Figure 2.

Phospholipids PS and PIP2 guide plasma membrane association of Bordetella effector BteA and its LRT motif in vivo. Localization of GFP-tagged LRT domain (LRT–GFP) and full-length BteA (BteA–GFP) of B. pertussis was analyzed after galactose induction in the following S. cerevisiae strains. A, wildtype S. cerevisiae BY4742 (WT) and its cho1Δ derivative deficient in PS synthesis. B, wildtype S. cerevisiae BY4741 (WT) and its dgk1Δ derivative deficient in PA synthesis. C, wildtype S. cerevisiae SEY6210 (WT) and its temperature-sensitive mss4^ts derivative exhibiting depleted levels of PIP2 after the shift from permissive (25 °C) to restrictive temperature (38 °C). White arrows indicate the relocalization of the LRT–GFP protein from the plasma membrane to internal puncta in the mss4^ts mutant. The expression of the GFP-tagged phospholipid probes GFP-Lact-C2 (PS-specific probe), GFP-Spo20^51–91 (PA-specific probe), and GFP-2xPH(PLCδ) (PIP2-specific probe) was used to visualize cellular PS, PA, and PIP2 levels, respectively. No difference in the localization of PA probe was observed in the dgk1Δ mutant (see text for further details). Representative images from two independent experiments with the same outcome are presented. Scale bar, 5 μm. LRT, lipid raft targeting; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PS, phosphatidylserine.