Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

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.

Activation Mechanisms of the VPS34 Complexes

“…In contrast to human complex I (see below), this Rab5a binding does not appear to cause a dislodging effect of the VPS34 kinase domain, indicating that the activation mechanism can be different depending on the complexes or their binding proteins [48]. The same study also found a new interaction between complex I and Rab1a and showed that membrane-immobilized Rab1a greatly increased complex I activity [31] (Figure 3C, left). Interestingly, GTP-bound Rab1a also binds to the VPS34 C2HH, the same region that complex II's VPS34 uses to interact with Rab5a.…”

“…Complex II is recruited to early endosomes and activated there by an early endosome-residing small GTPase, Rab5 [31,[43][44][45] (Figure 3B), which results in PtdIns(3)P enrichment in early endosomes [46,47]. While this recruitment and activation had been long known in the cell, the underlying mechanism was finally revealed recently using electron cryotomography (cryo-ET), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and unnatural amino acid (UAA)-mediated cross-linking [31]. Without Rab5, complex II lies in an inactive state in which the VPS34 kinase domain is autoinhibited by the VPS15 kinase domain.…”

A phosphoinositide and RAB switch controls early macropinocytosis

“…In addition, these vesicles gradually gain PtdIns3P, which can then recruit other effector proteins and ultimately controls the maturation of the endocytic vesicles to early and late endosomes [28, 37]. RAB5 binds to the VPS34 kinase complex, which generates PtdIns3P and regulates its activity [38, 52]. At the same time, PtdIns3P enhances membrane recruitment of RAB5 [47] and could potentially form a positive feedback loop.…”

The phosphoinositide code is read by a plethora of protein domains

“…The plant-specific remorin proteins contain a C-terminal helical domain that binds PI4P and sterol to mediate plasma membrane targeting [123]. The structure of the Vps34 complex II, which is involved in endocytic sorting, has recently been determined by cET and shows the position of a fused BATS domain near the membrane interacting surface [124].…”