Activation Mechanisms of the VPS34 Complexes

Abstract

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34-Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.

Keywords: ATG14L; Beclin 1; PtdIns(3)P; UVRAG; VPS15; VPS34; autophagy; endocytic pathway; lipids; membranes.

Figure 1

The class III PI3K VPS34/Vps34 is the most ancestral PI3K among the three classes of PI3Ks. The Class I PI3Ks phosphorylate PtdIns(4,5)P₂ to synthesize PtdIns(3,4,5)P₃, while the Class II PI3Ks phosphorylate PtdIns or PtdIns(4)P to synthesize PtdIns(3)P or PtdIns(3,4)P₂, respectively. The Class III PI3K (PIK3C3 or VPS34/Vps34 for metazoans/yeast) phosphorylates PtdIns to synthesize PtdIns(3)P. This phylogenetic tree was generated using MEGA (https://www.megasoftware.net/, accessed on 4 March 2021). The following proteins, listed with their Uniprot entry numbers, were used for the analysis: p110α (Human: P42336; Mouse: P42337; Zebrafish: F1QAD7; Xenopus: F6VXG1); p110β (Human: P42338; Mouse: Q8BTI9; Zebrafish: E7F251); p110γ (Human: O00329; Mouse: O35904; Zebrafish: F1RB17); p110δ (Human:P48736; Mouse: Q9JHG7; Zebrafish: Q6NTI2; Xenopus: A0A6I8RLG9); PIK3C2α (Human: O00443; Mouse: Q61194; Zebrafish: F6NMW9; Xenopus: B5DE87); PIK3C2β (Human: O00750; Mouse: E9QAN8; Zebrafish: F1QWN6); PIK3C2γ (Human: O75747; Mouse: O70167; Zebrafish: A0A0G2L6J0); and PIK3C3/VPS34 (Human: Q8NEB9; Mouse: Q6PF93; Zebrafish: F1Q9F3; Xenopus: F6ZM84; Yeast Vps34: P22543).

Figure 2

The role of each domain of VPS34/Vps34 complex I and II subunits in the assembly of the complexes. (A) Schematic representations of subunits in human numbers. (B) Structural schematic representations of complexes I and II. C2: C2 domain; CC: coiled-coil; BARA: β-α repeated, autophagy-specific (BARA) domain; BATS: Barkor/Atg14(L) autophagosome targeting sequence (BATS) domain; NTD: N terminal domain; CTD: C terminal domain; CXXCs: CXXC motifs; WD40: WD40 domain; PR: proline-rich domain; BH3: BH3 domain. The figures are modified from [34,35].

Figure 3

Activation mechanisms of VPS34/Vps34 complexes. (A) Schematic representations of the VPS34/Vp34 kinase domain. Left: A simplified structure of the kinase domain. An ATP molecule is sandwiched by the N- and C-lobes, which are connected by the hinge region. Right: A schematic representation of the kinase domain in human VPS34, modified from [35]. Residue numbering shown is for the human VPS34. (B) The activation mechanism of human complex II by Rab5a. Left: Complex II is recruited in an autoinhibition form to early endosomes by the early endosome specific small GTPase Rab5a in a GTP-dependent manner. Middle: The Rab5a binding not only recruits complex II to the membrane but releases the VPS34 kinase domain from autoinhibition by the VPS15 kinase domain. Rab5a binds mainly to the C2 helical hairpin insertion (C2HH) of VPS34, along with the small globular domain (SGD) and WD40 domain in VPS15. Along with Rab5a, three motifs in the Beclin 1 BARA domain are responsible for the membrane binding. Right: The membrane-anchored active complex II can tilt up and down to phosphorylate PtdIns and synthesize PtdIns(3)P. (C) Activation mechanisms of complex I. In addition to the Beclin 1 aromatic finger 1, the ATG14L BATS domain is essential for the membrane anchoring of complex I. Left: Complex I activation by Rab1a. GTP-bound Rab1a binds to the same C2HH in VPS34 as Rab5a–complex II, but not to the VPS15 SGD or WD40. Right: Complex I activation by the complex I-specific binding protein NRBF2.

Figure 4

VPS34 complex regulation by the ULK1 complex and mTORC1. (A) The ULK1 complex acts as a positive regulator of complex I during starvation, whereas mTORC1 acts as a negative regulator in nutrient-replete conditions. Their phosphorylation positions are indicated in the schematic structure of complex I. Green dots: activating phosphorylation by the ULK1 complex; red dots: inhibiting phosphorylation by mTORC1. (B) Left: VPS34 (or, putatively, complex II) acts as a positive regulator of mTORC1 in amino acid-replete conditions. Amino acids activate VPS34 to synthesize PtdIns(3)P, which activates PLD1. Phosphatidic acid (PA), the PLD1 product, binds to mTOR to activate it. Right: After long-term starvation, lysosomes are regenerated by autophagic lysosomal regulation (ALR). Then, accumulated autophagy substrates in autolysosomes are degraded, and amino acids are regenerated. This activates mTORC1 to phosphorylate UVRAG, the complex II-specific subunit, and activate complex II. This activity is necessary for the scission step of ALR, in which the tubulated autolysosome is detached by dynamin. (C) VPS34–mTORC1 activation by lysosome translocation. VPS34 (potentially complex II) is activated by amino acids to synthesize PtdIns(3)P, which is bound by FYCO1 via its FYVE domain. This also causes lysosome translocation to the cell periphery via the microtubule–kinesin–FYCO1 interaction. At the plasma membrane, mTORC1 is in close proximity to nutrient-signalling complexes to become activated.

Figure 5

Physicochemical parameters that affect activities and membrane binding of autophagy-related proteins. (A) A schematic structure of a phospholipid molecule. A phospholipid is composed of a polar head comprising a modified alcohol (R) and a phosphate, a backbone (glycerol here, also can be sphingosine), and fatty acids or acyl chains. R in the polar head defines the lipid species, such as serine for PS, ethanolamine for PE, choline for PC, and inositol for PtdIns. The polar head and backbone constitute hydrophilic head, facing the aqueous side of the membrane, whereas the acyl chains provide a hydrophobic barrier. (B) Three important physicochemical parameters that affect activities and membrane associations of autophagy-related proteins: packing, membrane curvature, and electrostatics.

Figure 6

Summary of membrane/lipid environments that are targeted by autophagy-related proteins. During starvation, PtdIns is synthesized in the ER by PIS, which is targeted by the ULK1 complex and essential for the generation of the autophagosome. Complex I, the ATG16–ATG12–ATG5 complex, and the ATG2/Atg2–WIPI4/Atg18 complex prefer unsaturated (unpacked) and highly curved membranes for their activations. ER membranes and ATG9/Atg9 vesicles fulfil these conditions. During expansion, phagophores are tethered and fused, which is facilitated by Atg8 family proteins. Also, curvatures are generated at least by the lipidated yeast Atg8, and the yeast Atg20–Atg24 complex might detect and stabilize membrane curvatures at the edge of the expanding phagophore to support expansion. Along with this, upon starvation, PS synthesis occurs at the ER–mitochondria contact site by PSS1, which is targeted by the ULK1 and complex I. This may also lead to the initiation of autophagosome formation.

Figure 7

Positive PtdIns(3)P feedback mechanisms via complexes I (A) and II (B). (A) Complex I is activated by its effector WIPI2d, which leads to a positive feedback loop of PtdIns(3)P synthesis. WIPI2d also facilitates the LC3B lipidation by interacting with the ATG12–ATG5–ATG16L E3 complex. GABARAP and GABARAPL1, Atg8 family proteins, are known to preferably interact with complex I, but the consequence of this interaction remains to be seen. (B) On early endosomes, Rab5 interacts with its GEF-effector complex, the Rabex5–Rabaptin5 complex. This interaction switches the Rab5 nucleotide binding from GDP to GTP and its binding partner from Rabex5 to Rabaptin5. This interaction causes the formation of Rab5 domain (clustering), which is enhanced by PtdIns(3)P, indicating positive feedback between Rab5–Rabex5–Rabaptin5 and complex II. These Rab5- and PtdIns(3)P-enriched clusters recruit more Rab5 effectors and PtdIns(3)P-binding proteins carrying PX and FYVE domains.