Autophagy is a non‐selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double‐membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin‐like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease‐related protein aggregates, respectively. Using X‐ray crystallography and NMR, we herein report the structural basis for Atg8–Atg19 and LC3–p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side‐chains of Trp and Leu in a four‐amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL‐containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.

Background: Vps30/Atg6 is responsible for both autophagy and vacuolar protein sorting.Results: Structure of the Vps30 BARA domain was determined and its function was characterized.Conclusion: BARA domain has a unique fold and is specifically required for autophagy.Significance: This study will be a basis for elucidating the various functions of Vps30 homologs.

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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.

The vacuole hydrolase aminopeptidase 1 (Ape1) is a cargo protein transported to the vacuole by the cytosol-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ape1 is processed into mature Ape1 (mApe1). mApe1 has been expressed, purified and crystallized in two crystal forms. Form I belongs to space group P2(1), with unit-cell parameters a = 120.6, b = 219.5, c = 133.1 A, beta = 116.5 degrees. Form II belongs to space group R3, with unit-cell parameters a = 141.2, c = 349.4 A. Diffraction data were collected from these crystals to a resolution of 2.5 A for form I and 1.83 A for form II. Self-rotation functions and the volume-to-weight ratio values suggest that forms I and II contain 12 and four mApe1 molecules per asymmetric unit, respectively, and that mApe1 exists as a tetrahedral dodecamer in both crystal forms.