Many peripheral proteins involved in cell signaling and membrane trafficking are targeted to specific cell membranes in response to the spatiotemporal dynamics of phosphorylated derivatives of phosphatidylinositiol (phosphoinositides; PI). However, the mechanisms by which different PIs mediate the membrane targeting and activation of their effector proteins are only beginning to unravel. Based on our previous structure-function studies on membrane targeting domains, which revealed that much of subcellular targeting by membrane targeting domains can be accounted for by the biophysical principles that govern their binding to model membranes in vitro, we have investigated the membrane targeting by recently discovered PI-binding domains, FYVE (Fab1p, YOTB, Vac1p, and EEA1) and PX (Phox) domains. These studies have shown that PIs specifically induce the membrane penetration of FYVE and PX domains by causing changes in protein conformation and electrostatic potential and that this process is essential for their membrane binding and cellular activities. The primary objective of this proposed research is to extend these studies to a broad range of PI-binding domains, including FYVE, PX, and ENTH (Epsin N-Terminal Homology) domains, and to fully elucidate the mechanisms by which various PIs induce their in vitro and cellular membrane targeting.
Specific aims for this proposed research are as follows: 1) Determination of the in vitro and cellular membrane targeting mechanisms of various FYVE domains with different membrane affinities; 2) Determination of the in vitro and cellular membrane targeting mechanisms of PX domains with different PI specificities; 3) Determination of the in vitro and cellular membrane targeting mechanisms of two distinct ENTH domains. The principal methodologies to be used include: (1) the biophysical analysis of interactions of PI-binding domains with various model membranes by monolayer, sedimentation, surface plasmon resonance, and fluorescence correlation spectroscopy measurements; (2) the quantitative analysis of fluorescent protein-tagged N-binding domains and their mutants transfected into mammalian cells to determine the rates of membrane translocation and the dissociation constants for membrane binding.
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