During the last decade it has become evident that autophagy is not simply a non-selective bulk degradation pathway for intracellular components. On the contrary, the discovery and characterization of autophagy receptors which target specific cargo for lysosomal degradation by interaction with ATG8 (autophagy-related protein 8)/LC3 (light-chain 3) has accelerated our understanding of selective autophagy. A number of autophagy receptors have been identified which specifically mediate the selective autophagosomal degradation of a variety of cargoes including protein aggregates, signalling complexes, midbody rings, mitochondria and bacterial pathogens. In the present chapter, we discuss these autophagy receptors, their binding to ATG8/LC3 proteins and how they act in ubiquitin-mediated selective autophagy of intracellular bacteria (xenophagy) and protein aggregates (aggrephagy).
- selective autophagy
Eukaryotic cells utilize two mechanistically distinct but largely complementary systems, the UPS (ubiquitin–proteasome system) and the lysosome (or vacuole in yeast and plants), to effectively degrade cellular components. Macroautophagy (hereafter autophagy) is characterized by formation of a double-membrane autophagosome that envelopes components of the cytoplasm and then fuses to a late endosome or lysosome for degradation of its content. During the 1990s genetic screens in yeast identified the ATG (autophagy-related protein) genes responsible for this process . Other chapters in this volume describe the molecular players in the autophagosome assembly pathway. Most likely, autophagy evolved both as a non-selective pathway for restoring intracellular nutrient supply during starvation, and as a quality control mechanism to facilitate selective removal of toxic or surplus structures .
Lipidated ATG8 [LC3 (light-chain 3) and GABARAP (γ-aminobutyric acid receptor-associated protein) proteins in mammals] is located both on the inner and outer membrane of autophagic vesicles, and acts as a perfect scaffold for specific recruitment of proteins to the phagophore (forming autophagosome). Selective autophagy is based on the recognition and degradation of a specific cargo, in a process depending on receptor proteins that bind ATG8/LC3 to facilitate enrichment of cargoes sequestrated for degradation (Figure 1). Most autophagy receptors bind ATG8/LC3 through a short LIR (LC3-interacting region) motif. The LIR motif is a degenerate sequence with a core motif corresponding to W/F/Y-XX-L/I/V (where X can be any amino acid). They target either ubiquitinated cargoes, interact directly with the cargo, or constitute ATG8-interacting ligands in the outer membrane of mitochondria. Studies of the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, which mediates selective import of two precursor enzymes to the yeast vacuole, have also contributed to the mechanistic understanding of autophagy as a selective process . However, the first selective autophagy receptor to be identified was the human protein p62/SQSTM1 (sequestosome 1) [4,5]. This protein was initially characterized as a scaffold protein acting in several important signalling pathways . In addition, p62 was known to accumulate in ubiquitin-containing protein bodies in a number of protein aggregation diseases , creating a link to human pathophysiology that instigated an increased interest in autophagy as a selective process.
Selective autophagy can be classified according to the cargoes involved (Table 1). In the present chapter we focus on the autophagy receptors, their interaction with ATG8/LC3 proteins and their roles in ubiquitin-mediated degradation of intracellular bacteria (xenophagy) and protein aggregates (aggrephagy).
Concomitant with an improved knowledge about cargoes comes identification of novel autophagy receptors orchestrating the process of recognition and delivery. Their function can be outlined by four common criteria: (i) autophagy receptors bind ATG8/LC3 through a conserved binding site, the LIR motif; (ii) autophagy receptors are degraded with the cargo; (iii) deletion of autophagy receptors should not interfere with the function of the basic autophagy machinery; and (iv) protein–protein binding domains or membrane-binding domains mediate interaction with the cargo.
It is important to mention that among a growing number of LIR-containing proteins interacting with ATG8/LC3/GABARAP proteins, many are not autophagy receptors .
On the basis of structural features and how they interact with the respective cargoes, autophagy receptors can be divided into the following four different groups (Figure 2): (i) SLRs (sequestosome 1-like receptors) share several similarities structurally and functionally with p62/SQSTM1 . The SLRs all contain dimerization or polymerization domains, bind ubiquitin and interact with members of the ATG8 family proteins. These autophagy receptors recognize ubiquitinated cargoes via C-terminal ubiquitin-binding domains with defined affinities for different ubiquitin moieties. However, in some cases they can also bind directly to the unmodified cargo [2,10]. (ii) Autophagic removal of damaged mitochondria (mitophagy) represents the best-studied example of selective autophagy mediated by membrane-associated receptors. Mammalian NIX (NIP3-like protein X), Bnip3, FUNDC1 and yeast Atg32 localize to the mitochondrial outer membrane, where they are anchored by transmembrane domains and mediate selective clearance of mitochondria through LIR-dependent interaction with ATG8 family proteins [11–15] (Figure 2). (iii) Recently, some specialized autophagy receptors have emerged as exemplified by the Stbd1 (starch-binding-domain-containing protein 1) and the E3-ubiquitin ligase Cbl. Stbd1 is suggested to act as a selective autophagy receptor for glycogen in a process named glycophagy  (Table 1). (iv) In the yeast Cvt pathway, the vacuolar hydrolases aminopeptidase 1 (Ape1p) and α-mannosidase (Ams1p) are selectively imported to the yeast vacuole through direct binding to the autophagy receptor Atg19. Binding initiates the formation of a multimeric, or aggregated, Cvt complex. Atg19 then interacts with the adaptor Atg11, which mediates translocation to the forming autophagosome where Atg19 binds to Atg8 to facilitate delivery of the hydrolases . Although Atg19 acts under nutrient-rich conditions, the homologous protein Atg34 acts under nutrient starvation-induced autophagy to transport Ape1p and Ams1p to the vacuole .
As mentioned above, p62 was the first selective autophagy receptor described in mammalian cells [4,5]. Kirkin et al. , who originally cloned p62, noted the ability of p62 to form aggregates, and coined the name SQSTM1 on the basis of this property. So far, four human ubiquitin-binding autophagy receptors have been described; p62/SQSTM1, NBR1 [neighbour of BRCA1 (breast cancer early-onset 1) gene 1] , NDP52 (nuclear dot protein 52)  and optineurin  (Figure 2).
Human p62 is 440 amino acids long and contains an N-terminal PB1 (Phox and Bem1p) domain, a LIR motif and a C-terminal UBA (ubiquitin-associated) domain (Figure 2). The PB1 domain has oppositely charged surface areas and allows p62 to self-interact, forming polymers in a front-to-back interaction. Polymerization of p62 is essential for selective degradation of p62 by autophagy, and among the human PB1 domain proteins only p62 has this ability. p62 binds ubiquitin via the C-terminal UBA domain. The polymerization- and ubiquitin-binding ability allows p62 to cluster ubiquitinated cargo. The LIR motif mediates interaction with LC3 and is necessary for the degradation of p62 and p62-containing structures by autophagy [5,21]. p62 has been instrumental for the understanding of receptor-mediated autophagy and has been most studied for its role in the clearance of protein aggregates (aggrephagy).
NBR1 and p62 display low sequence similarity, but share related domain architectures. They bind to each other via their PB1 domains, and act as partners in the clearance of protein aggregates, peroxisomes and midbody rings [2,18,22]. NBR1 cannot polymerize via PB1, but self-interacts via a coiled-coil domain. Homologues of NBR1 are found throughout the eukaryotic kingdom, whereas the presence of p62 is unique for metazoans and likely the result of a gene duplication event early in the metazoan lineage . During evolution p62 has lost several domains found in NBR1. Therefore these two proteins may also have some independent roles in selective autophagy. Supporting this, N-terminal to its UBA domain, NBR1 has a small amphipathic α-helix, the J-domain, that mediates interaction with cellular membranes. NBR1 was recently linked to J-domain-dependent degradation of peroxisomes in a process which includes, but does not depend on, p62 .
The two other SLRs, optineurin and NDP52, have both been linked to selective autophagy of bacteria (xenophagy) [19,20]. Optineurin has additionally been implicated in ubiquitin-independent selective autophagy of various protein aggregates . The domain architecture of both proteins comprises coiled-coil domains, functional LIR motifs and C-terminal ubiquitin-binding domains (Figure 2). Optineurin has been identified in protein aggregates of several neurodegenerative diseases, and given its ability to self-interact and oligomerize through coiled-coil domains it is likely to function as an aggregating cargo receptor like p62. Optineurin is also involved in ubiquitin-independent aggrephagy of huntingtin aggregates . Less is known about the aggrephagy properties of NDP52, but it has been implicated recently in selective autophagy of the microRNA processing DICER and the effector AGO2 (Argonaute 2) .
Interaction of autophagy receptors with ATG8/LC3
The interaction with ATG8 family proteins constitutes the pivotal link between autophagy receptors and the phagophore. Yeast has a single ATG8 protein, Drosophila melanogaster and Caenorhabditis elegans have two, Arabidopsis thaliana has nine and mammals have at least seven. Mammalian ATG8s can be grouped into two subfamilies which includes the LC3s (LC3A, LC3B and LC3C) and the GABARAPs (GABARAP, GABARAPL1 and GABARAPL2). Structurally, ATG8 proteins consist of two N-terminal α-helices and a ubiquitin-like C-terminal domain made up of four-stranded β-sheets with two α-helices on either side (Figure 3A). The ATG8 interaction between LC3B and p62 was mapped to the LIR motif . The functional LIR motif sequence in human p62 stretches from residues 335 to 441 (DDDWTHL). Structural studies of the complex between the p62 LIR peptide and LC3B have shown that the Trp338 and Lys341 bind in two hydrophobic pockets in the ubiquitin-like domain of LC3B. The first two aspartic acid residues in the LIR motif form electrostatic interactions with basic residues Arg10 and Arg11 in the N-terminal arm of LC3B (Figures 3B and 3C) . Following the initial characterization of the LIR motif in p62, similar motifs have been identified in numerous other ATG8 interactors, and a consensus LIR motif can be written as W/F/Y-X1X2-L/I/V, with an aromatic amino acid in the first position and a bulky hydrophobic amino acid in the fourth position. Acidic residues are frequently found in one to three positions preceding the aromatic residue. If there are no acidic residues at these positions there is usually an acidic residue at X1, like in yeast Atg19 .
A bioinformatics search for a short sequence like the LIR motif will yield a high number of candidate sequences, and a previous proteomics study identified 67 proteins that interact with the human ATG8 orthologues . An LIR motif alone is not sufficient for recruitment of a protein to the inner surface of the phagophore. Analogous to the oligomerization and aggregation of pre-Ape1 in the Cvt pathway, PB1-domain-mediated polymerization of p62 is absolutely required for its degradation by autophagy. In higher eukaryotes, such as mammals and plants, the occurrence of multiple ATG8 homologues adds additional layers of complexity to the regulation of selective autophagy since different autophagy receptors may bind to different ATG8 proteins. Supporting this notion, an atypical LIR motif has been identified in NDP52, composed of three consecutive hydrophobic amino acids (LVV) that recognize a hydrophobic patch on LC3C (Figure 3D). Only LC3C provides the right structure to compensate for the lack of aromatic residues in NDP52–LIR . In future studies it will be important to determine the exact roles of different ATG8 homologues and their interaction with autophagy receptors.
Ubiquitin modifications are involved in mediating cargo recognition by autophagy receptors in both aggrephagy and xenophagy. Ubiquitin is a small 76-amino-acid protein used as a secondary modifier by covalent attachment to other cellular proteins. Post-translational modification by ubiquitin acts as a versatile cellular signal controlling a wide range of biological processes including cell signalling, trafficking and the DNA damage response. Analogous to the proteasome, where ubiquitinated proteins are delivered by ubiquitin receptors, ubiquitin modification is also involved in determining cargo specificity in selective autophagy. Ubiquitination is mediated by a series of enzymatic reactions involving activating (E1), conjugating (E2) and ligating (E3) enzymes generating a covalent bond between the exposed C-terminal glycine residue in ubiquitin and a lysine residue in the cargo protein. Ubiquitin has seven internal lysine residues that can be linked to form polyubiquitin chains (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63), and conjugation on the N-terminal methionine (Met1) leads to formation of linear chains (Figure 4). The two most prevalent linkages are Lys48- and Lys63- linked chains. The linkage determines the structure of the ubiquitin chain, and signal specificity is achieved by alternative interactions with ubiquitin-binding proteins . Traditionally implied in the context of UPS-mediated protein degradation, Lys48-linked chains adopt compact conformations with the interaction surface partially buried within the fold of the chain (Figure 4). In contrast, Lys63- and Met1-linked chains adopt open conformations and are associated with autophagy as well as non-proteolytic pathways. Both chain conformations have been implicated in xenophagy , whereas Lys63-linked chains have been suggested to be important in aggrephagy [30,31].
Selective autophagy of protein aggregates: aggrephagy
Mutations, incomplete translation, aberrant protein modifications or failing complex formations can give rise to misfolded proteins. Most cells have a constitutive need for selective autophagy as a protein quality control mechanism acting together with the UPS to degrade misfolded proteins. These have an inherent tendency to aggregate owing to exposed hydrophobic patches that are normally concealed in the native folded state. Aggregation may compromise the role of functional proteins that are sequestered to the aggregate, and lead to a cascading problem for the cell. Actually, large protein aggregates are probably inert to the UPS leaving selective autophagy as the only available degradation route. The main source of post-translational damage to proteins is caused by ROS (reactive oxygen species) formed as natural by-products of normal metabolism of oxygen. Persistent or extensive oxidative damage promotes protein aggregation. The activity of different intracellular proteolytic systems decreases with aging, and deficient removal of oxidized proteins causes accumulation of toxic protein aggregates. This is a hallmark of several common neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, and protein misfolding disorders or proteinopathies in general [7,30]. The aggregates associated with these types of diseases will often contain intrinsically disordered proteins that are prone to aggregation, such as the hyperphosphorylated tau-containing neurofibrillary tangle in Alzheimer's disease brains, aggregated α-synuclein in the Lewy body in Parkinson's disease brains and the intracellular inclusion of the N-terminal fragments of mutant huntingtin in Huntington's disease brains .
Mice deficient for autophagy show tissue-specific accumulation of p62-positive structures containing ubiquitinated protein aggregates [33,34]. However, when mice with deficient autophagy in the liver were depleted for p62 a dramatic protein aggregation phenotype was reversed . These apparently opposing results suggested that p62, in addition to mediating degradation, is also required for aggregate formation. The fact that polymerization of p62 via the PB1 domain is required for aggregate formation and efficient autophagic degradation, supports this hypothesis . Formation of large protein aggregates is regarded as a cellular defence mechanism, as the occurrence of many small sub-microscopic protein aggregates can be more damaging than fewer and larger aggregates . The aggresome, formed in response to proteasomal inhibition or overexpression of aggregation prone proteins, is currently the best-studied protein aggregate with respect to formation and degradation mechanisms [30,37]. However, in this case it is likely not to be the single large aggresome localized at the microtubule-organizing centre of the cell that is degraded by autophagy, but rather smaller aggregates within or disentangled from the aggresome.
The Hsp70 (heat-shock protein 70) complex mediates quality control of newly synthesized proteins in the cytosol. Functional proteins are released or delivered to the Hsp90 chaperone complex, which monitors the condition of mature proteins. Hsp90 protects proteins from unfolding and aggregation, whereas Hsp70 is responsible for their degradation in cases when unfolding or aggregation cannot be prevented. In order to be degraded by the UPS, a cargo must be polyubiquitinated with chains consisting of four or more preferably Lys48-linked ubiquitin moieties. If the capacity of the proteasome is overwhelmed, ubiquitin chains on misfolded proteins can undergo remodelling by the combined activity of DUBs (de-ubiquitinating enzymes) and E3 ligases to remove and add ubiquitin chains of different linkages (Figure 5). The newly formed ubiquitin chains, which contain Lys63-linked chains, are recognized by p62 and HDAC6 (histone deacetylase 6), which direct protein aggregates to the aggresome and autophagy. HDAC6 facilitates dynein-mediated transport of ubiquitinated cargoes to the aggresome, and it is also important for the clearance of aggresomes by autophagy . In aggrephagy involving p62 and NBR1, the large adaptor protein ALFY (autophagy-linked FYVE) is also involved in facilitating aggregate formation and subsequent autophagic degradation of these aggregates  (Figure 5).
Pathogenic bacteria enter the cell through an invagination of the outer membrane, creating a phagosome targeted for lysosomal degradation in a process called LAP (LC3-associated phagocytosis). Following internalization, some bacteria escape the phagosome to proliferate and spread to neighbouring cells. The association of ubiquitin with cytosolic bacteria led to the hypothesis that antimicrobial autophagy requires an ubiquitin-dependent mechanism for cargo recognition and degradation, analogous to aggrephagy. Three of the SLRs, p62, NDP52 and optineurin, have been found to recognize ubiquitinated bacteria and facilitate sequestering to autophagosomes [19,20,39]. Depletion of either protein causes increased replication of Salmonella typhimurium and all three proteins are independently recruited to the same bacterium. To escape the phagosome, bacteria excrete peptides that damage the vacuolar membrane. NDP52 is recruited to Salmonella in damaged vacuoles through its binding to cytosolic galectin-8. The latter binds host vacuolar glycans exposed to the cytosol upon vacuole damage by bacteria. Since galectin-8 also signals sterile damage to endosomes or lysosomes, it serves as a versatile reporter of vesicle damage recruiting NDP52. After initial recruitment of NDP52, ubiquitination occurs, recruiting more NDP52 and the other SLRs for selective autophagic removal of damaged vesicles and pathogens . Perhaps the most important role of autophagy receptors during xenophagy is to reduce inflammation by degrading membrane remnants and limit inflammatory cytokine signalling. The professional intracellular pathogens have evolved mechanisms to exploit and interfere with the host's xenophagy response. For example, some bacteria survive and replicate in the phagosomes by excreting DUBs to counter recognition by the SLRs, whereas, i.e. Listeria monocytogenes, manipulate the host actin-nucleation machinery to promote intracellular motility and intercellular spreading. In this process the bacteria surround themselves with cellular proteins to escape host cell recognition .
Regulation of selective autophagy
The autophagy pathway is directly regulated by several kinases, including ULK1/2 (uncoordinated 51-like kinase 1/2) and mTORC1 [mammalian (also known as mechanistic) target of rapamycin complex 1]. The autophagy receptors p62 and optineurin, as well as LC3B, have been shown recently to be regulated by phosphorylation. TBK1 [TANK (tumour-necrosis-factor-receptor-associated factor-associated nuclear factor κB activator)-binding kinase 1] phosphorylates the UBA domain of p62 to increase its binding to ubiquitinated cargoes and boost their autophagic degradation [42,43]. TBK1 is also a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles . Optineurin is phosphorylated in its LIR motif by TBK1 to dramatically increase the affinity for binding to ATG8 family proteins, again to increase selective autophagy . LC3B has also been shown to be negatively regulated by phosphorylation in the N-terminal arm by protein kinase A .
Little is known about differential gene regulation occurring as a result of aggregate formation, but there is clearly a link between aggregate formation and oxidative stress responses. KEAP1 [Kelch-like ECH (erythroid cell-derived protein with cap ‘n’ collar homology)-associated protein 1] is an E3 ligase that mediates continuous degradation of NFR2 (NF-E2-related factor 2), a transcription factor responsible for activation of genes involved in the oxidative stress response. In a positive-feedback loop, p62 interacts with KEAP1 to disrupt the degradation of NRF2, thereby activating the oxidative stress response. Expression of p62 is itself under the control of NRF2, resulting in an amplification of the oxidative stress response as long as p62 levels rise .
In addition to regulation by phosphorylation, it is anticipated that other post-translational modifications including acetylation and ubiquitination will be implicated in the regulation of selective autophagy.
A distinction is usually made between basal housekeeping autophagy, important in quality control of proteins and organelles, and starvation- or stress-induced autophagy. In higher eukaryotes it seems likely that all basal autophagy is contributed by selective autophagy, whereas the starvation-induced and part of the stress-induced autophagy is more regularly associated with bulk degradation. However, our increased understanding of this process undoubtedly raises the question of the degree of selectivity involved. One could imagine that as a consequence of multi-cellularity autophagy has evolved to become increasingly more selective. A significant future challenge will be to define the spatiotemporal mechanisms by which ubiquitin and other signals control selective autophagy and the importance of selective autophagy in physiological and pathophysiological processes. Assessing the role of selective autophagy in cellular well-being will be vitally important to meet the acute challenges of age-related diseases that become more frequent as populations grow increasingly older.
• Selective autophagy plays a significant role in macroautophagy.
• In selective autophagy the forming autophagosome is enriched for specific cargoes, including organelles, protein aggregates, midbody rings and invading pathogens.
• Selective autophagy is mediated by autophagy receptor proteins that interact with cargo and the ATG8/LC3 family proteins.
• Autophagy receptors interact with ATG8 family proteins via LIR motifs, and ubiquitin is often involved in the labelling and recognition of cargo.
• Selective autophagy is regulated by signalling pathways mediating phosphorylation of several of the autophagy receptors.
We apologize to those whose work we were unable to include due to space constraints. This work was supported by grants from the Biology and Biomedicine (FRIBIO) program of the Norwegian Research Council and the Norwegian Cancer Society (to T.J.).
- © The Authors Journal compilation © 2013 Biochemical Society