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

Research output: Contribution to journalArticle

Authors

  • Frank T. Cooke
  • Gavin F. Painter
  • Andrew B. Holmes
  • Assia Shisheva
  • Yoshikuza Ohya
  • Peter J. Parker

Colleges, School and Institutes

External organisations

  • School of Biochemistry
  • Cancer Research UK
  • University of Cambridge
  • Wayne Stt. Univ. School of Medicine
  • Tokyo Metropolitan University

Abstract

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.

Details

Original languageEnglish
Pages (from-to)33905-33912
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number48
Publication statusPublished - 26 Nov 1999