| Phosphoinositides (PIs), important regulators of vesicle trafficking, the actin cytoskeleton, and proliferation, are modulated by the inositol polyphosphate 5-phosphatases (5-ptases). Saccharomyces cerevisiae has four 5-ptases encoded by INP51, INP52, INP53, and INP54 . This redundancy indicates the importance of these enzymes, but invites the question of why cells require multiple enzymes with the same activity. All four enzymes have 5-ptase activity that hydrolyzes PI(4,5)P2 to PI(4)P, while both Inp52p and Inp53p, as well as yeast Sac1p, also contain a SAC1-like domain with separate polyphosphoinositide phosphatase (PPIPase) activity that dephosphorylates PI(3)P, PI(4)P and PI(3,5)P2 all to PI. While deletion of individual INP5s is not lethal, double knockouts are growth inhibited, and simultaneous disruption of INP51, INP52, and INP53 results in inviability. Electron micrographs of inp5 mutants reveal plasma membrane invaginations, abnormal vesicular structures, and unusually thick cell walls. Complementation studies indicate that both the 5-ptase and the PPIPase domains found in these enzymes are crucial for cell viability, suggesting that the deregulation of the phosphoinositide signaling pathway is detrimental to the cell. Genetic studies revealed an interaction between Inp51p and Sac1p, suggesting that although these proteins exhibit different enzymatic activity, they may function in parallel pathways. A multi-copy suppression screen performed on the inp51 inp52 inp53 triple mutant to identify processes or proteins regulated by the 5-ptases identified SNAREs involved in secretion and regulators of MAP kinases implicated in cell wall biosynthesis. A putative ARF GAP that may modulate PI metabolism was also found. Additionally, the disruption of INP51 is able to overcome the polarized secretion defect exhibited by a CDC42 temperature sensitive mutant. These results indicate that at least one function of the Inp5p proteins is to regulate cellular secretion. Localization of a PH domain that specifically binds PI(4,5)P2 in various inp5 mutants demonstrates that distinct pools of PI(4,5)P2 exist in cells, providing some evidence that each 5-ptase functions in a different cellular compartment. These results provide strong evidence to suggest that the 5-ptases of S. cerevisiae perform discrete functions within the cell, and that these enzymes most likely regulate distinct steps involved in vesicle trafficking. |
