Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitincoated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin-or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process by which cells deliver bulk cytosolic components for degradation to the lysosome (1-4). Selectivity in cargo targeting is mediated via autophagy receptors that simultaneously bind cargoes and autophagy modifiers, autophagy-related protein 8 (ATG8)/ microtubule-associated protein light chain 3 (LC3)/γ-aminobutyric acid receptor-associated protein (GABARAP) proteins, which are conjugated to the autophagosomal membranes (5, 6). The regulatory mechanisms controlling the spatiotemporal dynamics of the autophagy receptor-target interaction in cells remain unclear (7). Multiple autophagy receptors have been identified with the yeast two-hybrid system (8, 9), which included an N-terminal fragment of optineurin (OPTN), a ubiquitin-binding protein also known as NF-κB essential modulator-related protein ( Fig. 1, A and B). The specific interactions between OPTN and LC3/GABARAP proteins were verified by pull-down assays in mammalian cells, directed yeast two-hybrid transformations, and in vitro using purified proteins ( Fig. 1C and fig. S1, A and B) (10). OPTN bound to ubiquitin chains and autophagy modifiers ATG8/LC3/GABARAP proteins but not to mono-ubiquitin or other ubiquitin-like proteins ( Fig. 1C and fig. S1C). Deletion mapping of the N-terminal region of OPTN identified an LC3 interacting motif (LIR), a linear tetrapeptide sequence present in known autophagy receptors that binds directly to LC3/GABARAP modifiers (9, 11, 12). The LIR was located between the coiled-coil domains of OPTN encompassing amino acids 169 to 209 (Fig. 1A) and was essential for in vitro and in vivo binding between OPTN and LC3/ GABARAP (Fig. 1, B and C, and figs. S1A and S2A). Single point mutations at either OPTN Phe 178 →Ala 178 (F178A) or I181A (13), corresponding to the WxxL of p62, were sufficient to abrogate the interaction with LC3/GABARAP proteins, whereas these mutants were still able to bind to linear ubiquitin chains fused to glutathione S-transferase (GST-4xUb) (...

Ubiquitination is the hallmark of protein degradation by the 26S proteasome. However, the proteasome is limited in its capacity to degrade oligomeric and aggregated proteins. Removal of harmful protein aggregates is mediated by autophagy, a mechanism by which the cell sequesters cytosolic cargo and delivers it for degradation by the lysosome. Identification of autophagy receptors, such as p62/SQSTM1 and NBR1, which simultaneously bind both ubiquitin and autophagy-specific ubiquitin-like modifiers, LC3/GABARAP, has provided a molecular link between ubiquitination and autophagy. This review explores the hypothesis that ubiquitin represents a selective degradation signal suitable for targeting various types of cargo, ranging from protein aggregates to membrane-bound organelles and microbes.

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.