**5. Recognition of ubiquitinated cargoes by p62 and NBR1**

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival. Defects in autophagy cause the accumulation of ubiquitin-positive protein inclusions, leading to severe liver injury (Komatsu et al., 2005) and neurodegeneration (Hara et al., 2006; Komatsu et al., 2006). The polyubiquitin-binding protein p62, also called sequestosome 1 (SQSTM1), is a common component of protein aggregates found in both the brain and the liver of patients suffering from protein aggregation diseases. These include Lewy bodies in Parkinson's disease, neurofibrillary tangles in Alzheimer's disease, and huntingtin aggregates (Kuusisto et al., 2001, 2002; Nagaoka et al., 2004; Zatloukal et al., 2002). In the liver, Mallory bodies, hyaline bodies in hepatocellular carcinoma, and α1 antitrypsin aggregate contain p62 (Zatloukal et al., 2002); all of these aggregates contain polyubiquitinated proteins. p62 interacts with ubiquitin via its C-terminal UBA domain (Vadlamudi et al., 1996) and self-assembles via its N-terminal PB1 domain (Ponting et al., 2002), thereby forming large aggregates containing ubiquitinated proteins. p62 further interacts with LC3, a mammalian homolog of Atg8, via the LC3 interacting region (LIR; residues 321-342), so that ubiquitinated protein aggregates containing p62 are selectively degraded by autophagy (Bjorkoy et al., 2005; Komatsu et al., 2007; Pankiv et al., 2007). Therefore, it is implied that p62 functions as a receptor protein for ubiquitinated proteins to be degraded in lysosomes. It is also hypothesized that p62 functions as a receptor for organelles such as peroxisomes and mitochondria (Kim et al., 2008; Kirkin et al., 2009), and for intracellular bacteria (Dupont et al., 2009; Yoshikawa et al., 2009; Zheng et al., 2009). Recently, neighbor of BRCA1 gene 1 (NBR1) has been identified as another autophagy receptor (Kirkin et al., 2009). The structure of NBR1 is similar to that of p62, and NBR1 can bind both LC3 via the LIR and ubiquitinated proteins via the UBA domain. Like p62, NBR1 is sequestered into the autophagosome via LC3-interaction and/or p62-interaction and markedly accumulates in autophagy-deficient tissues.

To clarify the molecular mechanism of ubiquitinated cargo recognition by p62 and NBR1, it is necessary to elucidate which proteins conjugated with either K48-linked or K63 linked polyubiquitin chains are targeted to the autophagy/lysosomal degradation pathway. Classically, proteins conjugated with K48-linked polyubiquitin chains are recognized as the proteolytic substrate by the UBD-containing proteasomal receptors. Recently, K63-linked chains have been implicated in proteolytic degradation of misfolded and aggregated proteins (Olzmann et al., 2007; Tan et al., 2008; Wooten et al., 2008). Given the reported preference of the known ubiquitin-binding autophagy receptors for K63 linked ubiquitin chains, cargoes conjugated with K63-linked ubiquitin chains may be preferentially targeted to the autophagy/lysosomal degradation pathway. However, p62 has been shown to compete for ubiquitinated cargo with the classical proteasomal receptors. Accumulation of p62 resulting from inhibition of autophagy compromised degradation of proteasomal substrates, most likely due to the excessive interaction between p62 and substrates conjugated with K48-linked polyubiquitin chains (Korolchuk et al., 2009). It remains to be clarified how p62 distinguishes between K48-linked and K63 linked polyubiquitin chains.

To date, several structural studies have been performed on p62. Isogai *et al.* determined the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form (Isogai et al., 2011). In crystals, the p62 UBA domain adopts a dimeric structure, which is distinct from that of other UBA domains. In solution, the domain exists in equilibrium between the dimer and monomer forms, and ubiquitin-binding shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The extreme C-terminal end of the p62 UBA domain is responsible for dimerization of the domain. Mutations that inhibit dimerization of the p62 UBA domain increase the affinity of p62 for ubiquitin. These results suggest an autoinhibitory mechanism in the p62 UBA domain to avoid self-degradation by the ubiquitin-proteasomal system. The interaction between the p62 LIR and LC3 is structurally and functionally well characterized (Ichimura et al., 2008; Noda et al., 2008). In the structure of the p62 LIR in complex with LC3, the tryptophan and leucine residues in the p62 LIR, DDDWTHL, interact with two hydrophobic pockets, the W-site and the L-site, where tryptophan and leucine residues respectively interact (Noda et al., 2010) on the surface of LC3. In agreement with the structure of the p62 LIR in complex with LC3, the tryptophan and leucine residues are involved in the turnover of p62 via autophagy. Recently, the structure of the NBR1 LIR in complex with GABARAP, a LC3 paralog, has also been determined (Rozenknop et al., 2011). Similar to the interaction between p62 LIR and LC3, the tyrosine and the third isoleucine

residues 321-342), so that ubiquitinated protein aggregates containing p62 are selectively degraded by autophagy (Bjorkoy et al., 2005; Komatsu et al., 2007; Pankiv et al., 2007). Therefore, it is implied that p62 functions as a receptor protein for ubiquitinated proteins to be degraded in lysosomes. It is also hypothesized that p62 functions as a receptor for organelles such as peroxisomes and mitochondria (Kim et al., 2008; Kirkin et al., 2009), and for intracellular bacteria (Dupont et al., 2009; Yoshikawa et al., 2009; Zheng et al., 2009). Recently, neighbor of BRCA1 gene 1 (NBR1) has been identified as another autophagy receptor (Kirkin et al., 2009). The structure of NBR1 is similar to that of p62, and NBR1 can bind both LC3 via the LIR and ubiquitinated proteins via the UBA domain. Like p62, NBR1 is sequestered into the autophagosome via LC3-interaction and/or p62-interaction and

To clarify the molecular mechanism of ubiquitinated cargo recognition by p62 and NBR1, it is necessary to elucidate which proteins conjugated with either K48-linked or K63 linked polyubiquitin chains are targeted to the autophagy/lysosomal degradation pathway. Classically, proteins conjugated with K48-linked polyubiquitin chains are recognized as the proteolytic substrate by the UBD-containing proteasomal receptors. Recently, K63-linked chains have been implicated in proteolytic degradation of misfolded and aggregated proteins (Olzmann et al., 2007; Tan et al., 2008; Wooten et al., 2008). Given the reported preference of the known ubiquitin-binding autophagy receptors for K63 linked ubiquitin chains, cargoes conjugated with K63-linked ubiquitin chains may be preferentially targeted to the autophagy/lysosomal degradation pathway. However, p62 has been shown to compete for ubiquitinated cargo with the classical proteasomal receptors. Accumulation of p62 resulting from inhibition of autophagy compromised degradation of proteasomal substrates, most likely due to the excessive interaction between p62 and substrates conjugated with K48-linked polyubiquitin chains (Korolchuk et al., 2009). It remains to be clarified how p62 distinguishes between K48-linked and K63-

To date, several structural studies have been performed on p62. Isogai *et al.* determined the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form (Isogai et al., 2011). In crystals, the p62 UBA domain adopts a dimeric structure, which is distinct from that of other UBA domains. In solution, the domain exists in equilibrium between the dimer and monomer forms, and ubiquitin-binding shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The extreme C-terminal end of the p62 UBA domain is responsible for dimerization of the domain. Mutations that inhibit dimerization of the p62 UBA domain increase the affinity of p62 for ubiquitin. These results suggest an autoinhibitory mechanism in the p62 UBA domain to avoid self-degradation by the ubiquitin-proteasomal system. The interaction between the p62 LIR and LC3 is structurally and functionally well characterized (Ichimura et al., 2008; Noda et al., 2008). In the structure of the p62 LIR in complex with LC3, the tryptophan and leucine residues in the p62 LIR, DDDWTHL, interact with two hydrophobic pockets, the W-site and the L-site, where tryptophan and leucine residues respectively interact (Noda et al., 2010) on the surface of LC3. In agreement with the structure of the p62 LIR in complex with LC3, the tryptophan and leucine residues are involved in the turnover of p62 via autophagy. Recently, the structure of the NBR1 LIR in complex with GABARAP, a LC3 paralog, has also been determined (Rozenknop et al., 2011). Similar to the interaction between p62 LIR and LC3, the tyrosine and the third isoleucine

markedly accumulates in autophagy-deficient tissues.

linked polyubiquitin chains.

residues in the NBR1 LIR, EDYIII, interact with GABARAP in a manner typical of the interaction of AIM with Atg8 homologs.
