Mammalian homologs of the yeast Atg8 protein (mAtg8s) are important in
autophagy, but their exact mode of action remains to be defined. Recently,
syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was shown to bind
mAtg8s. Here we broadened the analysis of potential mAtg8-SNARE interactions
and identified LC3-interacting regions (LIRs) in several SNAREs. Syntaxin 16
(Stx16), and its cognate SNARE partners all have LIR motifs and bind mAtg8s. A
knockout in STX16 caused defects in lysosome biogenesis whereas a double
STX16 and STX17 knockout completely blocked autophagic flux and decreased
mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses
revealed that mAtg8s and Stx16 maintained several aspects of lysosomal
compartments including their functionality as platforms for active mTOR. These
findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on
membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to
lysosome biogenesis.