[Abstract unavailable

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) (...

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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.

Selective autophagy ensures recognition and removal of various cytosolic cargoes. Hence, aggregated proteins, damaged organelles, or pathogens are enclosed into the double-membrane vesicle, the autophagosome, and delivered to the lysosome for degradation. This process is mediated by selective autophagy receptors, such as p62/SQSTM1. These proteins recognize autophagic cargo and, via binding to small ubiquitin-like modifiers (UBLs)--Atg8/LC3/GABARAPs and ATG5--mediate formation of selective autophagosomes. Recently, it was found that UBLs can directly engage the autophagosome nucleation machinery. Here, we review recent findings on selective autophagy and propose a model for selective autophagosome formation in close proximity to cargo.

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Deregulated nuclear factor jB (NF-jB) activation plays an important role in inflammation and tumorigenesis. ABIN proteins have been characterized as negative regulators of NF-jB signaling. However, their mechanism of NF-jB inhibition remained unclear. With the help of a yeast two-hybrid screen, we identified ABIN proteins as novel ubiquitin-interacting proteins. The minimal ubiquitin-binding domain (UBD) corresponds to the ABIN homology domain 2 (AHD2) and is highly conserved in ABIN-1, ABIN-2 and ABIN-3. Moreover, this region is also present in NF-jB essential modulator/IjB kinase c (NEMO/IKKc) and the NEMO-like protein optineurin, and is therefore termed UBD in ABIN proteins and NEMO (UBAN). Nuclear magnetic resonance studies of the UBAN domain identify it as a novel type of UBD, with the binding surface on ubiquitin being significantly different from the binding surface of other UBDs. ABIN-1 specifically binds ubiquitinated NEMO via a bipartite interaction involving its UBAN and NEMO-binding domain. Mutations in the UBAN domain led to a loss of ubiquitin binding and impaired the NF-jB inhibitory potential of ABINs. Taken together, these data illustrate an important role for ubiquitin binding in the negative regulation of NF-jB signaling by ABINs and identify UBAN as a novel UBD.

Protein dynamics plays an important role in protein function. Many functionally important motions occur on the microsecond and low millisecond time scale and can be characterized by nuclear magnetic resonance relaxation experiments. We describe the different states of a peptidyl carrier protein (PCP) that play a crucial role in its function as a peptide shuttle in the nonribosomal peptide synthetases of the tyrocidine A system. Both apo-PCP (without the bound 4'-phosphopantetheine cofactor) and holo-PCP exist in two different stable conformations. We show that one of the apo conformations and one of the holo conformations are identical, whereas the two remaining conformations are only detectable by nuclear magnetic resonance spectroscopy in either the apo or holo form. We further demonstrate that this conformational diversity is an essential prerequisite for the directed movement of the 4'-PP cofactor and its interaction with externally acting proteins such as thioesterases and 4'-PP transferase.