Abstract
Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12-ATG5 conjugate, a crucial player during Atg8-family protein 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 alters 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 autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our 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. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A1; GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/proteins.