DrrA containing the GEF and PI4P-binding domains (amino acids 201C647) localizes to both the Golgi and PM. protein was unable to bind to GST-Rab1 at a level detectable by this assay. (C) Representative images of the localization of the various GFP-DrrA constructs in HEK293 FcRII cells. Individual panels show endogenous staining of the Golgi maker GM130. DrrA made up of the GEF and PI4P-binding domains (amino acids 201C647) localizes to both the Golgi and PM. However, the GEF domain name alone (201C500) is sufficient for Golgi localization. The PI4P-binding domain name of DrrA (501C647) shows predominantly PM localization. This region is also the minimal region found to bind to plasma-membrane syntaxins (see Figure S3), however without the PI4P-binding region (amino acids 451C545) PM targeting does not occur. (D) Confocal xy images of HEK293 FcRII cells transfected with constructs expressing GFP-DrrA or EYFP-Lpg1101 or EYFP-Lpg2603 or mutant protein variants, and RFP-PALM. (E) Summary of GFP- or YFP-tagged truncation constructs tested for localization to the PM in HEK293 FcRII cells. Unshaded (white) constructs did not show plasma membrane (PM) localization. Constructs shaded grey or black gave a PM signal. In grey are the minimal C-terminal domain name constructs that gave a PM signal.(TIF) ppat.1004222.s001.tif (3.9M) GUID:?6D050152-F01D-4D6E-8BEA-239540D98FC7 Figure S2: Intracellular growth analysis HVH3 of single and triple LEPR mutants. Defects in intracellular replication of single or the triple LEPR mutants were not observed in macrophages or in amoeba. (A) Fold change in colony forming models over 72 hours of strains in A/J bone marrow-derived macrophages with an MOI of 1 1. Results are from two impartial experiments, with triplicate wells in each experiment. (B) Graph showing fold change in relative luminescence Phenoxodiol models (RLU) of strains in THP-1 cells using a 96-well plate format. Results shown Phenoxodiol are from two impartial experiments as indicated and represent the average of 8C12 wells per assay. (C) Fold change in colony forming models over 48 hours of strains in with an MOI of 1 1. Data represent the average from two impartial experiments performed in triplicate.(TIF) ppat.1004222.s002.tif (756K) GUID:?4E92307B-BD7D-4490-9540-317A4EF69DE9 Figure S3: Lpg1101 and Lpg2603 are functionally distinct compared to DrrA. Western-blot images showing co-immunoprecipitation of (A) GFP-tagged DrrA 200C500, 451C647, 501C647, 451C545 or 546C647, or (B) EYFP-tagged-Lpg1101 or EYFP-tagged 2603 proteins and FLAG-tagged SNARE proteins produced in HEK293 FcRII cells. Interactions were examined after precipitation of the SNARE proteins from cells extracts using anti-FLAG agarose. The antibodies indicated to the right of each blot show protein levels in the blots of the lysate (2.5C4% of input) and blots of the immunoprecipitate (IP). (C) Representative fluorescent micrographs (100) of CHO FcRII cells co-transfected with EYFP-Rab1a and mRFP-Lpg1101 or mRFP-LPg2603. The blue fluorescence is usually from 4,6-diamidino-2-phenylindole (DAPI) staining.(TIF) ppat.1004222.s003.tif (2.2M) GUID:?F641F7B5-544C-4586-9414-ACAAA70A196D Physique S4: The LEPR is usually important for localization to the PM. (A) Table summarizing the localization of EYFP-Lpg2603 site-mutants assessed by epifluorescence microscopy in HEK293 FcRII cells. (B) Examples from summary table A. Epifluorescent micrographs of CHO FcRII cells transfected with EYFP-Lpg2603 and mutant derivatives G354A and D355E,K358R. Shown in red is phalloidin staining. Arrows indicate fluorescence overlap between peripheral actin (phalloidin) and Lpg2603. (C) Representative images of HEK293 cells expressing GFP-tagged DrrA constructs 61C647, 451C647 and the minimal PM localization region 501C647. Data shows the effect of single and double amino acid substitutions at positions 565 and 568 within DrrA.(TIF) ppat.1004222.s004.tif (4.3M) GUID:?F39D5B25-D9FA-451B-A495-1A9DB7186E8A Figure S5: The MIM domain is important for PM-localization of DrrA. Micrographs of confocal Z-stacks of ectopically expressed GFPDrrA501C647 and lysine mutant variants (L610A, Phenoxodiol L614/615A, L617A, L610/614/615A, L614/615/716A, L610/614/615/617A).