harboring the indicated plasmid at an MOI of 10 for 2 days were probed for ADPR modification, tubulin was probed as a loading control. (C) Ceg3 expressed in COS-1 cells does not affect the protein level of PDHA1 regardless of CCCP treatment. COS-1 cells expressing the indicated proteins for 1 day were treated with 20 μM CCCP for another 24 hr, cells treated with DMSO were used as controls. PDHA1 was probed to evaluate mitophagy, tubulin was detected as a loading control. MOI, multiplicity of infection.