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The LIR motif – crucial for selective autophagy

Asa Birna Birgisdottir, Trond Lamark and Terje Johansen*Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway

*Author for correspondence (terje.johansen@uit.no)

Journal of Cell Science 126, 3237–3247� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126128

Summary(Macro)autophagy is a fundamental degradation process for macromolecules and organelles of vital importance for cell and tissuehomeostasis. Autophagy research has gained a strong momentum in recent years because of its relevance to cancer, neurodegenerativediseases, 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 keepup with the energy demand. This view has recently changed dramatically following an array of papers describing various forms ofselective autophagy. A main driving force has been the discovery of specific autophagy receptors that sequester cargo into formingautophagosomes (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 includecargo receptors, members of the basal autophagy apparatus, proteins associated with vesicles and of their transport, Rab GTPase-activating 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

IntroductionMacroautophagy (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, lipid-

storage 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 microtubule-

associated proteins 1A/1B light chain 3 (MAP1LC3 or LC3)

proteins (Fig. 1).

Members of the ATG8 family are the only known ubiquitin-

like (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

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(mitophagy and pexophagy), bacteria (xenophagy), ribosomes,

macromolecular structures, specific proteins and protein

aggregates (aggrephagy) by autophagy. The cytoplasm-to-

vacuole 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 selectiveautophagy receptors and the LIR motifA 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 protein-

aggregation 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).

Autophagosome

ULK1/2PI 3-kinase

complex

Phagophore Autolysosome

LysosomeEndosome

ATG9vesicle

?

Induction Nucleation ExpansionCargo recruitment

Closure MaturationCargo degradation

PtdIns(3)PDFCP1WIPI proteins

ATG12–ATG5–ATG16L1LC3-PE

Autophagy receptor (SLRs)

ATG9

ATG8-LIR interaction

Bacteria

Ubiquitin

Mitochondria

Peroxisome

Mitophagy receptor

Protein misfolded/aggregate

ATG12ATG5

ATG16L1

ATG5 ATG12

ATG7ATG10 ATG4B

ATG7ATG3

PEATG12ATG5

proLC3z

LC3-I

LC3-II

Key

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.

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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

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

LIR

4H EF SH2 Ring UBA 906Cbl

LIR

TM 358CBM20Stbd1

Specialized receptors

Coiled-CoilSKICHLIR

CC 789CC CC ZF ZF

LIR

CC CC CC UBAN ZF 577

Ub

OPTN

446SKICH CC ZF

LIR

NDP52

440PB1 ZZ

NESNLS1NLS2

UbLIR KIR

UBAp62

PB1 ZZ

LIR2

UBA

LIR1

966ZZ

Ub

CC1 FW UBACC2NBR1

TAX1BP1

LIR

ABD

CC ABD

LIR

Atg19

Atg34

415

412

Cvt cargo receptors

TM

LIR

529

219

LIR

TMBH3

LIR

TMTM TM 155FUNDC1

194BH3 TM

LIR

BNIP3

NIX

Atg32

Mitophagy receptors

Sequestosome-1-like receptors (SLRs)

Ub

Ub

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, LC3-

interacting 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.

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NDP52

L134

V135

V136

NBR1

K16

K20K24

K48

E730

D731

Y732

I735

Atg19 R28

R67

W412

E414

E413

L415

L343W340

R10

R11

p62

2

4

0

Bits

6

X–3 X–2X–1W0 X1 X2 L3 X4 X50

0

4

2

2

4

Bits

Bits

A

B C

HP1

HP2

LC3B Atg8

GABARAPL1 LC3C

N-terminal arm

Ub-like domain

W340

L343

R10

R11

D337

D338

D338

D337

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 N-

terminal 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).

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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-I44-

V70) 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).

Different strategies have been used to identify proteins thatinteract 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 (Mohrluder 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 usingdeletion mapping and protein–protein interaction assays, and by

testing deletion and point-mutated constructs. We have found thatpeptide array analysis is a specific and efficient method foridentification of LIR motifs (Alemu et al., 2012).

Cargo receptors in selective autophagyFollowing discovery of p62 as a selective autophagy receptor, therelated neighbor of BRCA1 gene 1 (NBR1) was found to act asan aggrephagy receptor (Kirkin et al., 2009b). Subsequently,

nuclear dot protein 52 kDa (NDP52) was found to be animportant xenophagy receptor (Thurston et al., 2009) togetherwith optineurin (Wild et al., 2011). These and the otherautophagy 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 selectiveautophagy, these proteins have also recently been shown to

regulate innate immunity signaling pathways and, thus, weresuggested to represent a new class of pattern recognitionreceptors, 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 LIRdomain (an atypical LIR motif in the case of NDP52 and

TAX1BP1) and an ubiquitin-binding domain. These threefeatures of SLRs are required for the efficient execution oftheir role as autophagic cargo receptors (Pankiv et al., 2007;

Ichimura et al., 2008b; Itakura and Mizushima, 2011; Deosaranet al., 2013). Studies of selective autophagy in mammalian cellsand of the Cvt pathway in yeast revealed that the cargo must

either be aggregated or represent a reasonably large structure thatenables the binding of many receptor molecules; alternatively,the autophagy receptors themselves need to be able tomultimerize 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 ofautophagy obviously impacts on signaling that involves SLRs. Todiscuss these signaling pathways is beyond the scope of this

Commentary, but it is worth noting that accumulation of SLRsoccurs during cellular stresses, including infection andinflammation, oxidative stress, ER-stress and metabolic stress

(Johansen and Lamark, 2011; Deretic, 2012). This accumulationmay 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 poorlyunderstood. One exception is the regulation of the KEAP1–NRF2oxidative-stress-response pathway, in which p62 binds to and

sequesters KEAP1, leading to its autophagic degradation and theconcomitant induction of NRF2 (Komatsu et al., 2010; Jain et al.,2010; Taguchi et al., 2012). A positive feedback loop is

Box 1. Specificities of the LIR-ATG8 proteininteraction

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).

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established in that increased p62 levels activate NRF2, which, inturn, further increases p62 levels (Jain et al., 2010). Recently, it

has been found that the liver toxicity of accumulated p62 is dueto constitutive upregulation of the NRF2 oxidative-stress-response 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 nostructural similarities. Substrates targeted by p62 includeubiquitylated protein aggregates and membrane-embedded

structures, such as intracellular bacteria and peroxisomes. Thesingle feature that unites the various structures appears to be thatthey become ubiquitylated before they are degraded. The LIR

motif is absolutely required for targeting of SLRs and boundcargoes into the lumen of autophagosomes (Johansen and Lamark,2011). Selective autophagy depends on a direct interactionbetween the LIR motif and ATG8 homologs that are conjugated

to the inner, concave membrane of the phagophore. However, theLIR motif by itself does not bring a protein to the inner surface of aphagophore, and the majority of LIR-motif-containing proteins are

not substrates for selective autophagy. Selective autophagy of p62depends on its PB1-domain-driven polymerization, but for thedelivery of p62-associated cargos, ubiquitin binding and

interactions with other proteins are also important. For theselective autophagy of protein aggregates, p62 collaborates withautophagy-linked FYVE protein (ALFY), a nuclear scaffoldingprotein that is recruited to cytosolic protein aggregates in a p62-

dependent manner. ALFY interacts directly with ATG5 andphosphatidylinositol (3)-phosphate [PtdIns(3)P], and may act as ascaffold protein that induces the assembly of an autophagy-

compatible 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 binddirectly to each other through their PB1 domains, and collaboratein selective autophagy of misfolded proteins and probably alsomidbody 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 NBR1in a process that depends on the coincident membrane binding of

its amphipathic J domain and the adjacent UBA domain (Deosaranet al., 2013). Specialized intracellular pathogens have oftendeveloped strategies to avoid or use autophagy for their own

purposes, but other pathogens are efficiently degraded by selectiveautophagy if they are released into the cytoplasm or uponmembrane rupture (Mostowy and Cossart, 2012). Microbes thatare 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 canalso be polyubiquitylated and targeted for autophagic degradation

by p62 (Dupont et al., 2009). Furthermore, p62 promotesautophagic killing of intracellular microbes. Cytoplasmicprecursors of antimicrobial peptides (ubiquitin or ribosomal

precursor proteins) are transported by p62 into autolysosomes ormicrobe-containing autolysosomes. Here, the precursors areconverted into peptides that have been shown to kill

Mycobacterium tuberculosis (Ponpuak et al., 2010); and thesepeptides might also be potent against other microbes. Viruses canalso act as substrates and p62 has been implicated in xenophagic

elimination of Sindbis virus (Orvedahl et al., 2010). Efficientxenophagy of Salmonella enterica serotype Typhimurium (S.

typhimurium) is mediated by p62, NDP52 and optineurin, with thep62-containing microdomains on ubiquitin-coated bacteriaappearing to be physically separated from areas that areoccupied by NDP52 and optineurin (Cemma et al., 2011;

Mostowy et al., 2011; Wild et al., 2011). However, althoughNDP52 is recruited to ubiquitin-coated bacteria through its C-terminal Zinc-finger (ZF) domain (Fig. 2), it is initially targeted to

damaged Salmonella-containing vacuoles that are marked bygalectin-8, which binds exposed b-galactoside-containing glycans.In this way, cytosolic galectin-8 functions as an ubiquitin-

independent ‘danger’ receptor and ‘eat-me’ signal (Thurstonet al., 2012). Galectin 8 also detects non-bacteria induced damageto endosomes or lysosomes, suggesting that membrane rupture isthe initial common event detected during invasion by microbes

(Thurston et al., 2012). The atypical LIR motif in NDP52, termedCLIR, comprises the tripeptide Leu-Val-Val and binds specificallyto LC3C (Fig. 3A). Efficient recruitment of the other ATG8 family

members to bacteria-degrading autophagosomes depends on bothNDP52 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 miRNAeffector AGO2 by selective autophagy (Gibbings et al., 2012).An ubiquitin-independent role of optineurin in aggrephagy hasrecently 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 specificmitochondrial membrane receptors, as discussed below.

Mitophagy receptors

Both yeast and mammalian cells can selectively eliminate

damaged or superfluous mitochondria by mitophagy (reviewedby Ashrafi and Schwarz, 2013). In yeast, mitophagy isorchestrated by Atg32, an integral protein of the outermitochondrial 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 interactwith Atg8 indirectly through Atg11 and directly through its LIR

motif in the N-terminal cytosolic domain. Atg32 recruits Atg8and Atg11 to the mitochondria surface to form an initiatorcomplex essential for mitophagy (Kondo-Okamoto et al., 2012).

In mammalian cells, three integral OMM proteins that all have aLIR motif in their cytosolic N-terminal domain are implicated inmitophagy (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 byZhang and Ney, 2009). Bnip3 induces the removal of bothmitochondria and endoplasmic reticulum (Hanna et al., 2012).

Homodimerization of Bnip3 through the transmembrane domainfacilitates the interaction between the LIR motif of Bnip3 andLC3B. Nix also facilitates LIR-dependent mitophagy (Novak

et al., 2010) (supplementary material Table S1). During erythroidcell maturation, Nix mediates the complete removal ofmitochondria (Schweers et al., 2007; Sandoval et al., 2008).

Additionally, Nix is involved in depolarization-inducedmitophagy. Nix has a core LIR motif identical to Bnip3.However, in contrast to Bnip3, Nix does not interact with

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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 hypoxia-

induced 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).

Specialized autophagy receptors

So far few autophagy receptors are known to only interact with

one substrate under certain circumstances. Starch-binding-

domain-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, non-

receptor, 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 ascargo receptorsThe presence of functional LIR motifs in components of the core

autophagy machinery demonstrates that LIR-motif-mediated

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

Cargo recruitment Maturation

Phagophore Autophagosome Autolysosome+

–

Transport

Microtubule

ULK1–ULK2 complex

LC3-PE (LC3-II)

SLRs (p62, NBR1, NDP52, OPTN)

Mitochondria

Mitophagy receptor

Ubiquitylated misfolded protein

ATG4

Kinesin

FYCO1

TBC1D5, TBC1D25

ATG8–LIR-motif interaction

DOR and TP53INP1

Key

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.

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et al., 2012). It is possible that LIR-ATG8 interactions of ULKcomplex proteins facilitate and/or stabilize tethering of the ULK

complex to the phagophore.

Other members of the core autophagy apparatus, Atg3 in yeastand ATG4B in mammals, undergo LIR-motif-dependentinteractions with ATG8 that potentially serve a regulatory role

(Satoo et al., 2009; Yamaguchi et al., 2010). Among the fourATG4 homologs (ATG4A, ATG4B, ATG4C, ATG4D), ATG4Bis the main human ATG4 homolog that efficiently processes

ATG8 precursors and ATG8-PE (Li et al., 2011). The crystalstructure of the human ATG4B–LC3B complex indicatesconformational changes in ATG4B upon binding of the LC3

substrate that facilitate access of LC3 to the catalytic site ofATG4B (Satoo et al., 2009). Interestingly, in the crystal structure,the N-terminal LIR motif of ATG4B interacts with a LIR-bindingsite on an adjacent (non-substrate) LC3. This interaction with the

LIR motif stabilizes an open conformation of the N-terminal tailof ATG4B, which presumably favors membrane targeting. SinceATG4 also mediates deconjugation of ATG8 proteins, a process

that requires membrane targeting, the conformation of the N-terminal tail might, therefore, regulate the deconjugation activityof ATG4 (Satoo et al., 2009). The yeast E2-like enzyme Atg3

contains a canonical LIR motif (WEDL) that is essential for theefficient transfer of Atg8 from Atg3 to PE. The Atg3 LIR motif isrequired for the Cvt pathway but not for starvation-induced

autophagy. The interaction between the LIR motif of Atg3 andAtg8 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 autophagosomesand other vesicles

Although some steps of autophagosome formation are well

understood, the membrane origins of autophagosomes are stilldebated. Multiple membrane sources were found to be involved,such as endoplasmic reticulum (ER), mitochondria, ER-

mitochondria contact sites and plasma membrane (Hamasakiet al., 2013; Weidberg et al., 2011). The plasma membrane cancontribute directly to the formation of ATG16L1-positiveautophagosome precursors that depend on interactions between

ATG16L1 and the clathrin heavy chain (Ravikumar et al., 2010).Hence, clathrin-mediated endocytosis might be involved inregulating the initial stages of autophagosome formation

(Ravikumar et al., 2010). Interestingly, the clathrin heavy chainalso interacts with GABARAP through a LIR motif on a surface-exposed a-helix in the flexible linker region (Mohrluder et al.,

2007a). Structural studies show that LIR motifs adopt a b-conformation when bound to ATG8-proteins and form anintermolecular parallel b-sheet (Fig. 3B) (Noda et al., 2010). Itwill, therefore, be interesting to learn which conformation the

clathrin LIR has upon binding to GABARAP. Clathrin andGABARAP 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 whetherthis interaction impacts on autophagosome formation. Calreticulin,which competes with clathrin for binding to GABARAP

(Mohrluder et al., 2007a), also has a LIR motif very similar tothat of clathrin (supplementary material Table S1) (Mohrluderet al., 2007b). Calreticulin is a luminal Ca2+-dependent chaperone

of the ER, but is also involved in variety of cytosolic functions as aregulator of intracellular Ca2+ homeostasis (Wang et al., 2012). Itis 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 clathrin-

dependent endocytosis might represent phagophore precursors.During and after phagophore formation, proteins are recruited tothe forming autophagosome in a ‘retrieve–recycle’ manner. The

tumor protein 53-induced nuclear protein 2 (TP53INP2; alsoknown as and, hereafter, referred to as DOR) exits the nucleus inresponse to cellular stress or the activation of autophagy (Nowak

et al., 2009; Mauvezin et al., 2010). Cytoplasmic DOR thenlocalizes to autophagosomes where it interacts with thetransmembrane protein VMP1 (Nowak et al., 2009). On

autophagosomes, DOR interacts with LC3B through its LIRmotif (Sancho et al., 2012), but it does not colocalize with theautolysosome-associated protein LAMP1, indicating that DORlocalizes only to early autophagosomes (Mauvezin et al., 2010).

Through its interaction with VMP1, DOR presumably acts as ascaffold protein that recruits ATG8 proteins to theautophagosome (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 itsnuclear exit in response to autophagy activation. Interestingly,

DOR and its homolog TP53INP1 share two highly conservedregions, including the LIR motif (Sancho et al., 2012). LIR-mediated localization of TP53INP1 to autophagosomes induces

autophagy- and caspase-dependent cell death, and it has beensuggested that TP53INP1 displaces p62 from LC3B, which thenpromotes cell death (Seillier et al., 2012).

The mechanisms that regulate membrane trafficking in

autophagy are poorly understood. The Rab GTPases (a largefamily of monomeric, small GTPases) in their active form arespatially 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 autophagosomesthrough direct interactions between its LIR motif and ATG8homologs, and its GAP activity regulates the fusion betweenautophagosomes and lysosomes (Itoh et al., 2011). TBC1D25

inhibits Rab33B, a Golgi-resident Rab (Itoh et al., 2011). ActiveRab33B binds to ATG16L1 and is involved in recruitment of theATG12-ATG5-ATG16L complex to preautophagosomal structures

(Itoh et al., 2008). The authors suggest a model whereby TBC1D25uses ATG8 proteins as scaffolds to regulate autophagosomalmaturation (Itoh et al., 2011).

Recently, 14 of 36 human TBC (Tre2, Bub2, Cdc16)-domain-containing Rab GAPs were shown to interact with ATG8 proteinsin 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 toautophagosomes upon starvation-induced autophagy (Popovicet al., 2012). TBC1D5 is involved in retrograde traffic from

endosomes to the Golgi. Interestingly, the N-terminal LIR ofTBC1D5 interacts with the Vps29 subunit of the retromercomplex, a recycling endosome sorting complex responsible for

vesicle delivery from early endosomes to the Golgi. The binding ofTBC1D5 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 ofTBC1D5 can tether the endosome and autophagosome, therebymediating autophagosome maturation (Popovic et al., 2012).

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The examples above demonstrate crosstalk betweenendocytosis and autophagy, which then converge in lysosomal

degradation. Rab7 is involved in maturation of bothautophagosomes and endosomes, as well as in the transport ofautophagosomes and endosomes towards lysosomes for

degradation. The Rab7 effector FYVE and coiled-coil-domain-containing 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 connectingloop between its FYVE and GOLD domains. This flexible loop ispredicted to be folded in a way that blocks the interaction

between the FYVE domain and PtdIns(3)P. Binding to LC3B onautophagic structures releases this inhibition and targets FYCO1exclusively to PtdIns(3)P-containing membranes that contain

LC3B (Pankiv et al., 2010). FYCO1 couples autophagosomes andother Rab7-positive vesicles to molecular motors. Depending onthe direction of vesicle movement, FYCO1 is coupled to kinesinmolecular 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 anotherprotein that localizes to autophagosomes through an interactionbetween its LIR motif and ATG8 proteins (supplementary materialTable S1) (Colecchia et al., 2012). The kinase activity of MAPK15

is known to affect the rate of both basal and starvation-inducedautophagy (Colecchia et al., 2012) but the substrates of MAPK15that are involved in autophagy are unknown.

LIR-containing signaling proteins that act as substrates forselective 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 Wntsignaling (Gao et al., 2010). The C-terminal DEP domain of Dvl2contains a LIR motif that binds to ATG8 proteins (Gao et al., 2010;Zhang et al., 2011). The N-terminal DIX domain mediates the self-

oligomerization of Dvl2 that is necessary for its ubiquitylation, andfacilitates 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 toautophagosomes. Thus, p62 mediates the indirect association ofDvl2 with LC3B and GABARAP (Gao et al., 2010). This is also

likely to be the case for the interaction of Dvl2 with GABARAP-L1 (Zhang et al., 2011). Consequently, Dvl2 is degraded byautophagy through its LIR-dependent interaction with ATG8

proteins and by means of the autophagy receptor p62. Veryrecently, it was shown that b-catenin is selectively degraded byautophagy during nutrient deprivation via the formation of a b-catenin-LC3 complex depending on a LIR motif in b-catenin. A

regulatory feedback mechanism is at work, in which active Wnt/b-catenin signalling represses autophagy and p62 expression, whileb-catenin is itself targeted for autophagic clearance in

autolysosomes upon autophagy induction (Petherick et al., 2013).

Regulation of the interaction between LIR andATG8 through phosphorylationSince 25% of the known LIR motifs harbour an S or T residue as the

‘any amino acid residue’ at position –1 immediately N-terminal tothe aromatic residue of the LIR motif (supplementary materialTable S1), it is conceivable that binding affinity of LIR motifs is

regulated through phosphorylation. Indeed, NDP52 recruits TANK-

binding 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-motif-

containing proteins. For instance, phosphorylation of S12 in the N-

terminal 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 N-

terminal 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.

AcknowledgementsWe thank members of our group for critical reading of themanuscript, and Steingrim Svenning for help with Fig. 3.

FundingThis work was funded in part by grants from the FUGE and FRIBIOprograms of the Norwegian Research Council, the NorwegianCancer Society and the Blix foundation to T.J.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126128/-/DC1

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