Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with Gproteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with a SacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain.Inositol phosphates play a role in a variety of eukaryotic cellular processes, including chemotaxis and membrane trafficking. They are regulated by a number of enzymes. The group of phosphatidylinositol 3-kinases (PI3K) 1 phosphorylates the lipid substrates PI, PI(4)P, and PI(4,5)P 2 at the 3-position of the inositol ring (1). The lipid product PI(3,4,5)P 3 has been strongly implicated to be important in chemotaxis in neutrophils and fibroblasts (2, 3). PTEN, identified as a tumor suppressor gene (4), reverses the action of PI3K by dephosphorylation of PI(3,4,5)P 3 and PI(3,4)P 2 at the 3-position (5). Another group of enzymes, the inositol 5-phosphatases, can remove the phosphate group at the 5-position of the inositol ring (6 -8). The importance of inositol 5-phosphatase activity in PI(3,4,5)P 3 regulation is demonstrated by SHIP1. In stimulated B-cells, SHIP1 accounts for the major phosphatase activity toward PI(3,4,5)P 3 , and inactivation of SHIP1 leads to an increased and prolonged PI(3,4,5)P 3 production (9). Other inositol 5-phosphatases have been shown to play important roles in a number of cellular processes. Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome in human (10), and deletion of the presynaptic inositol 5-phosphatase Synaptojanin leads to neurological abnormalities and early death of mice (11).In the social amoeba, Dictyostelium discoideum chemotaxis toward folic acid and cAMP is an essential strategy for survival (12). Several observations suggest that phosphatidylinositol phosphates mediate chemotaxis and, in particular, the localization of the signal inside D. discoideum cells. The PH domains of a number of proteins involved in chemotaxis, including CRAC, Akt/PKB, and PhdA, have been shown to transiently localize at the leading edge of cells moving in a chemotactic gradient (13-15). As these PH domains bind to PI(3,4,5)P 3 and PI(3,4)P 2, an asymmetric lipid distribution is implicated by...