Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast.

A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins.

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report, we describe a functional LC3 interacting region (LIR), present in the amino terminal of most Cx protein family members, which can mediate the autophagy degradation of Cx43 without the need of ubiquitin.

How RB1CC1/FIP200 claws its way to autophagic engulfment of SQSTM1/p62-ubiquitin condensates.

Macroautophagy/autophagy mediates the degradation of ubiquitinated aggregated proteins within lysosomes in a process known as aggrephagy. The cargo receptor SQSTM1/p62 condenses aggregated proteins into larger structures and links them to the nascent autophagosomal membrane (phagophore). How the condensation reaction and autophagosome formation are coupled is unclear.

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape.

Studies of Receptor-Atg8 Interactions During Selective Autophagy.

Autophagy research frequently requires the determination of protein-protein interactions. The experimental system described in this chapter allows a simple, versatile, and quantitative in vitro analysis of interactions between recombinant cargo receptor and Atg8 proteins by fluorescence microscopy. The assay can be easily modified to study other protein-protein interactions.

Accessory Interaction Motifs in the Atg19 Cargo Receptor Enable Strong Binding to the Clustered Ubiquitin-related Atg8 Protein.

Selective autophagy contributes to cellular homeostasis by delivering harmful material into the lysosomal system for degradation via vesicular intermediates referred to as autophagosomes. The cytoplasm-to-vacuole targeting pathway is a variant of selective autophagy in Saccharomyces cerevisiae during which hydrolases such as prApe1 are transported into the vacuole. In general, selectivity is achieved by autophagic cargo receptors that link the cargo to autophagosomal membranes because of their ability to simultaneously interact with the cargo and Atg8 proteins that coat the membrane.

Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy.

Autophagy is a major pathway for the clearance of harmful material from the cytoplasm. During autophagy, cytoplasmic material is delivered into the lysosomal system by organelles called autophagosomes. Autophagosomes form in a de novo manner and, in the course of their formation, isolate cargo material from the rest of the cytoplasm.

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19.

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed.

Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane-cargo apposition during selective autophagy.

Autophagy protects cells from harmful substances such as protein aggregates, damaged mitochondria and intracellular pathogens, and has been implicated in a variety of diseases. Selectivity of autophagic processes is mediated by cargo receptors that link cargo to Atg8 family proteins on the developing autophagosomal membrane. To avoid collateral degradation during constitutive autophagic pathways, the autophagic machinery must not only select cargo but also exclude non-cargo material.