Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular proce sses, including signal transduction and membrane trafficking. In order to s tudy the intracellular localization of the PI3K product, phosphatidylinosit ol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-bi nding 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 fibroblas ts, a tagged probe localized to endosomes, as detected by fluorescence micr oscopy, Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of mult ivesicular endosomes, While yeast cells deficient in PI3K activity (vps15 a nd vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesic les of endosomes and vacuoles of wild-type yeast. vps27 Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vac uolar labelling and increased endosome labelling, Thus PI(3)P follows a con served intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endoso me and the subsequent steps leading to multivesicular endosome formation.