Structural Basis for Receptor-Mediated Selective Autophagy of Aminopeptidase I Aggregates

Abstract

Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity.

A Diversity of Selective Autophagy Receptors Determines the Specificity of the Autophagy Pathway

“…Significantly, the in vitro results were replicated in cells, where p62/SQSTM1 bodies are liquid-like, dynamic, and governed by the same principles as those identified using the recombinant proteins (Sun et al, 2018). Oligomerization and phase separation were reported for the Atg19:prApe1 complex in yeast (Yamasaki et al, 2016), as well as SEPA-1, the protein required for P granule production in Caenorhabditis elegans (Zhang et al, 2018).…”

Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein

“…GST pulldown assay was performed as previously described ( Yamasaki et al, 2016 ). Briefly, 50 μg of GST-fused ScHfl1 variants were incubated with 7.5 μl of GST-accept resin in 300 μl of PBS for 1 hr.…”

Mechanism of cargo-directed Atg8 conjugation during selective autophagy

“…Atg5 and Atg5-Atg16 showed robust interaction with all Atg19 truncations including the C-terminal domain encompassing amino acids 365–415 (Figure 2E and Figure 2—figure supplement 2C). The presence of Atg12, either in context of the Atg12~Atg5 conjugate or the Atg12~Atg5-Atg16 complex, changed the properties of the interaction and required the presence of amino acids 124–254, which include the cargo binding domain of Atg19 (Yamasaki et al, 2016). We corroborated the results of the pull down experiments for the full Atg12~Atg5-Atg16 complex in a microscopy-based assay under equilibrium conditions (Figure 2F).…”