Legionella effector LpPIP recruits protein phosphatase 1 to the mitochondria to induce dephosphorylation of outer membrane proteins
Fig 1
L. pneumophila targets mitochondria using T4SS effectors with tail anchors (TA).
(A) Schematic representation of the bioinformatics workflow used to screen for L. pneumophila T4SS effectors (see S1 Text) that contain a TA with net positive tail charges (see S1 Table). (B) Features of the four effectors predicted to contain a TA with a net positive charge in their C-tail. TMHMM and TOPCONS were used to predict the number and location of transmembrane domains (TMDs) present within the 302 effectors screened. A black dot indicates the prediction of one TMD, while a white dot indicates no predicted TMD. The length of the predicted TMD and C-tail, along with the net C-tail charge, is indicated. (C) Representative images of HeLa cells transiently transfected with FLAGLpg1625, FLAGLpg1803, FLAGLpg2344, or FLAGLpg2444. Transfected cells were fixed and processed for immunofluorescence with antibodies against FLAG (green) and NDUFAF2 (mitochondria; magenta). Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. (D) Mitochondrial sub-fractionation and carbonate extraction using mitochondria isolated from HeLa cells transiently transfected with FLAGLpg1625. 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 (+). Mitochondria were also subjected to sodium carbonate extraction, with pellet (P) and supernatant (S) fractions (lanes 7 and 8) separated via 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). (E) HeLa cells transiently transfected with FLAGLpg1625, FLAGLpg16251-108, FLAGLpg162585-130, FLAGLpg1625K128A, and FLAGLpg1625AAA (K97A/R100A/K128A) were processed for immunofluorescence with antibodies against FLAG (green) and NDUFAF2 (mitochondria; magenta). Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm.