How the Atg1 complex assembles to initiate autophagy

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Autophagy is a fundamental cellular process for degrading and recycling cellular components.
• The Atg1 complex is a critical initiator of autophagy, regulating its early stages.
• The Atg17-Atg31-Atg29 subcomplex is stably localized at the phagophore assembly site (PAS).

Purpose of the Study:

• To elucidate the energetics and dynamics of Atg1 complex assembly.
• To understand the molecular mechanisms governing the initiation of autophagy.
• To identify regulatory checkpoints in the autophagy pathway.

Main Methods:

• Isothermal titration calorimetry (ITC) to measure binding affinities.
• Sedimentation velocity analytical ultracentrifugation to assess complex formation and stoichiometry.
• Hydrogen-deuterium exchange (HDX) to probe protein dynamics and conformational changes.

Main Results:

• The Atg1EAT domain is dynamic but rigidifies upon binding Atg13 with high affinity (∼100 nM).
• Atg1EAT and Atg13 form a stable 2:2 dimer.
• This dimer associates with the Atg17-Atg31-Atg29 complex with lower affinity (∼10 μM).

Conclusions:

• A detailed model for Atg1 complex assembly is proposed.
• The Atg13-Atg17 binding interaction represents the weakest link in the assembly pathway.
• This interaction serves as a critical regulatory checkpoint for autophagy initiation.