ITC

The peptides used in ITC are listed in Fig 4A. ITC experiments were performed using a Microcal iTC200 calorimeter (Malvern Panalytical), with stirring at 1,000 rpm at 25°C. TEX264 peptides were prepared as injection samples from the syringe. The concentration of TEX264 peptides was 0.5 mM. In total, the cell was filled with 200 μl LC3B and GABARAP with a 10‐times lower concentration than the syringe samples. The titration involved 18 injections of 2 μl syringe sample at intervals of 120 s into a cell after one injection of 0.4 μl syringe sample. The datasets obtained from the titration of the same syringe sample to the cell filled with buffer were used as reference data for subtraction of the heat of the dilution. MicroCal Origin 7.0 software was used for data analysis. Thermal measurement of the first injection of the syringe samples was removed from the analysis. Thermal titration data were fitted to a single‐site binding model, which determines the thermodynamic parameters enthalpy (ΔH), dissociation constant (K _d), and stoichiometry of binding (N). When the fitting was not convergent due to weak interactions, N was fixed to 1.0 or that of the control experiment. The error of each parameter shows the fitting error.