Analysis of phosphoinositide-binding proteins using liposomes as an affinity matrix.

Phosphorylated derivatives of phosphatidylinositol (PI), collectively called phosphoinositides, regulate many important cellular signaling pathways. Lipid signaling disorders are linked to the development of several severe diseases in humans (1). Consequently, there is an increasing interest in the identification of proteins that interact with these lipids. The recent discovery of phosphoinositide-binding domains such as pleckstrin homology (PH), Fab1, YOTB, Vac1, and EEA1 (FYVE), phox homology (PX), and epsin N-terminal homology (ENTH) domains (2) has sped up the discovery of proteins that bind to phosphorylated lipids. Nitrocellulose strips containing immobilized lipids have been used to assess the phosphoinositide-binding properties of proteins (3). Likewise, phosphoinositides covalently coupled to Sepharose® beads have been employed to identify novel lipidbinding proteins (4,5). However, there are several disadvantages connected with these approaches. Extensive chemical synthesis is required to obtain phosphoinositide derivatives that can be linked to affinity matrices (4,5). Furthermore, a high number of nonspecific binding partners usually attach to the affinity matrix as well. Binding assays that use lipids immobilized on nitrocellulose membranes often produce rather moderate affinity and low specificity associations with the immobilized lipids, which may inaccurately reflect the in vivo characteristics of a particular protein-lipid interaction (3). Connected with these problems is the recent discovery that the binding specificities of lipid-interacting domains is not determined by their lipid-binding domains alone, but requires additional interactions (6,7). Several liposome-based assays for analyzing binding specificities of recombinant proteins have also been described (8–12). However, the published examples required expression and usually immobilization of recombinant proteins on solid support. Consequently, these methods require construction of respective expression vectors, and more importantly, they are not suitable for purification or identification of additional lipid binding factors. Here we describe a novel affinity binding protocol that uses phosphoinositide-containing liposomes as a matrix for protein binding. Different from other liposome-based assays, this method allows affinity purification of lipid-binding proteins from crude subcellular fractions. The