Atg12 is a ubiquitin-like protein that plays an essential role in cellular homeostasis by regulating the degradation and recycling of cytoplasmic organelles and macromolecules. Atg12 is one of two ubiquitin-like protein systems that is required during the early steps of autophagosome formation. Upon the initiation of phagopore assembly Atg12 is activated by binding to the E1-like enzyme Atg7 and is then transferred to the E2 enzyme Atg10. Finally, Atg12 is conjugated to a lysine residue in Atg5 allowing the formation of a large multimeric complex with Atg16. This Atg12-Atg5-Atg16 complex localizes to the surface of the expanding phagopore where it functions as an E3 ligase for the lipidation of Atg8, the other autophagy-specific ubiquitin-like protein. Atg8, or LC3 in mammals, is conjugated to the lipid phosphatidylethanolamine and is then incorporated into the double-membrane phagopore structure to regulate its expansion. Defects in either of these autophagy-specific ubiquitin-like systems can result in various diseases including tumor formation or neurodegeneration.
Researchers have used Atg12 antibodies to examine the assembly of Atg12 into higher order structures and its subcellular localization. Mechanistic details of the Atg12-Atg5-Atg16 complex in autophagosome initiation were investigated by Romanov et al. in EMBO (1). The group used a monoclonal Atg12 antibody to monitor Atg12-Atg5 complex formation and identified amino acid residues essential for membrane binding. They first used an in vitro liposomal binding assay to identify Atg5 mutants defective in membrane binding and then showed the Atg5 mutants were still conjugated to Atg12 through immunoprecipitation and subsequent blotting with the monoclonal Atg12 antibody. These membrane binding results were confirmed in vivo using the Atg12 antibody for the analysis of yeast cell fractionation experiments examining the membrane association of Atg5-Atg12. This research demonstrated the effectiveness of the monoclonal Atg12 antibody to study ubiquitin-like protein systems and their essential role in the early steps of autophagosome assembly.
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