Legionella effector LpPIP recruits protein phosphatase 1 to the mitochondria to induce dephosphorylation of outer membrane proteins
Fig 3
Recruitment of PP1 by LpPIP dephosphorylates mitochondrial proteins.
(A) Schematic of the phosphoproteomics workflow used in this study. Wild-type Flp-In T-REx 293 cells and stable cell lines expressing FLAGLpPIP or FLAGRVxFdead were induced with 1 µg/mL tetracycline for 24 h. Proteins from isolated mitochondria were trypsinized and cleaned by solid phase extraction before phosphopeptide enrichment using titanium dioxide (TiO2) beads. Global proteomics and phosphopeptide-enriched samples were analyzed by quantitative mass spectrometry and processed using Spectronaut and Perseus. (B) Scatterplot comparing mitochondrial protein abundance between Flp-In T-REx 293 stable cells expressing FLAGLpPIP or FLAGRVxFdead (n = 3 technical replicates). Log2 fold change of mean LFQ intensity is plotted against −Log10 p-value. Significance thresholds were set at ± 1.5-fold change and p-value = 0.01. Corresponding data are available in the Supporting information (S5 Table). (C) Scatterplot comparing mitochondrial phosphopeptide abundances between Flp-In T-REx 293 cells expressing FLAGLpPIP and FLAGRVxFdead (n = 3 technical replicates). Data was filtered for mitochondrial and mitochondrial-interacting proteins using the MitoCarta3.0 annotation and restricted to serine/threonine phosphopeptides. Significance thresholds were set at ± 1.5-fold change and p-value of 0.01 using Student t test. Phosphopeptides from mitochondrial outer membrane proteins are labeled in orange. Hollow circles indicate phosphopeptides with imputed values; solid circles represent those without. Corresponding data are available in the Supporting information (S5 Table). (D) Heatmap of the mitochondrial phosphopeptides significantly reduced in Flp-In T-REx 293 stable cells expressing FLAGLpPIP relative to wild-type (n = 4 technical replicates) and cells expressing FLAGRVxFdead (n = 3 technical replicates). Average of the Log2 abundance of phosphopeptide for each group was used to generate the heatmap in Prism 10. Missing values are indicated by crosses. Corresponding data are available in the Supporting information (S5 Table). (E) pLogo [56] sequence motif analysis of the seven residues flanking 39 phosphoserine/phosphothreonine (pSer/pThr) sites significantly dephosphorylated in FLAGLpPIP expressing cells (Fig 3C). Arginine (R) at position −3 relative to pSer/pThr was significantly enriched (log-odd probability of 6.213 and p-value of 1.715 e−4). (F) Gene ontology (GO) biological process enrichment analysis of mitochondrial proteins with significantly downregulated phosphopeptides (Fig 3D), performed using ShinyGO 0.80 [57]. The top 10 enriched GO terms are shown. Corresponding data are available in the Supporting information (S2 Data).