The ATG5 Interactome Links Clathrin Vesicular Trafficking With The ATG8 Lipidation Machinery For Autophagosome Assembly

Abstract

Autophagosome formation involves the sequential actions of conserved ATG family proteins that regulate the lipidation of the ubiquitin-like modifier ATG8 at the nascent isolation membrane.Although the molecular steps driving this process are well understood, the source of membranes supplied for the expanding autophagosome and their mode of delivery remain uncertain. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12~ATG5 conjugate that is crucial for ATG8 lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins caused defects in autophagic flux in cell-based starvation assays. To determine whether the ATG12~ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagycompetent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. Proteins whose surface expression increased contingent on autophagic capability included EPHB2, SLC12A4, and JAG1. Those whose surface expression was decreased included CASK, SLC27A4 and LAMP1. These data provide evidence for direct regulatory coupling between the ATG12~ATG5 conjugate and the clathrin membrane trafficking system, and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery.

Atg8-Family Proteins—Structural Features and Molecular Interactions in Autophagy and Beyond

“…This interaction was proposed to be mediated by AIM/LIR and stimulate Atg8 conjugation. On the other hand, clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors, have been identified in the Atg5/Atg12 interactome [46], and earlier studies identified clathrin heavy chain as a direct interactor for GABARAP [47]. Additional work is required to clarify the exact interaction surfaces between the UBLs in the Atg12-Atg5 conjugate and partner proteins.…”

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition

“…In parallel, deficiency of the GABARAP-type subfamily may additionally directly disturb the intracellular distribution of surface proteins, especially those containing functional interaction motifs [ 52 ]. Interestingly, recent results from a yet unpublished study show altered surfaceome composition of Atg5 -/- mouse embryonic fibroblasts [ 57 ]. ATG5 is a key component of the LC3/GABARAP lipidation machinery which is essential for their integration into autophagy-related and unrelated membranes [ 53 ].…”