EVIDENCE FOR PHOSPHATIDYLINOSITOL 3-KINASE AS A REGULATOR OF ENDOCYTOSIS VIA ACTIVATION OF RAB5

Phosphatidylinositol (PI) 3-kinases have been implicated in several as pects of intracellular membrane trafficking, although a detailed mecha nism is yet to be established, In this study we demonstrated that wort mannin, a specific inhibitor of PI 3-kinases, inhibited constitutive e ndocytosis of horseradish peroxidase and transferrin in BHK-21 and TRV b-1 cells, The IC50 was approximate to 40 ng/ml (93 nM), In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective , constitutively activated Rab5 Gln(79) --> Leu mutant (Rab5:Q79L), pr oviding further evidence that PI 3-kinase activity is essential for th e early endocytic process. To further investigate the mechanism, we ex amined the effect of wortmannin on early endosome fusion in vitro, Wor tmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 re versed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly ind icate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.