View the Current Issue

Article

  • The EMBO Journal (2000) 19, 4577 - 4588
  • doi:10.1093/emboj/19.17.4577

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells

David J. Gillooly1, Isabel C. Morrow2,3, Margaret Lindsay2,3, Robert Gould3, Nia J. Bryant2, Jean-Michel Gaullier1, Robert G. Parton2,3,4 and Harald Stenmark1,4

  1. Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
  2. Centre for Molecular and Cellular Biology, University of Queensland, Queensland 4072, Australia
  3. Centre for Microscopy and Microanalysis and Department of Physiology and Pharmacology, University of Queensland, Queensland 4072, Australia
  4. R.G.Parton and H.Stenmark contributed equally to this work

Correspondence to:

Robert G. Parton, E-mail: R.Parton@mailbox.uq.edu.au

Harald Stenmark, E-mail: stenmark@ulrik.uio.no

Received 10 April 2000; Accepted 10 July 2000; Revised 3 July 2000

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

  • Keywords:

    • electron microscopy,
    • membrane traffic,
    • multivesicular body,
    • phosphoinositide,
    • PI 3-kinase