GFP-ATG3 GUV Assay 

GUV Preparation

Clean the coverglass

Coat cleaned coverslips with 100 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore). Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.

Spread a lipid mixture at 1 mg/ml with a molar composition of 70% DOPC, 20% DOPE, 5% DO-PI(3)P, 5% DOPS, 0.3% Atto647N DOPE uniformly onto the PVA film.

Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.

Use 100 μL 330 mOsm sucrose solution for swelling for 1 h at room temperature

Harvest the GUVs and use them with 12 h.

GUV Assay

Set up the reaction in an eight-well observation chamber (Lab Tek) at room temperature.

Coat the chamber with 1 mg/ml β casein for 10 min.

Wash the coated chamber with reaction buffer (25 mM HEPES at pH 7.4, 150 mM NaCl and 1 mM TCEP).

Make a 120 µL reaction mixtures with the proteins and 5 mM ATP and 1 mM MgCl2. The final concentration of WIPI2d 200 nM, E3 complex is 50 nM, ATG7 is 100 nM, GFP-ATG3 or Mutant is 100 nM, and mCherry-LC3B is 500 nM.

Add 3 µL GUVs to initiate the reaction.

Pick random views for imaging within 5 min.