Legionella effector LpPIP recruits protein phosphatase 1 to the mitochondria to induce dephosphorylation of outer membrane proteins
Fig 5
LpPIP does not specify substrates selectivity for PP1.
(A) HeLa cells transiently transfected with FLAGLpPIP or FLAGLpPIP-CYB5ATA were stained with 100 nM MitoTracker Deep Red FM (mitochondria; MTDR) and immunostained with antibodies against FLAG (green) and PDI (ER; magenta). Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. (B) Pearson correlation analysis of FLAG or PDI and PPP1CB fluorescence signals was performed on FLAGLpPIP-CYB5ATA expressing HeLa cells and untransfected wild-type HeLa cells (n = 3 independent experiments, 16 cells per experiment). Data are shown as the mean ± SEM of three independent experiments. Larger circles represent mean values from each experiment, and smaller circles represent individual cell values. Unpaired t test was performed on the means, revealing a significant difference of 0.35 ± 0.03 between the two groups, with a p-value of 0.0005. Representative images are shown in S4B Fig. Corresponding raw data are available in the Supporting information (S1 Data). (C) Whole cell FLAG co-immunoprecipitation was performed using untransfected HeLa cells, HeLa cells expressing FLAGLpPIP, or FLAGLpPIP-CYB5ATA (n = 3 technical replicates). The Log2 difference of proteins enriched in FLAGLpPIP and FLAGLpPIP-CYB5ATA relative to untransfected cells is shown. Proteins with significant fold change (± 1.5-fold change with a p-value ≤ 0.05) are represented as filled circles, while non-significant differences are shown as hollow circles. Mitochondrial proteins based on MitoCarta3.0 are indicated in orange, and ER proteins based on the Human Protein Atlas are indicated in blue. Corresponding data are available in the Supporting information (S6 Table). (D) Volcano plot showing proteins enriched following FLAG co-immunoprecipitation from HeLa cells expressing FLAGLpPIP-CYB5ATA compared to untransfected cells (n = 3 technical replicates). Mitochondrial proteins based on MitoCarta3.0 are indicated in orange, and ER proteins based on the Human Protein Atlas are indicated in blue. Corresponding data are available in the Supporting information (S6 Table). (E) Scatterplot comparing phosphopeptides abundances in whole cell samples from HeLa cells transfected with FLAGLpPIP to empty vector pCDNA5 (n = 4 technical replicates). Data were filtered for serine and threonine phosphopeptides. The significance thresholds were set to a ±1.5-fold change with a p-value of 0.01 using Student t test. Phosphopeptides derived from mitochondrial outer membrane proteins (MitoCarta3.0) and ER proteins (Human Protein Atlas) are labeled in orange and blue, respectively. Non-imputed phosphopeptides are shown as filled circles, while phosphopeptides with imputed values are shown as hollow circles. Corresponding data are available in the Supporting information (S7 Table). (F) Scatterplot comparing phosphopeptides abundances in whole cell samples from HeLa cells transfected with FLAGLpPIP-CYB5ATA to empty vector pCDNA5 (n = 4 technical replicates), processed as described in Fig 5E. Corresponding data are available in the Supporting information (S7 Table).