Autophagosome formation depends on a carefully orchestrated interplay between membraneassociated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III
phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/
VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR
motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High
resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were
obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family.
Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked
colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of
S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR
motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of
PtdIns3K-C1 on membranes for efficient autophagosome formation
