Complementation analysis in PtdInsP kinase-deficient yeast mutants demonstrates that Schizosaccharomyces pombe and murine Fab1p homologues are phosphatidylinositol 3-phosphate 5-kinases

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) is widespread in eukaryotic cells. In Saccharomyces cerevisiae, PtdIns(3,5)P₂ synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P₂ is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P₂ synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P₂ is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P₂ in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1 homologues both restored basal PtdIns(3,5)P₂ synthesis in Δfab1 cells and made PtdIns(3,5)P₂ in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I Pipkin supported no PtdIns(3,5)P₂ synthesis. Thus, FAB1 and its homologues constitute a distinct class of Type III Pipkins dedicated to PtdIns(3,5)P₂ synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Δfab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P₂ synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.