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Commentary 3237 The LIR motif – crucial for selective autophagy Åsa Birna Birgisdottir, Trond Lamark and Terje Johansen* Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway *Author for correspondence ([email protected]) Journal of Cell Science 126, 3237–3247 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.126128 Journal of Cell Science Summary (Macro)autophagy is a fundamental degradation process for macromolecules and organelles of vital importance for cell and tissue homeostasis. Autophagy research has gained a strong momentum in recent years because of its relevance to cancer, neurodegenerative diseases, muscular dystrophy, lipid storage disorders, development, ageing and innate immunity. Autophagy has traditionally been thought of as a bulk degradation process that is mobilized upon nutritional starvation to replenish the cell with building blocks and keep up with the energy demand. This view has recently changed dramatically following an array of papers describing various forms of selective autophagy. A main driving force has been the discovery of specific autophagy receptors that sequester cargo into forming autophagosomes (phagophores). At the heart of this selectivity lies the LC3-interacting region (LIR) motif, which ensures the targeting of autophagy receptors to LC3 (or other ATG8 family proteins) anchored in the phagophore membrane. LIR-containing proteins include cargo receptors, members of the basal autophagy apparatus, proteins associated with vesicles and of their transport, Rab GTPaseactivating proteins (GAPs) and specific signaling proteins that are degraded by selective autophagy. Here, we comment on these new insights and focus on the interactions of LIR-containing proteins with members of the ATG8 protein family. Key words: ATG8, LC3, GABARAP, LIR, p62, Selective autophagy Introduction Macroautophagy (hereafter referred to as autophagy) is an intracellular degradation process, in which a double membrane structure called the phagophore expands and closes upon itself to sequester part of the cytoplasm to form an autophagosome (varying in diameter from 0.5 to 1.5 mm). First, the autophagosome fuses either with a late endosome forming an amphisome or directly with a lysosome forming an autolysosome. Amphisomes also fuse with lysosomes to form autolysosomes (Mizushima et al., 2011) (Fig. 1). This process can degrade all kinds of molecules and supramolecular structures in the cytoplasm, including organelles such as peroxisomes and mitochondria (Johansen and Lamark, 2011). Because of its fundamental importance in cellular homeostasis and cellular signaling, autophagy is highly relevant for a number of diseases, including cancer, neurodegenerative diseases, muscular dystrophy, lipidstorage disorders and processes such as development, ageing and innate immunity (Levine and Kroemer, 2008; Levine et al., 2011; Mizushima and Komatsu, 2011; Deretic, 2012). In addition to (macro)autophagy, microautophagy and chaperone-mediated autophagy represent distinct autophagy pathways (Arias and Cuervo, 2011; Mijaljica et al., 2011). Pioneering genetic studies in yeast have revealed a number of AuTophaGy (ATG) genes (Nakatogawa et al., 2009; Mizushima et al., 2011). Currently, 38 Atg proteins are known in yeast. Of these, 17 (Atg1 to Atg10, Atg12 to Atg16, Atg18 and Atg22) are part of the core autophagy machinery used by all the different autophagy pathways. The components of the core autophagy machinery are well conserved from yeast to mammals and appear to act in a similar hierarchical manner. In mammals the core machinery consists of (i) the complex of uncoordinated 51-like kinase 1 and 2 (ULK1–ULK2 ); (ii) a class III phosphatidylinositol 3-kinase (PI 3-kinase) complex; (iii) ATG2A and ATG2B, and the mammalian Atg18 homologs WD-repeat protein interacting with phosphoinositides 1, 2, 3 and 4 (WIPI1, WIPI2, WIPI3 and WIPI4, respectively); (iv) ATG9; (v) a complex of the ATG12–ATG5 conjugate and Atg16L1 and; (vi) ATG8 or the microtubuleassociated proteins 1A/1B light chain 3 (MAP1LC3 or LC3) proteins (Fig. 1). Members of the ATG8 family are the only known ubiquitinlike (Ubl) proteins that are conjugated to a lipid, namely phosphatidylethanolamine (PE). ATG8-PE is present both on the outer and inner membranes of the phagophore. During autophagosome maturation, ATG8 is deconjugated from the outer membrane by ATG4. This is necessary for autophagosome biogenesis (Mizushima et al., 2011). In mammals, two subfamilies of at least seven ATG8 proteins exist: the LC3 proteins LC3A, LC3B and LC3C, with two N-terminal splice variants of LC3A, and GABARAP (c-amino butyric acid receptor-associated protein), GABARAPL1 and GABARAPL2. In mammals, LC3B is the most prevalent and well-established autophagosome marker. Yeast has only one Atg8 homolog, Caenorhabditis elegans and Drosophila melanogaster have two, whereas Arabidopsis thaliana has nine ATG8 homologs (reviewed by Shpilka et al., 2011). Autophagy was traditionally regarded as a non-selective, bulk degradation process mainly induced to replenish energy stores upon starvation. A distinction is usually made between basal, housekeeping autophagy that is important for quality control of proteins and organelles, and starvation- or stress-induced autophagy. During the last decade evidence has accumulated that autophagy can be highly selective (Kirkin et al., 2009a; Kraft et al., 2010; Johansen and Lamark, 2011). Selective autophagy refers to the selective degradation of, for instance, organelles 3238 Journal of Cell Science 126 (15) Induction Nucleation Expansion Cargo recruitment Closure Maturation Cargo degradation Endosome Lysosome PI 3-kinase complex ULK1/2 ? ATG9 vesicle Autophagosome Phagophore ATG5 ATG12 ATG7 ATG10 Journal of Cell Science ATG5 ATG12 proLC3z ATG4B LC3-I ATG7 ATG3 ATG5 ATG12 ATG16L1 Key LC3-II PE PtdIns(3)P DFCP1 Autolysosome Protein misfolded/aggregate Ubiquitin WIPI proteins Peroxisome ATG9 Bacteria ATG12–ATG5–ATG16L1 Mitophagy receptor LC3-PE Mitochondria Autophagy receptor (SLRs) ATG8-LIR interaction Fig. 1. Overview of selective autophagy in mammalian cells. Activation of the complex between uncoordinated 51-like kinases 1 and 2 (ULK1–ULK2) and the scaffold proteins ATG13, FIP200 and ATG101 is essential for the induction of autophagy. At the nucleation step, proteins and lipids are recruited to the phagophore. ATG9, a multi-spanning transmembrane protein, is located on vesicles that dynamically traffic to and from the phagophore. The class III phosphatidylinositol 3-kinase (PI 3-kinase) complex, with the catalytic subunit Vps34, the Ser/Thr kinase Vps15 and the regulatory subunits beclin-1 and ATG14L, generates PtdIns(3)P at the phagophore. PtdIns(3)P is required for the recruitment of WD-repeat proteins that interact with phosphoinositides (WIPIs) and double-FYVE-containing protein 1 (DFCP1). WIPIs, in turn, recruit ATG2A and ATG2B into a complex, which can communicate with ATG9. Expansion of the phagophore depends on two ubiquitin-like (Ubl) conjugation systems (boxed). Conjugation of ATG5 to ATG12, which requires the E1 enzyme ATG7 and the E2 enzyme ATG10, generates an oligomeric complex between the ATG12–ATG5 conjugate and ATG16L1. ATG8/LC3 proteins are subsequently conjugated to phosphatidylethanolamine (PE) following cleavage by the cysteine protease ATG4 acting on nascent ATG8s (proLC3) to expose a C-terminal glycine residue required for covalent attachment to PE. The exposed glycine of ATG8 (LC3-I) is activated by ATG7 (E1), activated ATG8 is transferred to ATG3 (E2-like enzyme) forming an ATG8,ATG3 thioester intermediate, before ATG8 is conjugated to PE by the E3-like ATG12–ATG5–ATG16 complex. The cargo for selective autophagy is recruited to the inner, concave, surface of the growing phagophore by autophagy receptors that are associated both with the cargo and with lipidated ATG8/LC3 (LC3 II). The phagophore expands and encloses its cargo to form the double-membrane autophagosome. Fusion of autophagosomes with late endosomes or lysosomes (maturation) forms autolysosomes where the enclosed cargo is degraded. (mitophagy and pexophagy), bacteria (xenophagy), ribosomes, macromolecular structures, specific proteins and protein aggregates (aggrephagy) by autophagy. The cytoplasm-tovacuole targeting (Cvt) pathway, which selectively directs aggregated precursors of aminopeptidase 1 and a-mannosidase to the vacuole, is the only biosynthetic pathway that uses the autophagy core machinery (Lynch-Day and Klionsky, 2010). Together with the discovery of bona fide selective autophagy receptors in mammalian cells, studies of the Cvt pathway have helped elucidate some of the molecular basis for selective autophagy. Emerging selectivity – discovery of selective autophagy receptors and the LIR motif A selective autophagy receptor needs to be able to bind specifically to cargo and to dock onto the forming phagophore enabling autophagic sequestration and degradation of the cargo. The first selective autophagy receptor to be identified was p62 [also known as sequestosome-1 (SQSTM1)] (Bjørkøy et al., 2005; Komatsu et al., 2007; Pankiv et al., 2007). p62 was well known to act as a scaffold protein in signaling pathways involving NF-kB (Moscat et al., 2007), but to also accumulate in ubiquitin-containing protein inclusions in many proteinaggregation diseases including Alzheimer disease, Pick disease, dementia with Lewy bodies, Parkinson disease and multiple system atrophy (Kuusisto et al., 2001; Zatloukal et al., 2002). We found that p62 is both a selective autophagy substrate and a cargo receptor for autophagic degradation of ubiquitylated protein aggregates (Bjørkøy et al., 2005; Pankiv et al., 2007). Consistently, knockout of autophagy in the liver of mice demonstrated that p62, which binds both ubiquitin and LC3, regulates the formation of protein aggregates and is removed by autophagy (Komatsu et al., 2007). The authors showed that blocking of autophagy resulted in a failure to degrade p62 and lead to extensive accumulation of protein aggregates, severe hepatomegaly and liver dysfunction (Komatsu et al., 2007). The LIR motif in selective autophagy 3239 cargo receptor Atg19 (Pankiv et al., 2007; Ichimura et al., 2008b; Noda et al., 2008). The motif has also been called Atg8-family interacting motif (AIM) (Noda et al., 2010). The structures of p62 and Atg19 peptides bound to LC3B and Atg8, respectively, revealed a common W-x-x-L motif (x5any amino acid) (Ichimura et al., 2008b; Noda et al., 2008) (Fig. 3) (see also p62 consists of 440 amino acids and contains an N-terminal PB1 domain, followed by a ZZ-type zinc-finger domain and a C-terminally located ubiquitin-binding UBA domain (Fig. 2). Detailed deletion mapping and point mutation analyses, together with X-ray crystallography and NMR lead to the elucidation of the LC3-interacting region (LIR) motifs of p62 and of the Cvt Sequestosome-1-like receptors (SLRs) LIR KIR p62 ZZ PB1 Ub UBA NLS2 440 NLS1 NES LIR2 NBR1 CC1 ZZ ZZ PB1 FW LIR NDP52 ZF OPTN CC Ub CC Coiled-Coil CC CC LIR Journal of Cell Science 966 446 LIR SKICH Ub UBA UBA Ub CC SKICH TAX1BP1 LIR1 CC2 ZF ZF 789 Ub CC CC UBAN ZF 577 Mitophagy receptors LIR FUNDC1 TM TM TM 155 LIR BH3 BNIP3 TM 194 LIR BH3 NIX TM 219 LIR Atg32 TM 529 Specialized receptors 4H Cbl EF SH2 LIR UBA Ring 906 LIR Stbd1 CBM20 TM 358 Cvt cargo receptors LIR Atg19 CC 415 ABD LIR Atg34 ABD 412 Fig. 2. Domain architecture of selective autophagy cargo receptors known to date. The sequestosome-1-like receptors (SLRs) constitute of p62, NBR1, NDP52, TAX1BP and OPTN (optineurin) in mammals. The known mitophagy receptors FUNDC1, BNIP3, NIX (BNIP3L) in mammals, and Atg32 in yeast, are shown. The specialized receptors Cbl and Stbd1, characterized in mammals, are involved in selective autophagy of Src kinase and glycogen, respectively. The Cvt cargo receptors, Atg19 and Atg34 in yeast, are essential for the Cvt pathway. PB1, Phox and Bem1 domain (dark pink); ZZ, ZZ-type zink finger domain (blue); CC, coiled-coil domain (light pink); NLS1 and NLS2, nuclear localization signals 1 and 2 (dark gray); NES, nuclear export signal (dark gray); LIR, LC3interacting region (dark red); KIR, Keap interacting region (green); UBA, ubiquitin-associated domain (yellow); FW, four tryptophan domain (dark yellow); SKICH, SKIP carboxyl homology domain (light green); ZF, Zinc-finger domain (yellow); UBAN, ubiquitin binding in ABIN and NEMO domain (yellow); TM, transmembrane domain (light blue); BH3, Bcl-2 homology (BH) domain 3 (light purple); 4H, four-helix bundle domain (light gray); EF, EF-hand-fold domain (light gray); SH2, Src-homology 2 domain (light gray); Ring, really-interesting-new-gene-finger domain (blue); CBM20, family 20 carbohydrate-binding module domain (light gray); ABD, Ams1-binding domain (orange). The size of the receptors (in numbers of amino acids) is indicated. 3240 Journal of Cell Science 126 (15) A p62 Atg19 R28 HP1 W340 L343 E414 D338 HP2 R11 L415 W412 D337 E413 R67 R10 NBR1 LC3B Atg8 NDP52 K24 K20 V135 E730 K16 Y732 I735 L134 V136 D731 K48 LC3C GABARAPL1 C B 4 Bits 2 R11 D338 W340 0 6 D337 Bits R10 4 2 L343 0 4 Ub-like domain Bits Journal of Cell Science N-terminal arm 2 0 X–3 X–2 X–1W0 X1 X2 L3 X4 X5 Fig. 3. LIR motif consensus and structural determinants of LIR–ATG8 interactions. (A) Surface representation of LC3B bound to the p62-LIR peptide (top left), yeast Atg8 bound to the Atg19-LIR peptide (top right), GABARAP-L1 bound to the NBR1-LIR peptide (bottom left) and LC3C bound to the NDP52-LIR peptide (non-canonical LIR-motif) (bottom right). The hydrophobic pockets (HP1 and HP2) of LC3B, Atg8 and GABARAP-L1 as well as the hydrophobic patch of LC3C are indicated in bright yellow. The amino acids (yellow) of the different LIR peptides that bind in the pockets are shown as well as the amino acids (red) that interact with basic residues of the ATG8 proteins (blue). (B) Ribbon diagram of LC3B with the N-terminal arm (blue) and the Ubl domain (gray). The bound p62-LIR peptide is depicted in red. Amino acids D337 and D338 in the p62-LIR peptide interact with the basic residues R10 and R11 in the N-terminal arm of LC3B. Amino acids W340 and L343 in the p62-LIR peptide binding to hydrophobic pockets in LC3B are also indicated. (C) Sequence logos that are a graphical representation of amino acid residues as stacks at each position in multiple sequence alignments of LIR motifs. The overall height of the stack indicates the sequence conservation at that position, whereas the height of symbols within the stack indicates the relative frequency of each amino at that position. The sequence logos were created on the basis of 42 verified LIR motifs (upper panel) and were split into 22 W-type LIRs (middle panel) and 15 F-type LIRs (lower panel). The analysis of these 42 LIRs (33 of which are published, see supplementary material Table S1) confirms the core consensus sequence [W/F/Y]xx[L/I/V], in which alternative letters are placed in square brackets with a solidus between them. Only five LIRs have Tyr (Y) at the aromatic position binding to the HP1 pocket. W-type LIRs prefer Leu (L) in HP2 (13 out of 22). Such a preference is not seen among the 15 F-type LIRs, in which I, L and V are similarly distributed. F-type LIRs have a significantly higher average number of acidic residues than W-type LIRs. The average number of E, D, S, or T in the three positions Nterminal to the core hydrophobic residue (positions X21 to X23) is 1.7 and 2.5 for W- and F-type LIRs, respectively. The Seq2Logo-1.0 server (http://www.cbs. dtu.dk/biotools/Seq2Logo-1.0/) was used with Kullback-Leibler logo type and Hobohm1 clustering (threshold 0.63 and 0 weight on prior pseudo counts) (Thomsen and Nielsen, 2012). The LIR motif in selective autophagy Journal of Cell Science Box 1. Specificities of the LIR-ATG8 protein interaction A compilation of verified LIR motifs reveals a core consensus sequence [W/F/Y]xx[L/I/V] (see Fig. 3C). Most LIR motifs have a W or an F at the aromatic position binding to the HP1 pocket, but a few have Y at this position. Structural data show that the Y-type LIR1 of NBR1 binds in a manner similar to W-type LIRs, but mutation of the core Y residue into W or F demonstrated that W results in higher binding affinity than F or Y (Rozenknop et al., 2011). In addition to the core motif, the importance of an acidic charge (E, D, S or T), either N- or C-terminal to the conserved aromatic residue, is evident. The prevalent use of S and T flanking the core motif indicates a regulation by phosphorylation. Electrostatic interactions may determine the substrate specificity since they often involve residues found only in a subset of the ATG8 homologs. For F-type LIRs, a higher number of electrostatic interactions appear to compensate for a lower affinity between F and HP1. The choice of amino acid at position X1 is also more important for F-type LIRs than for W-type LIRs. The F-type LIRs of ULK1 and ATG13 have a preference for the GABARAP subfamily. Mutagenesis of these LIRs showed V (Val), C (Cys), I (Ile), E (Glu) and F as the only amino acids acceptable in position X1 (Alemu et al., 2012). The LC3C-specific LIR of NDP52 represents a more-specialized variant, because it has lost the aromatic residue and the binding to the HP1 pocket (see Fig. 3A). Lacking this aromatic residue, it is unable to bind to most ATG8 proteins but does interact with LC3C because a rotation of the b-strand of the LIR improves shape complementarity and creates additional interstrand hydrogen bonds with the binding cleft in LC3C (von Muhlinen et al., 2012). LIR-independent interactions involving ATG8 proteins also exist. For example, C. elegans autophagy receptors do not contain LIR motifs (Lin et al., 2013), and several of the proteins identified by Behrends and colleagues interact with ATG8 in a manner that is not affected by mutations in the LIR docking site (e.g. ATG16L, ATG7 and ATG5) (Behrends et al., 2010). Box 1), and the importance of the acidic residues N-terminal to the core of the DDDWTHL LIR motif of p62 was verified by alanine substitutions (Pankiv et al., 2007; Ichimura et al., 2008a; Noda et al., 2008). The LIR motif of p62 presents as an extended b-strand that forms an intermolecular parallel b-sheet with the b2 strand of LC3B. The ATG8 family proteins have a C-terminal, ‘core’ Ubl domain that contains the conserved ‘ubiquitin fold’ and an additional N-terminal arm with two a-helices that are closed onto the core Ubl domain (Fig. 3B). The LIR-containing peptide is located in the interface of the N-terminal arm and the Ubl domain. In this LIR docking site, two hydrophobic pockets HP1 and HP2 in the Ubl domain of LC3 accommodate the side chains of the W and L residues (Ichimura et al., 2008b; Noda et al., 2008; Noda et al., 2010) (Fig. 3A). The two pockets are located on the opposite side of the hydrophobic patch (L8-I44V70) of ubiquitin. Electrostatic interactions, which involve two of the three aspartic acid residues of the LIR motif and basic residues in the N-terminal arm and Ubl domain of LC3 (R10, R11, K49 and K50), are also important for the interaction between p62 and LC3 (Fig. 3A,B). The importance of the basic residues in the N-terminal arm of LC3B for binding and autophagic degradation of p62 has been demonstrated by domain swap experiments (Shvets et al., 2008; Shvets et al., 2011). 3241 Different strategies have been used to identify proteins that interact with ATG8 proteins through LIR motifs, including candidate approaches (Pankiv et al., 2007; Noda et al., 2008; Sancho et al., 2012), bioinformatics searches (Kraft et al., 2012), proteomics (Behrends et al., 2010; Pankiv et al., 2010), phage display (Mohrlüder et al., 2007b) and yeast two-hybrid assays (Kirkin et al., 2009b; Novak et al., 2010; Wild et al., 2011; Popovic et al., 2012). LIR motifs have been identified by using deletion mapping and protein–protein interaction assays, and by testing deletion and point-mutated constructs. We have found that peptide array analysis is a specific and efficient method for identification of LIR motifs (Alemu et al., 2012). Cargo receptors in selective autophagy Following discovery of p62 as a selective autophagy receptor, the related neighbor of BRCA1 gene 1 (NBR1) was found to act as an aggrephagy receptor (Kirkin et al., 2009b). Subsequently, nuclear dot protein 52 kDa (NDP52) was found to be an important xenophagy receptor (Thurston et al., 2009) together with optineurin (Wild et al., 2011). These and the other autophagy receptors discussed below use LIR-motif-dependent interactions to target their cargos for autophagic degradation. Sequestosome-1-like receptors In addition to the role of p62, NDP52 and optineurin in selective autophagy, these proteins have also recently been shown to regulate innate immunity signaling pathways and, thus, were suggested to represent a new class of pattern recognition receptors, the sequestosome-1-like receptors (SLRs) (Deretic, 2012). The SLRs currently consists of p62, NBR1, NDP52, optineurin and Tax1-binding protein 1 (TAX1BP1) (Fig. 2). They all contain a dimerization or multimerization domain, a LIR domain (an atypical LIR motif in the case of NDP52 and TAX1BP1) and an ubiquitin-binding domain. These three features of SLRs are required for the efficient execution of their role as autophagic cargo receptors (Pankiv et al., 2007; Ichimura et al., 2008b; Itakura and Mizushima, 2011; Deosaran et al., 2013). Studies of selective autophagy in mammalian cells and of the Cvt pathway in yeast revealed that the cargo must either be aggregated or represent a reasonably large structure that enables the binding of many receptor molecules; alternatively, the autophagy receptors themselves need to be able to multimerize the cargo (Lynch-Day and Klionsky, 2010; Johansen and Lamark, 2011). The dual nature of SLRs as autophagy receptors and scaffolding proteins that act in signaling pathways is intriguing. Since the levels of SLRs are regulated by autophagy, the rate of autophagy obviously impacts on signaling that involves SLRs. To discuss these signaling pathways is beyond the scope of this Commentary, but it is worth noting that accumulation of SLRs occurs during cellular stresses, including infection and inflammation, oxidative stress, ER-stress and metabolic stress (Johansen and Lamark, 2011; Deretic, 2012). This accumulation may have dramatic effects on stress-related signaling pathways, but the exact role of autophagy in the control of signal transduction – beyond its effect on receptor levels – is poorly understood. One exception is the regulation of the KEAP1–NRF2 oxidative-stress-response pathway, in which p62 binds to and sequesters KEAP1, leading to its autophagic degradation and the concomitant induction of NRF2 (Komatsu et al., 2010; Jain et al., 2010; Taguchi et al., 2012). A positive feedback loop is Journal of Cell Science 3242 Journal of Cell Science 126 (15) established in that increased p62 levels activate NRF2, which, in turn, further increases p62 levels (Jain et al., 2010). Recently, it has been found that the liver toxicity of accumulated p62 is due to constitutive upregulation of the NRF2 oxidative-stressresponse pathway (Inami et al., 2011). One of the striking features of SLRs is their ability to mediate the selective autophagy of substrates that apparently have no structural similarities. Substrates targeted by p62 include ubiquitylated protein aggregates and membrane-embedded structures, such as intracellular bacteria and peroxisomes. The single feature that unites the various structures appears to be that they become ubiquitylated before they are degraded. The LIR motif is absolutely required for targeting of SLRs and bound cargoes into the lumen of autophagosomes (Johansen and Lamark, 2011). Selective autophagy depends on a direct interaction between the LIR motif and ATG8 homologs that are conjugated to the inner, concave membrane of the phagophore. However, the LIR motif by itself does not bring a protein to the inner surface of a phagophore, and the majority of LIR-motif-containing proteins are not substrates for selective autophagy. Selective autophagy of p62 depends on its PB1-domain-driven polymerization, but for the delivery of p62-associated cargos, ubiquitin binding and interactions with other proteins are also important. For the selective autophagy of protein aggregates, p62 collaborates with autophagy-linked FYVE protein (ALFY), a nuclear scaffolding protein that is recruited to cytosolic protein aggregates in a p62dependent manner. ALFY interacts directly with ATG5 and phosphatidylinositol (3)-phosphate [PtdIns(3)P], and may act as a scaffold protein that induces the assembly of an autophagycompatible structure (Clausen et al., 2010; Filimonenko et al., 2010). In flies that lack the ALFY ortholog Blue cheese (Bchs), accumulation of the p62 ortholog Ref(2)P in ubiquitin-positive protein aggregates has been observed (Clausen et al., 2010) suggesting a conserved role for ALFY. Redundancy and/or collaboration clearly exist between different SLRs, although this is not very well studied. NBR1 and p62 bind directly to each other through their PB1 domains, and collaborate in selective autophagy of misfolded proteins and probably also midbody rings (Kirkin et al., 2009b; Pohl and Jentsch, 2009; Kuo et al., 2011). These two proteins also collaborate in pexophagy. Here, binding and clustering of peroxisomes is mediated by NBR1 in a process that depends on the coincident membrane binding of its amphipathic J domain and the adjacent UBA domain (Deosaran et al., 2013). Specialized intracellular pathogens have often developed strategies to avoid or use autophagy for their own purposes, but other pathogens are efficiently degraded by selective autophagy if they are released into the cytoplasm or upon membrane rupture (Mostowy and Cossart, 2012). Microbes that are released into the cytosol are ubiquitylated and then recognized by SLRs (Dupont et al., 2009; Thurston et al., 2009; Zheng et al., 2009). Membrane remnants associated with exposed microbes can also be polyubiquitylated and targeted for autophagic degradation by p62 (Dupont et al., 2009). Furthermore, p62 promotes autophagic killing of intracellular microbes. Cytoplasmic precursors of antimicrobial peptides (ubiquitin or ribosomal precursor proteins) are transported by p62 into autolysosomes or microbe-containing autolysosomes. Here, the precursors are converted into peptides that have been shown to kill Mycobacterium tuberculosis (Ponpuak et al., 2010); and these peptides might also be potent against other microbes. Viruses can also act as substrates and p62 has been implicated in xenophagic elimination of Sindbis virus (Orvedahl et al., 2010). Efficient xenophagy of Salmonella enterica serotype Typhimurium (S. typhimurium) is mediated by p62, NDP52 and optineurin, with the p62-containing microdomains on ubiquitin-coated bacteria appearing to be physically separated from areas that are occupied by NDP52 and optineurin (Cemma et al., 2011; Mostowy et al., 2011; Wild et al., 2011). However, although NDP52 is recruited to ubiquitin-coated bacteria through its Cterminal Zinc-finger (ZF) domain (Fig. 2), it is initially targeted to damaged Salmonella-containing vacuoles that are marked by galectin-8, which binds exposed b-galactoside-containing glycans. In this way, cytosolic galectin-8 functions as an ubiquitinindependent ‘danger’ receptor and ‘eat-me’ signal (Thurston et al., 2012). Galectin 8 also detects non-bacteria induced damage to endosomes or lysosomes, suggesting that membrane rupture is the initial common event detected during invasion by microbes (Thurston et al., 2012). The atypical LIR motif in NDP52, termed CLIR, comprises the tripeptide Leu-Val-Val and binds specifically to LC3C (Fig. 3A). Efficient recruitment of the other ATG8 family members to bacteria-degrading autophagosomes depends on both NDP52 and LC3C (von Muhlinen et al., 2012). TAX1BP1 (T6BP) is a cargo receptor with homology to NDP52. TAX1BP1 binds ubiquitin and contains the same atypical LIR motif as NDP52 (Newman et al., 2012) but its role in xenophagy is unknown. NDP52 is required for degradation of the micro RNA (miRNA)-processing enzyme DICER, and the main miRNA effector AGO2 by selective autophagy (Gibbings et al., 2012). An ubiquitin-independent role of optineurin in aggrephagy has recently been reported (Korac et al., 2013). It should also be noted that p62 has a role in mitophagy (Johansen and Lamark, 2011), although this process is primarily mediated by specific mitochondrial membrane receptors, as discussed below. Mitophagy receptors Both yeast and mammalian cells can selectively eliminate damaged or superfluous mitochondria by mitophagy (reviewed by Ashrafi and Schwarz, 2013). In yeast, mitophagy is orchestrated by Atg32, an integral protein of the outer mitochondrial membrane (OMM) with a N-terminus that faces the cytosol and C-terminus located in the intermembrane space (Kanki et al., 2009; Okamoto et al., 2009). Atg32 can interact with Atg8 indirectly through Atg11 and directly through its LIR motif in the N-terminal cytosolic domain. Atg32 recruits Atg8 and Atg11 to the mitochondria surface to form an initiator complex essential for mitophagy (Kondo-Okamoto et al., 2012). In mammalian cells, three integral OMM proteins that all have a LIR motif in their cytosolic N-terminal domain are implicated in mitophagy (Fig. 2). Two homologous BCL2 homology 3 (BH3)only proteins, Bnip3 and Nix (also known as Bnip3L), are able to induce mitophagy and can also activate cell death (reviewed by Zhang and Ney, 2009). Bnip3 induces the removal of both mitochondria and endoplasmic reticulum (Hanna et al., 2012). Homodimerization of Bnip3 through the transmembrane domain facilitates the interaction between the LIR motif of Bnip3 and LC3B. Nix also facilitates LIR-dependent mitophagy (Novak et al., 2010) (supplementary material Table S1). During erythroid cell maturation, Nix mediates the complete removal of mitochondria (Schweers et al., 2007; Sandoval et al., 2008). Additionally, Nix is involved in depolarization-induced mitophagy. Nix has a core LIR motif identical to Bnip3. However, in contrast to Bnip3, Nix does not interact with The LIR motif in selective autophagy LC3B but with GABARAP-L1 during mitochondrial stress (Schwarten et al., 2009; Novak et al., 2010). Hence, residues flanking the core LIR motif might be involved in determining specificity. Bnip3 and Nix are both involved in hypoxia-induced mitophagy (Zhang et al., 2008; Bellot et al., 2009). The third mitophagy receptor in the OMM is FUNDC1; it acts in hypoxiainduced mitophagy, but with a different mechanism that involves the dephosphorylation of its LIR motif, which enhances its binding to LC3B (see below) (Liu et al., 2012). Journal of Cell Science Specialized autophagy receptors So far few autophagy receptors are known to only interact with one substrate under certain circumstances. Starch-bindingdomain-containing protein 1 (Stbd1) and the E3-ubiquitin ligase Cbl represent such specialized receptors (Fig. 2). Stbd1 binds glycogen in vitro and is associated with glycogen in cells; it binds more tightly to abnormal glycogen that is poorly branched (Jiang et al., 2011). Stbd1 binds to GABARAP-L1 through a LIR motif and has been proposed to act as an autophagy receptor for glycogen in a process termed glycophagy (Jiang et al., 2011). Kinase activity can also be regulated by selective autophagy that involves interaction with the LIR motif. For instance, Cbl has been identified as an autophagy receptor for the active, nonreceptor, membrane-associated tyrosine kinase Src (Sandilands et al., 2012). Increased Src activity promotes tumorigenesis but excessive Src signaling can be cytotoxic (Yeatman, 2004). When integrin signaling through the focal adhesion kinase (FAK)–Src pathway is disrupted in cancer this can lead to excessive and cytotoxic Src activity. By using its LIR motif to bind LC3B, Cbl is able to switch the targeting of Src from the proteasome to autophagic degradation, thereby promoting cancer cell survival (Sandilands et al., 2012). Many LIR-containing proteins do not act as cargo receptors The presence of functional LIR motifs in components of the core autophagy machinery demonstrates that LIR-motif-mediated Cargo recruitment interactions do not only help targeting cargo receptors to autophagosomes but are also involved in regulating autophagosome formation and maturation. In addition to the core autophagy machinery, several other LIR-motif-containing proteins are involved in autophagosome formation, transport and maturation (fusion to lysosomes) (see Fig. 4) (supplementary material Table S1). LIR-motif-containing proteins in the core autophagy machinery The ATG proteins of the core autophagy machinery are involved in all steps of autophagosome formation (Fig. 1) (Mizushima et al., 2011). The yeast serine/threonine kinase Atg1 (ULK1 in mammals) forms a large complex with Atg13 and the Atg17– Atg31–Atg29 ternary complex. Recently, two independent studies reported a LIR-motif-dependent interaction between Atg1 and Atg8 (Kraft et al., 2012; Nakatogawa et al., 2012). Atg1 is present on autophagosomes in an Atg8-dependent manner before it is transported to the vacuole for its degradation. Kraft et al. (Kraft et al., 2012) also showed that Atg13, in complex with Atg1, is degraded by autophagy. Similarly, Atg1 and Atg13 are degraded by autophagy during nutrient starvation in Arabidopsis (Suttangkakul et al., 2011), but the role of the interaction between their LIR motifs and ATG8 is currently unknown. Mutations in the LIR motif of Atg1 result in reduced autophagy but do not influence its functions during initiation of autophagosome formation (Nakatogawa et al., 2012). This indicates that Atg1 is also involved in late events of autophagy. Kraft et al. showed that the Atg1–Atg8 interaction is conserved and maintained in mammals, by demonstrating that ULK1 associates with autophagosomes in a LIR-motif-dependent manner (Kraft et al., 2012). We identified that the same LIR motif in ULK1 is required for its starvation-induced association with autophagosomes (Alemu et al., 2012). In contrast to that in Atg1, the LIR motif of ULK1 does not significantly mediate its degradation. We also mapped LIR motifs in ULK2, and the ULK complex proteins ATG13 and FIP200, and demonstrated their binding to ATG8 proteins with a preference for the GABARAP-subfamily (Alemu Maturation + Phagophore Autolysosome Autophagosome Transport Microtubule – Key ULK1–ULK2 complex SLRs (p62, NBR1, NDP52, OPTN) ATG4 LC3-PE (LC3-II) Ubiquitylated misfolded protein TBC1D5, TBC1D25 Kinesin DOR and TP53INP1 Mitophagy receptor Mitochondria 3243 ATG8–LIR-motif interaction FYCO1 Fig. 4. Involvement of LIR-ATG8 interaction in selective autophagy. Selective recruitment of cargo to the inner membrane of the phagophore is mediated by interaction between a LIR-motif containing autophagy receptor and lipidated ATG8 (shown here LC3-PE). Transport of autophagosomes towards plus ends of microtubules involves the interaction of the LIR motif of FYCO1 with LC3-PE on the outer autophagosomal membrane. Maturation of the autophagosome is dependent on interaction between the LIR motif of factors involved in the autophagy machinery (e.g. the ULK1–ULK2 complex or ATG4, or regulatory factors, such as TBC1D5, TBC1D25, DOR and TP53INP1) with ATG8 proteins, which then recruit effector proteins to the outer membrane. Journal of Cell Science 3244 Journal of Cell Science 126 (15) et al., 2012). It is possible that LIR-ATG8 interactions of ULK complex proteins facilitate and/or stabilize tethering of the ULK complex to the phagophore. Other members of the core autophagy apparatus, Atg3 in yeast and ATG4B in mammals, undergo LIR-motif-dependent interactions with ATG8 that potentially serve a regulatory role (Satoo et al., 2009; Yamaguchi et al., 2010). Among the four ATG4 homologs (ATG4A, ATG4B, ATG4C, ATG4D), ATG4B is the main human ATG4 homolog that efficiently processes ATG8 precursors and ATG8-PE (Li et al., 2011). The crystal structure of the human ATG4B–LC3B complex indicates conformational changes in ATG4B upon binding of the LC3 substrate that facilitate access of LC3 to the catalytic site of ATG4B (Satoo et al., 2009). Interestingly, in the crystal structure, the N-terminal LIR motif of ATG4B interacts with a LIR-binding site on an adjacent (non-substrate) LC3. This interaction with the LIR motif stabilizes an open conformation of the N-terminal tail of ATG4B, which presumably favors membrane targeting. Since ATG4 also mediates deconjugation of ATG8 proteins, a process that requires membrane targeting, the conformation of the Nterminal tail might, therefore, regulate the deconjugation activity of ATG4 (Satoo et al., 2009). The yeast E2-like enzyme Atg3 contains a canonical LIR motif (WEDL) that is essential for the efficient transfer of Atg8 from Atg3 to PE. The Atg3 LIR motif is required for the Cvt pathway but not for starvation-induced autophagy. The interaction between the LIR motif of Atg3 and Atg8 liberates Atg8 from being bound by the LIR motif of Atg19, thus allowing Atg8–PE conjugation (Yamaguchi et al., 2010). LIR-containing proteins associated with autophagosomes and other vesicles Although some steps of autophagosome formation are well understood, the membrane origins of autophagosomes are still debated. Multiple membrane sources were found to be involved, such as endoplasmic reticulum (ER), mitochondria, ERmitochondria contact sites and plasma membrane (Hamasaki et al., 2013; Weidberg et al., 2011). The plasma membrane can contribute directly to the formation of ATG16L1-positive autophagosome precursors that depend on interactions between ATG16L1 and the clathrin heavy chain (Ravikumar et al., 2010). Hence, clathrin-mediated endocytosis might be involved in regulating the initial stages of autophagosome formation (Ravikumar et al., 2010). Interestingly, the clathrin heavy chain also interacts with GABARAP through a LIR motif on a surfaceexposed a-helix in the flexible linker region (Mohrlüder et al., 2007a). Structural studies show that LIR motifs adopt a bconformation when bound to ATG8-proteins and form an intermolecular parallel b-sheet (Fig. 3B) (Noda et al., 2010). It will, therefore, be interesting to learn which conformation the clathrin LIR has upon binding to GABARAP. Clathrin and GABARAP are both involved in trafficking of the GABAA receptor, suggesting that the LIR-mediated interaction has a physiological relevance. However, it has not been studied whether this interaction impacts on autophagosome formation. Calreticulin, which competes with clathrin for binding to GABARAP (Mohrlüder et al., 2007a), also has a LIR motif very similar to that of clathrin (supplementary material Table S1) (Mohrlüder et al., 2007b). Calreticulin is a luminal Ca2+-dependent chaperone of the ER, but is also involved in variety of cytosolic functions as a regulator of intracellular Ca2+ homeostasis (Wang et al., 2012). It is presently not known whether these LIR interactions are relevant for both autophagosome formation and trafficking of the GABAA receptor, or for only the latter. The autophagosome precursor that is generated by clathrindependent endocytosis might represent phagophore precursors. During and after phagophore formation, proteins are recruited to the forming autophagosome in a ‘retrieve–recycle’ manner. The tumor protein 53-induced nuclear protein 2 (TP53INP2; also known as and, hereafter, referred to as DOR) exits the nucleus in response to cellular stress or the activation of autophagy (Nowak et al., 2009; Mauvezin et al., 2010). Cytoplasmic DOR then localizes to autophagosomes where it interacts with the transmembrane protein VMP1 (Nowak et al., 2009). On autophagosomes, DOR interacts with LC3B through its LIR motif (Sancho et al., 2012), but it does not colocalize with the autolysosome-associated protein LAMP1, indicating that DOR localizes only to early autophagosomes (Mauvezin et al., 2010). Through its interaction with VMP1, DOR presumably acts as a scaffold protein that recruits ATG8 proteins to the autophagosome (Nowak et al., 2009). The LIR motif in DOR overlaps with its nuclear export signal (Sancho et al., 2012). Hence, mutation of the core LIR residues of DOR blocks its nuclear exit in response to autophagy activation. Interestingly, DOR and its homolog TP53INP1 share two highly conserved regions, including the LIR motif (Sancho et al., 2012). LIRmediated localization of TP53INP1 to autophagosomes induces autophagy- and caspase-dependent cell death, and it has been suggested that TP53INP1 displaces p62 from LC3B, which then promotes cell death (Seillier et al., 2012). The mechanisms that regulate membrane trafficking in autophagy are poorly understood. The Rab GTPases (a large family of monomeric, small GTPases) in their active form are spatially organized into distinct membrane regions, where they recruit effectors to regulate intracellular vesicle trafficking events (Stenmark, 2009). Rab GTPase-activating proteins (GAPs) negatively regulate the activity of Rab GTPases. The Rab GAP TBC1D25 is recruited to phagophores and autophagosomes through direct interactions between its LIR motif and ATG8 homologs, and its GAP activity regulates the fusion between autophagosomes and lysosomes (Itoh et al., 2011). TBC1D25 inhibits Rab33B, a Golgi-resident Rab (Itoh et al., 2011). Active Rab33B binds to ATG16L1 and is involved in recruitment of the ATG12-ATG5-ATG16L complex to preautophagosomal structures (Itoh et al., 2008). The authors suggest a model whereby TBC1D25 uses ATG8 proteins as scaffolds to regulate autophagosomal maturation (Itoh et al., 2011). Recently, 14 of 36 human TBC (Tre2, Bub2, Cdc16)-domaincontaining Rab GAPs were shown to interact with ATG8 proteins in yeast two-hybrid screens (Popovic et al., 2012). One of these, TBC1D5 contains two LIR motifs, which are both required for ATG8 binding and its co-localization with ATG8 to autophagosomes upon starvation-induced autophagy (Popovic et al., 2012). TBC1D5 is involved in retrograde traffic from endosomes to the Golgi. Interestingly, the N-terminal LIR of TBC1D5 interacts with the Vps29 subunit of the retromer complex, a recycling endosome sorting complex responsible for vesicle delivery from early endosomes to the Golgi. The binding of TBC1D5 to Vps29 can be titrated out by LC3 (Popovic et al., 2012), indicating that TBC1D5 acts as a molecular switch between endosomes and autophagy. Furthermore, the C-terminal LIR of TBC1D5 can tether the endosome and autophagosome, thereby mediating autophagosome maturation (Popovic et al., 2012). Journal of Cell Science The LIR motif in selective autophagy The examples above demonstrate crosstalk between endocytosis and autophagy, which then converge in lysosomal degradation. Rab7 is involved in maturation of both autophagosomes and endosomes, as well as in the transport of autophagosomes and endosomes towards lysosomes for degradation. The Rab7 effector FYVE and coiled-coil-domaincontaining protein 1 (FYCO1) is localized on phagophores, autophagosomes and late endosomes and, in addition to Rab7, interacts with LC3 and PtdIns(3)P (Pankiv et al., 2010). FYCO1 binds to LC3 through a LIR motif in the middle of the connecting loop between its FYVE and GOLD domains. This flexible loop is predicted to be folded in a way that blocks the interaction between the FYVE domain and PtdIns(3)P. Binding to LC3B on autophagic structures releases this inhibition and targets FYCO1 exclusively to PtdIns(3)P-containing membranes that contain LC3B (Pankiv et al., 2010). FYCO1 couples autophagosomes and other Rab7-positive vesicles to molecular motors. Depending on the direction of vesicle movement, FYCO1 is coupled to kinesin molecular motors. This is further supported by the identification of a potential kinesin-binding site in FYCO1 (Pankiv et al., 2010). Mitogen-activated protein kinase 15 (MAPK15) is another protein that localizes to autophagosomes through an interaction between its LIR motif and ATG8 proteins (supplementary material Table S1) (Colecchia et al., 2012). The kinase activity of MAPK15 is known to affect the rate of both basal and starvation-induced autophagy (Colecchia et al., 2012) but the substrates of MAPK15 that are involved in autophagy are unknown. LIR-containing signaling proteins that act as substrates for selective autophagy One signaling pathway regulated by autophagy is Wnt signaling; autophagy enhances the degradation of Dishevelled2 (Dvl2), a transducer of the Wnt pathway and, thus, negatively affects Wnt signaling (Gao et al., 2010). The C-terminal DEP domain of Dvl2 contains a LIR motif that binds to ATG8 proteins (Gao et al., 2010; Zhang et al., 2011). The N-terminal DIX domain mediates the selfoligomerization of Dvl2 that is necessary for its ubiquitylation, and facilitates its binding to LC3B and GABARAP (Gao et al., 2010). Ubiquitylation of Dvl2 is enhanced during starvation, and is essential for its interaction with p62 and subsequent targeting to autophagosomes. Thus, p62 mediates the indirect association of Dvl2 with LC3B and GABARAP (Gao et al., 2010). This is also likely to be the case for the interaction of Dvl2 with GABARAPL1 (Zhang et al., 2011). Consequently, Dvl2 is degraded by autophagy through its LIR-dependent interaction with ATG8 proteins and by means of the autophagy receptor p62. Very recently, it was shown that b-catenin is selectively degraded by autophagy during nutrient deprivation via the formation of a bcatenin-LC3 complex depending on a LIR motif in b-catenin. A regulatory feedback mechanism is at work, in which active Wnt/bcatenin signalling represses autophagy and p62 expression, while b-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction (Petherick et al., 2013). Regulation of the interaction between LIR and ATG8 through phosphorylation Since 25% of the known LIR motifs harbour an S or T residue as the ‘any amino acid residue’ at position –1 immediately N-terminal to the aromatic residue of the LIR motif (supplementary material Table S1), it is conceivable that binding affinity of LIR motifs is 3245 regulated through phosphorylation. Indeed, NDP52 recruits TANKbinding kinase 1 (TBK1) to the bacterial surface (Thurston et al., 2009). Optineurin also recruits TBK1 to ubiquitylated Salmonella, resulting in the subsequent phosphorylation of optineurin at S177 located at the ‘any amino acid residue’ at position –1, which strongly enhances the binding to LC3B (Wild et al., 2011). Optineurin and NDP52 occupy the same microdomains on the bacteria. Thus, NDP52-bound TBK1 can also phosphorylate the LIR of optineurin, thereby enhancing the response. The phosphorylation state of the LIR motif of Bnip3 has been shown to determine whether it executes pro-survival mitophagy or apoptosis. Phosphorylation of S17 and S24 that flank the LIR increases binding of Bnip3 to LC3B and GABARAP-L2, and induces mitophagy. When its LIR motif is unphosphorylated, Bnip3 functions as a BH3-only protein and promotes apoptosis (Zhu et al., 2013). Furthermore, the interaction of FUNDC1 with LC3B is enhanced during hypoxia through the dephosphorylation of the tyrosine residue (Y18 binding to HP1) in its LIR, which facilitates mitophagy (Liu et al., 2012). Not only the LIR motifs but also the binding surface of ATG8 proteins may be phosphorylated to regulate binding of LIR-motifcontaining proteins. For instance, phosphorylation of S12 in the Nterminal arm of rat LC3B by protein kinase A (PKA) negatively affects autophagy (Cherra et al., 2010). This residue is adjacent to the two Arg residues (R10 and R11) that bind to two aspartic acid residues (bold) in the DDDWTHL LIR motif of p62, but it has not been studied whether phosphorylation of S12 affects the docking of p62. Further studies are required to thoroughly address the question to which extent LIR motifs and LIR docking sites are regulated by posttranslational modifications. Concluding remarks The interaction between LIR motifs and ATG8 proteins is crucial for the recruitment of cargo to the inner surface of the phagophore, and for the recruitment of effector proteins to the outer autophagosomal membrane where these effectors mediate transport and maturation of autophagosomes (Fig. 4). The characterization of LIR-motif-containing proteins and the elucidation of their roles in autophagy are still at an early stage. Additional examples on how LIR–ATG8 interactions are regulated through phosphorylation or other post-translational modifications are clearly anticipated in future studies. It will be interesting to see whether an interaction of LIR motifs with ATG8 proteins is also involved in processes other than autophagy. The binding of TBC1D5 to Vps29 through its Nterminal LIR motif suggests that there are binding partners for LIR-motifs other than ATG8 proteins. Finally, it will also be interesting to investigate whether these LIR–ATG8 interactions can be explored as druggable targets. Acknowledgements We thank members of our group for critical reading of the manuscript, and Steingrim Svenning for help with Fig. 3. Funding This work was funded in part by grants from the FUGE and FRIBIO programs of the Norwegian Research Council, the Norwegian Cancer Society and the Blix foundation to T.J. 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Known LIR-containing proteins and details of their LIR motifs and interaction partners LIR protein LIR motif LIRpos ATG8 ortholog interaction Role in autophagy References Sequestosome-1-like receptors (SLRs) p62 SGGDDDWTHLSS 338 Binds to LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2. Autophagy receptor for degradation of ubiquitylated cargo: protein aggregates, soluble proteins, midbody rings, damaged mitochondria, peroxisomes and phagocytic membrane remnants. Also involved in xenophagy. (Pankiv et al., 2007; Ichimura et al., 2008b) SASSEDYIIILP 732 Binds to LC3A, LC3B, LC3C, GABARAP, GAPARAP-L1 and GAPARAP-L2. Autophagy receptor; targets ubiquitylated cargo. Involved in aggrephagy. Acts as an autophagy receptor for peroxisomes. Also involved in xenophagy. (Kirkin et al., 2009b; Behrends et al., 2010) LCGVSEWDPILE 661 Binds to six of the eight AtATG8 family members that were tested. Plant (Arabidopsis thaliana) NBR1 that is a functional hybrid of mammalian p62 and NBR1. (Svenning et al., 2011) ENEEDILVVTT 134 Strong preference for LC3C. Note the non-canonical LIR motif. Autophagy receptor; targets invasive bacteria for degradation (xenophagy) (Thurston et al., 2009; von Muhlinen et al., 2012) EGNSDMLVVTT 141 Binds to LC3C and GABARAP. Very weak binding to LC3B. *Note non-canonical LIR motif. Recently identified cargo receptor where the N- terminal part shows homology with NDP52. Role in xenophagy has not been studied. (Newman et al., 2012) GSSEDSFVEIRM 178 Binds to LC3A, LC3B, GABARAP, GABARAP-L1 and GABARAP-L2. (LC3C not tested). Autophagy receptor; targets ubiquitin-coated bacteria (xenophagy). Also targets various protein aggregates in an ubiquitin-independent manner. (Wild et al., 2011) (Q13501) NBR1 (Q14596) AtNBR1 (Q9SB64) NDP52 (Q13137) Tax1bp1/T6BP (Q86VP1) Optineurin (Q96CV9) Mitophagy receptors Nix/Bnip3L (O60238) AGLNSSWVELPM 36 Binds to LC3A, LC3C, GABARAP, GABARAP-L1 and –L2. Relatively weak interaction A mitophagy receptor required for selective mitochondrial clearance after mitochondrial damage and during (Schwarten et al., 2009; LIR protein Bnip3 LIR motif LIRpos References with LC3B. erythroid differentiation. Novak et al., 2010) 18 Binds to LC3B and GABARAPL2. A mitophagy receptor that induces removal of both mitochondria and ER. (Hanna et al., 2012; Zhu et al., 2013) ESDDDSYEVLDL 18 Binds to LC3A, LC3B, GABARAP and GABARAP-L2.* A mitophagy receptor; mediates hypoxia-induced mitophagy. (Liu et al., 2012) DSISGSWQAIQP 86 Binds to ScAtg8. A mitophagy receptor; regulates selective degradation of mitochondria in yeast (Okamoto et al., 2009; KondoOkomoto et al., 2012) (Q8IVP5) ScAtg32 Role in autophagy ESLQGSWVELHF (Q12983) FUNDC1 ATG8 ortholog interaction (P40458) Specialized receptors Stbd1 RVDHEEWEMVPR 203 Binds to LC3B, LC3C, GABARAP, GABARAP-L1 and L2. (LC3A not tested). Specialized autophagy receptor; involved in degradation of glycogen particles (Behrends et al., 2010; Jiang et al., 2011) ASSSFGWLSLDG 802 Binds to LC3B*. Specialized autophagy receptor. An E3 ubiquitin ligase that targets active tyrosine kinase Src for autophagy (Sandilands et al., 2012) NEKALTWEEL 412 Binds to ScAtg8. A cargo receptor for aminopeptidase I (Ape1) and αmannosidase in the Cvt pathway in yeast. (Noda et al., 2008) LSRPFTWEEI 409 Binds to ScAtg8. A cargo receptor (Atg19 homolog) that acts cooperatively with Atg19 in the Cvt pathway in yeast; mediates the delivery of α-mannosidase to the vacuole but not of aminopeptidase I (Ape1). (Suzuki et al., 2010) (O95210) c-Cbl (P22681) Cvt cargo receptors ScAtg19 (P35193) ScAtg34 (Q12292) Components of the core autophagy machinery ULK1 SCDTDDFVMVPA 357 Preference for GABARAP and GABARAP-L1. Also binds to Member of the core autophagy machinery. Required for (Alemu et al., 2012; LIR protein LIR motif LIRpos (O75385) ULK2 ATG8 ortholog interaction autophagosome biogenesis. Kraft et al., 2012) SCDTDDFVLVPH 353 Same as ULK1. Member of the core autophagy machinery. Strongly related to ULK1. Not needed for autophagy in cells expressing ULK1. (Alemu et al., 2012) HEDSDDFVLVPK 391 Binds to DmATG8A. Member of the core autophagy machinery in D. melanogaster. Important for autophagosome biogenesis. (Alemu et al., 2012) RSFEREYVVVEK 429 Binds to ScAtg8. Member of the core autophagy machinery in yeast. Required for autophagosome biogenesis. Atg8 binding triggers vacuolar degradation of the Atg1–Atg13 complex in yeast. (Nakatogawa et al., 2012; Kraft et al., 2012) GNTHDDFVMIDF 444 Preference for GABARAP and GABARAP-L1. Also binds to GABARAP-L2, LC3A and LC3C. Very weak binding to LC3B. Member of the core autophagy machinery; A component of the ULK1–ULK2 complex. (Alemu et al., 2012) DAHTFDFETIPH 702 Binds to GABARAP and GABARAP-L1. Member of the core autophagy machinery; A component of the ULK1–ULK2 complex. (Alemu et al., 2012) DAATLTYDTLRF 8 Binds to LC3B and LC3C. Discrepancy in references regarding binding to GABARAP and GABARAP-L2. Member of the core autophagy machinery. A cysteine protease used for processing ATG8 family proteins prior to conjugation of ATG8/LC3 to phosphatidylethanolamine (PE). (Satoo et al., 2009; Behrends et al., 2010; Li et al., 2011) LDGVGDWEDLQD 270 Binds to ScAtg8. Member of the core autophagy machinery in yeast. An E2-like enzyme involved in conjugation of Atg8 to phosphatidylethanolamine. (Yamaguchi et al., 2010) KIVDNDWLLPSY 105 Binds to PfAtg8. A LIR-like motif in Plasmodium falciparum (Pf) Atg3 has been described that interacts with PfAtg8. The importance of the interaction for PfAtg 8 lipidation and autophagy has (Hain et al., 2012) (Q8MQJ7) ScAtg1 (P53104) ATG13 (O75143) FIP200 (Q8TDY2) ATG4B (Q9Y4P1) ScAtg3 (P40344) PfAtg3 (C0H519) References GABARAP-L2, LC3A and LC3C. Weak binding to LC3B. (Q8IYT8) DmATG1B Role in autophagy LIR protein LIR motif LIRpos ATG8 ortholog interaction Role in autophagy References not been investigated. Proteins associated with autophagosomes and other vesicles Clathrin HC VGYTPDWIFLLR 514 Binds to GABARAP.* It has been proposed that clathrin mediated endocytosis contributes to the formation of early autophagic precursors. The role of the LIR interaction in autophagosome formation has not been addressed. (Mohrlüder et al., 2007a) GSLEDDWDFLPP 200 Binds to GABARAP.* Calreticulin competes with clathrin for binding to GABARAP. Calreticulin might have an important regulatory role for the GABARAP interaction with clathrin. Direct role in autophagy is not known. (Mohrlüder et al., 2007a and 2007b) EDEVDGWLIIDL 35 Binds to LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2. Required for autophagosome development. (Nowak et al., 2009; Sancho et al., 2012) (Q00610) Calreticulin (P27797) TP53INP2/DOR (Q8IXH6) TP53INP1 EKEDDEWILVDF 31 Binds to LC3A, LC3B, LC3C GABARAP, GABARAP-L1 and GABARAP-L2. Localizes to autophagosomes mediated by LIR binding and induces autophagy-dependent cell death. (Seillier et al., 2012; Sancho et al., 2012) SPLLEDWDIISP 136 Binds to LC3B, GABARAP and GABARAP-L2.* GTPase-activating protein for Rab33B. Regulates the interaction of autophagosomes with lysosomes by inactivating Rab33B. (Itoh et al., 2011) NSYRKEWEELFV 58 Binds to LC3A, LC3B and GABARAP-L1.* A Rab GTPase-activating protein recruited from endosomes to autophagosomes during starvation-induced autophagy. LIR 1 mediates binding to Vps29 in the retromer complex and this binding can be titrated out by LC3. (Popovic et al., 2012) SSKDSGFTIVSP 788 Binds to LC3A, LC3B and GABARAP-L1.* TBC1D5 LIR2 can bridge endosomes and autophagosomes. TBC1D5 appears to mediate autophagosome maturation (Popovic et al., 2012) (Q96A56) TBC1D25 (Q3MII6) TBC1D5 LIR1 (Q92609) TBC1D5 LIR2 (Q92609) Interacts with VMP1 and presumably acts as a scaffold protein recruiting ATG8 proteins to the autophagosome membrane. LIR protein FYCO1 LIR motif LIRpos ATG8 ortholog interaction PPDDAVFDIITD 1280 Preference for LC3A and LC3B. Weak binding to LC3C, GABARAP, GABARAP-L1 and L2. Involved in movement of autophagosomes to lysosomes using microtubuli plus end-directed transport. (Pankiv et al., 2010; Alemu et al., 2012) EYRSRVYQMILE 340 Binds to LC3B, GABARAP and GABARAPL1.* A MAP kinase that stimulates autophagy. (Colecchia et al., 2012) (Q9BQS8) MAP15K (Q8TD08) Role in autophagy References Signaling proteins that acts as a substrates for selective autophagy Dvl2 EVRDRMWLKITI 444 Binds to LC3B and GABARAP. Does not bind GABARAP-L2. * Important adaptor protein in the Wnt signaling pathway. Autophagy negatively regulates Wnt signaling by accelerating Dvl degradation under stress conditions. Functional role in autophagy is unknown. (Gao et al., 2010) LHPPSHWPLIKA 504 Binds to LC3B. Other ATG8 proteins were not tested. Wnt signalling negatively regulates autophagy and p62 expression while β-catenin, like Dvl2 is degraded by autophagy upon nutrient deprivation revealing a regulatory feedback mechanism. (Petherick et al., 2013) (O14641) β-catenin (P35222) *Only these were tested for interaction. Bold amino acids indicate the core consensus sequence.

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