Phosphoinositides (PIs) control numerous cellular processes such as cell signaling, proliferation, organization of cytoskeleton, membrane trafficking, ion channel activity, transcription and mRNA trafficking. Mis-regulated PI metabolism has been linked to a number of human hereditary diseases, including certain cancers, diabetes, Lowe's syndrome, Bipolar disorder, Charcot-Marie-Tooth disease (CMT) and Amyotrophic Lateral Sclerosis (ALS). One class of PI metabolizing enzymes contains a conserved PI phosphatase module named Sac. Despite considerable attention, little is known about the molecular properties of this family of PI phosphatases. For example, Sac domains in different proteins prefer a specific subgroup of PIs as substrates, but how the substrate specificity is determined by the otherwise homologous Sac domains is unresolved. Moreover, the mechanisms for the regulation of enzymatic activity are still largely unknown. Our overall Research Goal is to elucidate the molecular mechanisms underlying substrate specificity, catalytic function and regulation, and intra-family diversity of this essential PI phosphatases family. Towards this goal, we have recently solved the crystal structure of the conserved Sac domain from yeast Sac1, the first structure of the Sac domain-containing phosphatase family. Our crystal structure of the Sac phosphatase domain reveals a striking configuration of the catalytic motif and a large positively charged groove at the catalytic site. The crystal structure of the Sac domain, as well as our preliminary biochemical data also suggests that Sac phosphatases may form a dimer and the dimerization of Sac domain may play a role in functional regulation. Based on these structural features, we propose to further pursue the molecular mechanisms of the Sac protein family with the following three Specific Aims: (1) Delineate the catalytic mechanism and the substrate specificity of Sac phosphatases; (2) Probe the membrane interaction and the mechanism for interfacial catalysis of Sac1; (3) Elucidate the mechanism for the regulation of enzymatic activity of Sac1. We will apply a multi-disciplinary approach, including structural biology, molecular biology, biochemistry, and cell biology tools to address our specific aims. We expect with our long term efforts, we will gain new knowledge about the molecular basis for the function of this Sac domain-containing phosphatase family. Given that these enzymes play a house-keeping role in normal cellular function and that mutations of these enzymes are associated with neuronal degeneration diseases, we also anticipate a close Relevance to Human Health. The relevance to public health also comes from the fact that some PI phosphatases in pathogenic bacteria have been found to be used as weapons to invade and thrive in host cells and the fact that the in vivo PI levels affect host cell defense against viral infections. Establishing the molecular basis for the Sac phosphatases will be extremely valuable in the understanding of their basic cell biology and may also provide candidate targets for therapeutic intervention under conditions of endogenous genetic mutations or exogenous pathogenic infections.