Role of Rab5 in the recruitment of hVps34/p150 to the early endosome.
PI 3-kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3-kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo. We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP-dependent Rab5-p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope-tagged hVps34 and p150 to Rab5-positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.
Murray
James T
JT
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.
Panaretou
Christina
C
Stenmark
Harald
H
Miaczynska
Marta
M
Backer
Jonathan M
JM
D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000502:physiology*
D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; C094981:p150 protein kinase; D020696:rab5 GTP-Binding Proteins