Activation of lipid kinase VPS34 by Rab GTPases
VPS34 is a lipid kinase that uses phosphatidylinositol (PI) as a substrate to produce the signalling lipid phosphatidylinositol-3-phosphate (PI(3)P). In the cell, VPS34 has to team up with other proteins in order to increase its enzymatic activity. It forms primarily two hetero-tetrameric complexes, complexes I and II, which act as key regulators of autophagy and endocytic trafficking, respectively. Complex I consists of VPS34, VPS15, Beclin 1 and ATG14L, whereas complex II contains UVRAG instead of ATG14L. VPS34 complex activity is regulated by membrane properties and by members of the Rab family of small G proteins, which are important for orchestrating intracellular vesicle sorting and transport. However, their detailed activation mechanisms have been poorly understood. Firstly, we found that high curvature, negative charge, lipid unsaturation and specific phosphoinositides activate both VPS34 complexes. While the autophagic complex I relies on an amphipathic helix in ATG14L for membrane association, the endocytic complex II uses mainly three aromatic loops in the Beclin 1 subunit to engage with membranes. I have found that Rab GTPases act as specific regulators for each complex. While Rab5 is a strong activator for endocytic complex II, Rab1 specifically activates the autophagic complex I. To gain insight into the activation mechanism, I obtained a 9.8 Å resolution structure of complex II bound to Rab5 on membranes, using electron cryo-tomography and subtomogram averaging. Complex II is dynamic on membranes with only the UVRAG/Beclin 1 “adaptor arm” permanently contacting the outer leaflet of the lipid bilayer. In contrast, the VPS15/VPS34 “catalytic arm” hovers over the membrane and is able to tilt up and down. Rab5 binds on the “adaptor arm” of the complex to a tripartite motif made of VPS34 C2, VPS15 WD40 and FF domain. These interactions increase membrane recruitment and PI(3)P production of complex II specifically on early endosomes.
electron cryo microscopy, VPS34, Rab5, Rab1
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