ABSTRACT
The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
Subject(s)
Cell Membrane/metabolism , Class III Phosphatidylinositol 3-Kinases/chemistry , Class III Phosphatidylinositol 3-Kinases/metabolism , rab1 GTP-Binding Proteins/chemistry , rab1 GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/chemistry , rab5 GTP-Binding Proteins/metabolism , Beclin-1/chemistry , Beclin-1/genetics , Beclin-1/metabolism , Class III Phosphatidylinositol 3-Kinases/genetics , Endosomes/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Structure, Secondary , Tomography , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Vacuolar Sorting Protein VPS15/chemistry , Vacuolar Sorting Protein VPS15/genetics , Vacuolar Sorting Protein VPS15/metabolism , rab1 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/geneticsABSTRACT
The GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5, Rab9 and Rab11. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.
