Vps34 Kinase Domain Dynamics Regulate the Autophagic PI 3-Kinase Complex

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Macroautophagy is a fundamental cellular process essential for homeostasis.
• The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is crucial for initiating macroautophagy.
• The precise mechanism of allosteric regulation of VPS34 within PI3KC3-C1 remains elusive.

Purpose of the Study:

• To elucidate the allosteric regulation of the PI3KC3-C1 complex.
• To identify the key conformational changes governing VPS34 activity in autophagy.
• To determine the role of VPS15 in transmitting signals to VPS34.

Main Methods:

• Cryo-electron microscopy (EM) to resolve complex structures.
• Crosslinking mass spectrometry to map protein interactions.
• Biochemical assays to assess enzyme activity in vitro.
• Yeast cell-based autophagy induction assays.

Main Results:

• Five distinct conformational substates of PI3KC3-C1 were identified.
• A specific substate revealed the VPS34 catalytic domain dislodged but tethered by a linker.
• A "leashed" construct, preventing VPS34 dislodging, abolished enzyme activity and autophagy.
• VPS15 was implicated in regulating the tethering of the VPS34 catalytic domain.

Conclusions:

• The dislodging and tethering of the VPS34 catalytic domain is a critical allosteric switch for autophagy initiation.
• Regulation of this switch by VPS15 is essential for PI3KC3-C1 function.
• This finding provides a novel mechanistic insight into the control of macroautophagy.