Liposome-Based Methods to Study GTPase Activation by Phosphoinositides.

ABSTRACT

Phosphoinositides (PIPs) are lipid messengers with different functions according to their localization. After their local production by the action of lipid kinases or phosphatases, PIPs regulate various biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, or gene expression through binding of their phosphorylated inositol head group with different protein domains such as PH, PX, and FYVE. It is well known that PIPs regulate the activity of small GTPases by interacting with and activating Guanyl-nucleotide Exchange Factor (GEF) proteins through specific domains such as the ones mentioned above. However, most of the in vitro assays to assess the activation of GTPases focus on the GTPase only and neglect the fact that co-activators, such as membranes and protein activators, have a significant effect in vivo. Herein, we describe not only the classical protein-lipid overlay and liposome sedimentation methods but also an assay we have developed, which contains three partners a liposome which composition reproduces the membrane of the target of the GTPase, the recombinant specific DH-(PIP affinity) GEF domain, and the recombinant GTPase to be tested by different PIPs. This assay allows us to clearly quantify the GTPase activation.