Pathophysiology of Plasma Membrane PI4P Generation

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 DESCRIPTION (provided by applicant): Phosphorylated metabolites of phosphatidylinositol, called phosphoinositides, play major constitutive and regulatory roles in a wide variety of physiological processes. Their generation and interconversions, via phosphorylation and dephosphorylation of the 3, 4 and 5 position of the inositol ring, are mediated by a large number of kinases and phosphatases. An especially important enzyme for this metabolism is phosphatidylinositol 4-kinase type IIIa (PI4KIIIa), as this protein is responsible for the generatin of the bulk of phosphatidylinositol-4-phosphate (PI4P) at the plasma membrane. This PI4P pool is a key determinant of plasma membrane identity and the precursor of the majority of cellular PI (4, 5) P2 and thus also its downstream metabolites, such as PI (3, 4, 5) P3, DAG and IP3. Thus, the function of PI4KIIIa impacts numerous fundamental processes, including exo-endocytosis, actin dynamics, transport of ions and other substances across the plasma membrane, signal transduction and regulation of the cell cycle. PI4KIIIa is part of a protein complex conserved from yeast to humans. The kinase PI4KIIIa itself (expressed by a single gene in all species) is essential for cell life, whereas defects in accessory factors (EFR3, TTC7 and FAM126, all three encoded by a pair of genes in mammals) have been implicated in human diseases. The fundamental importance of the PI4KIIIa complex for cellular physiology together with its emerging connections to pathological conditions argues forcefully for the need to understand its structure and function. We will use a comprehensive approach that combines cell biology, structural biology and biochemistry to investigate the function and regulation of the PI4KIIIa complex at molecular, cellular, and organismal levels. In Aim 1, to obtain mechanistic insights as to how the PI4KIIIa accessory factors target the kinase to the PM and regulate its activity there, we will investigate the molecular architecture of the PI4KIIIa complex. We will obtain crystal structures for complex components or their subassemblies and then piece together the entire complex from these, making use of biochemical and electron microscopy (EM) information. Structures for EFR3, TTC7 and a TTC7/FAM126 subassembly already available from preliminary work, together with biochemical and cell-based experiments, define the molecular mechanism for PI4KIIIa recruitment to the PM. They further suggest a mechanism for regulating complex assembly that we will investigate, along with the roles of the accessory factors in modulating the catalytic activity of PI4KIIIa. Aim 2 is complementary, with biochemistry and imaging studies of these proteins in cell extracts and living cells (mouse and human derived cells) to determine the dynamics of the complex and of its subunits in their natural context in mammalian organisms. In this aim, we will test our hypotheses that depletion of these subunits causes a defect in plasma membrane PI4P synthesis, and, further, that this defect underlies disease pathogenesis in humans.