Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs.

ABSTRACT

Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 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. Abbreviations ATG autophagy-related; BafA1 bafilomycin A1; GABARAP GABA type A receptor-associated protein; GABARAPL1 GABA type A receptor associated protein like 1; GFP enhanced green fluorescent protein; KO knockout; LDS LIR docking site; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; PIK3C3 phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4 phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; SQSTM1/p62 sequestosome 1; VPS Vacuolar protein sorting; ULK unc-51 like autophagy activating kinase.