Fig. 7.

Extensive surfaces of Atg8 and the Atg3 catalytic domain are required for activation of the Atg3~Atg8 intermediate. a E3-dependent activation of Atg3~Atg8 intermediate testing roles of indicated surfaces through multiple-Ala scanning mutagenesis over Atg3′s catalytic domain. Quantification is of WT or indicated mutant versions of Atg3~Atg8 remaining after 2.5 min as a function of NH₂OH concentration in pulse-chase assays, with Atg12–Atg5-Atg16 as E3. b E3-dependent activation of Atg3~Atg8 intermediate testing roles of indicated surfaces through multiple-Ala scanning mutagenesis over Atg8, performed as in (a). c Effects of covalent Atg3 complex formation with Atg8, as detected by comparing ¹⁵N, ¹H] TROSY spectra of ¹⁵N-labeled Atg8 G116C (red) alone and disulfide-bonded complex with Atg3^cat as a proxy for Atg3^cat–Atg8 intermediate (cyan), with chemical shift perturbations per Atg8 residue shown below. Stripes indicate resonances with line-broadening due to intermediate exchange