Abstract

The ATG8 conjugation machinery supports both canonical autophagy, in which ATG8 proteins are lipidated on forming autophagosomes, and CASM, in which ATG8 proteins are conjugated to stressed single-membrane compartments. How cells allocate this shared machinery between these competing membrane programs remains unclear. Here, we identify ATG16L2 as a specialized regulator of V-ATPase-responsive ATG8 lipidation (atg8ylation). ATG16L2 does not form stable homodimers and is inactive in the absence of ATG16L1, but it becomes functional through heterodimerization with ATG16L1. The ATG16L1-ATG16L2 heterodimer constitutes a VAIL-competent E3-like complex that is excluded from WIPI2-positive autophagic membranes but readily recruited to stressed lysosomes. ATG16L2 also confers VAIL activity onto the otherwise VAIL-deficient ATG16L1 isoform. We propose that ATG16L1 homodimers and ATG16L1-ATG16L2 heterodimers represent alternative assemblies of the ATG8 conjugation machinery: ATG16L1 homodimers are dedicated to WIPI2-positive autophagic membranes, whereas ATG16L1-ATG16L2 heterodimers containing either ATG16L1 isoform form a VAIL-selective assembly.