Legionella pneumophila Exploits PI(4)P to Anchor Secreted Effector Proteins to the Replicative Vacuole
Figure 5
Binding of L. pneumophila Icm/Dot–Secreted Proteins to PIs In Vitro
(A) Binding of affinity-purified GST fusion proteins of SidC, SdcA, SidD, or SdhB (160 pmol) to different lipids (100 pmol; left panels) or 2-fold serial dilutions of PIs (100–1.56 pmol; right panels) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti-GST antibody. Lysophosphatidic acid is denoted by LPA; lysophosphocholine is denoted by LPC; sphingosine-1-phosphate is denoted by SP; phosphatidic acid is denoted by PA; and phosphatidylserine is denoted by PS. The experiment was reproduced at least three times with similar results.
(B) PL vesicles (20 μl, 1 mM lipid) composed of PC (65%), PE (30%), and 5% (1 nmol) either PI(4)P, PI(3)P or PI(4,5)P2 were incubated with affinity-purified GST-SidC or GST-SidD (40 pmol), centrifuged, and washed. Binding of GST fusion proteins to PL vesicles was assayed by Western blot with an anti-GST antibody. Similar results were obtained in three separate experiments.