Legionella effector LpPIP recruits protein phosphatase 1 to the mitochondria to induce dephosphorylation of outer membrane proteins
Fig 2
Lpg1625 recruits PP1 to mitochondria via an RVxF motif.
(A) Mitochondria were isolated from wild-type HeLa cells or cells transiently transfected with FLAGLpg1625 and solubilized in 1% digitonin prior to immunoprecipitation with anti-FLAG conjugated agarose beads. Eluates were analyzed by mass spectrometry. The Log2 fold change of the mean LFQ intensity is plotted against −Log10 p-value (n = 3 technical replicates). The curve indicates significantly enriched proteins (FDR = 0.05, s0 = 1). Mitochondrial outer membrane proteins are labeled in orange with the annotation from MitoCarta 3.0. Corresponding data are available in the Supporting information (S3 Table). (B) AlphaFold [43] was used to predict the structure of Lpg1625, which was labeled with the RVxF motif and transmembrane domain (TMD) using ChimeraX [44]. The model has an average per-residue model confidence score (pLDDT) of 65.94, indicating low model confidence. (C) Mitochondria sub-fractionation and carbonate extraction were performed using mitochondria isolated from HeLa cells transiently transfected with FLAGRVxFdead. Mitochondria were either left intact (lanes 1 and 2), subjected to hypotonic swelling (lanes 3 and 4), or solubilized with 0.5% Triton X-100 (lanes 5 and 6). Samples treated with Proteinase K (PK) are indicated (+). For carbonate extraction, mitochondria were washed in a fresh solution of 0.1 M sodium carbonate and integral (pellet, P) and soluble (supernatant, S) proteins were separated using ultracentrifugation. Samples were analyzed using SDS-PAGE and immunoblotting using the indicated antibodies. Corresponding raw images are available in the Supporting information (S1 Raw Images). (D) Mitochondria isolated from wild-type HeLa cells or cells transiently transfected with FLAGRVxFdead were solubilized in 1% digitonin buffer prior to immunoprecipitation with anti-FLAG conjugated agarose beads. Eluates were processed for mass spectrometry. The Log2 fold change of mean LFQ intensity is plotted against −Log10 p-value (n = 3 technical replicates). The curve indicates significantly enriched proteins (FDR = 0.05, s0 = 1). Mitochondrial outer membrane proteins are labeled in orange with the annotation from MitoCarta3.0. Corresponding data are available in the Supporting Information (S3 Table). (E) HeLa cells expressing FLAGLpPIP, or FLAGRVxFdead were stained with 100 nM MitoTracker Deep Red FM (mitochondria; MTDR) and, following fixation, processed for immunofluorescence using antibodies against FLAG (green) and PPP1CB (magenta). Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. Fluorescence signal profiles of FLAG and PPP1CB were measured across the indicated line of interest. Corresponding raw data are available in the Supporting information (S1 Data). (F) The correlation between PPP1CB and FLAGLpPIP was assessed using the Pearson correlation coefficient (r) in HeLa cells expressing FLAGLpPIP or FLAGRVxFdead (n = 3 independent experiments, 45 cells in total). Data represent mean ± SEM of the three experiments represented in three different colors. Larger circles represent the mean values from each experiment, while smaller circles are values from individual cells. An unpaired t test on the means revealed a significant difference of 0.59 ± 0.02 between groups (p-value < 0.0001). Corresponding raw data are available in the Supporting information (S1 Data). (G) Proportion of PPP1CB localized to mitochondria in untransfected and HeLa cells transiently transfected with FLAGLpPIP or FLAGRVxFdead, was calculated using raw integrated density in Fiji. Representative images are shown in Fig 2E. Data are presented as mean ± SEM (n = 3 independent experiments, 5 cells each). Ordinary one-way ANOVA showed no significant difference between the groups. Corresponding raw data are available in the Supporting information (S1 Data). (H) AlphaFold3 predicted structure of LpPIP-PPP1CB complex (ipTM = 0.86; pTM = 0.85) labeled with the RVxF motif on LpPIP (green) and known RVxF binding pocket on PPP1CB (red). Image generated using ChimeraX [44]. (I) AlphaFold3 structure of LpPIP-PPP1CB complex labeled with the active site (purple) and three substrate binding grooves (yellow) of PPP1CB.