Methods in Molecular Biology Volume 954, 2013, pp 367-380

Interactions of Legionella Effector Proteins with Host Phosphoinositide Lipids

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Abstract

By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anchors the effector proteins SidC and SidM, which promote the interaction of LCVs with the ER and the secretory vesicle trafficking ­pathway. In this chapter, we describe protocols to (1) identify PI-binding proteins in Legionella lysates using PI-beads, (2) determine PI-binding specificities and affinities of recombinant Legionella effector proteins by protein–lipid overlays, and (3) use Legionella effectors to identify cellular PI lipids.