Abstract
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
Highlights
Autophagy is an evolutionarily conserved intracellular renovation process, responsible for degradation and recycling of dysfunctional or misfolded proteins as well as aged and/or damaged organelles
Given the multiple roles of the PtdIns3K-C1s core components PIK3C3, phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4) and BECN1, in several cellular processes, we focused on ATG14 as the sole autophagy specific component of the complex to directly address a functional role for the LC3-interacting region (LIR) motifs
We identified single functional LIR motifs in PIK3C3, BECN1 and ATG14 of the autophagy-specific PtdIns3K complex I which all display a preference for binding to GABA type A receptorassociated protein (GABARAP) and GABARAPL1
Summary
Autophagy is an evolutionarily conserved intracellular renovation process, responsible for degradation and recycling of dysfunctional or misfolded proteins as well as aged and/or damaged organelles. More than 40 autophagyrelated (Atg) genes encoding components of the autophagy machinery have been identified by yeast genetics [2] Most of these are conserved in mammals and regulate the key steps in autophagosome formation, including initiation, nucleation, elongation, lysosome fusion, and degradation. Initiation of autophagy is mediated by the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex and the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) [3]. In both yeast and mammals there are at least 2 PtdIns3K complexes responsible for generating phosphatidylinositol 3-phosphate (PtdIns3P)-rich membranes [4,5]. PtdIns3P-rich membranes act as platforms for autophagosome nucleation and recruitment of downstream effectors such as ZFYVE1/DFCP1 (double FYVE-containing protein 1) and WIPIs (WD repeat domain, phosphoinositide interacting) [7,8,9]
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