Research Article 2685 The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes

Elena Shvets*, Ephraim Fass*,‡, Ruthie Scherz-Shouval and Zvulun Elazar§ Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel *These authors contributed equally to this work. ‡Present address: Department of Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, Campus Box 8230, St Louis, MO 63110, USA. §Author for correspondence (e-mail: [email protected])

Accepted 26 May 2008 Journal of Cell Science 121, 2685-2695 Published by The Company of Biologists 2008 doi:10.1242/jcs.026005

Summary LC3 belongs to a novel ubiquitin-like protein family that is within the ubiquitin core. Knockdown of LC3 isoforms and involved in different intracellular trafficking processes, overexpression of LC3 mutants that fail to interact with p62 including autophagy. All members of this family share a unique blocked the incorporation of p62 into autophagosomes. The three-dimensional structure composed of a C-terminal ubiquitin accumulation of p62 was accompanied by elevated levels of core and two N-terminal α-helices. Here, we focus on the specific polyubiquitylated detergent-insoluble structures. p62, however, contribution of these regions to autophagy induced by amino is not required for LC3 lipidation, autophagosome formation acid deprivation. We show that the ubiquitin core by itself is and targeting to lysosomes. Our results support the proposal sufficient for LC3 processing through the conjugation that LC3 is responsible for recruiting p62 into autophagosomes, machinery and for its consequent targeting to the a process mediated by phenylalanine 52, located within the autophagosomal membrane. The N-terminal region was found ubiquitin core, and the N-terminal region of the protein. to be important for interaction between LC3 and p62/SQSTM1 (hereafter termed p62). This interaction is dependent on the first 10 amino acids of LC3 and on specific residues located Key words: Autophagy, LC3, p62

Introduction 1994; Sagiv et al., 2000; Wang et al., 1999). The modification of Autophagy is an intracellular trafficking pathway that mediates LC3 at its C-terminus, similarly to that of Atg8, is an essential step delivery of cytoplasmic constituents (proteins and organelles) for in the formation of autophagosomes in mammals (Tanida et al.,

Journal of Cell Science bulk degradation within the lysosome/vacuole compartments. This 2004). All members of the Atg8 family share a similar structure pathway, initiated predominantly in response to nutrient starvation, made of a ubiquitin-like core decorated with two unique N-terminal is involved in diverse physiological functions, such as cell α-helices. It has been recently found (Nakatogawa et al., 2007) that development, programmed cell death, cancer, pathogen infection Atg8 serves as a membrane-tethering and a hemifusion factor during and degradation of ubiquitylated protein aggregates formed in many the expansion of autophagosomal membranes and that truncation pathological conditions (Boland and Nixon, 2006; Huang and of its N-terminus impairs this activity. However, the exact role of Klionsky, 2007). During autophagy, cytoplasm is first enwrapped mammalian Atg8, especially that of its different regions, has not by a double-membrane vesicle, called an autophagosome. Following yet been fully determined. Recent reports showed that LC3 directly movement and targeting to lysosomes, the autophagosome outer interacts with sequestosome-1 (SQSTM; also known as p62) membrane fuses with the lysosome/vacuole, releasing a single- (Bjorkoy et al., 2005; Pankiv et al., 2007), a protein involved in membrane vesicle (autophagic body) into this compartment to cell signaling, receptor internalization and protein turnover (Moscat generate an autolysosome. Finally, lysosomal hydrolases degrade et al., 2007; Seibenhener et al., 2007), via a newly identified LC3- the autophagosome inner membrane and its sequestered contents interacting region (LIR), located between its ZZ and ubiquitin- (autophagic body). associated (UBA) domains. However, the region of LC3 involved Two conserved ubiquitin-like (UBL) conjugation systems, Atg12 in this interaction is not known. p62 binds polyubiquitin via the and Atg8, are essential for early stages of autophagosome biogenesis UBA domain and is commonly present in protein inclusions in yeast and mammals (Ohsumi, 2001). Atg8 is first cleaved by associated with neurodegenerative diseases (Babu et al., 2005; Atg4, a specific cysteine protease, thereby exposing a C-terminal Seibenhener et al., 2004; Zatloukal et al., 2002). Thus, p62 glycine residue. This allows Atg8 to become conjugated to interaction with LC3 is required for the degradation of phosphatidylethanolamine (PE), a unique modification not found polyubiquitylated protein aggregates by autophagy (Bjorkoy et al., in other UBL proteins. The Atg8-PE conjugate plays a crucial role 2005). The question remains, however, whether this interaction is in subsequent dynamic rearrangement of membranes, leading to essential for autophagy or merely required for delivery of the formation of an autophagosome (Abeliovich et al., 2000; polyubiquitylated protein aggregates for lysosomal degradation. Kirisako et al., 2000; Lang et al., 1998; Mizushima et al., 2001). In the present study, we determined the minimal structure Atg8 shows a high level of sequence identity with a family of UBL required for conjugation and the consequent targeting of LC3 to mammalian proteins, including MAP1-LC3 (LC3, official symbol the autophagosomal membrane. We found that LC3 N-terminal α- MLP3B), GATE-16 and GABARAP (Mann and Hammarback, helix together with a specific site located within the ubiquitin core 2686 Journal of Cell Science 121 (16)

Fig. 1. Effect of N-terminal truncation in LC3 on its processing and autophagosomal localization. (A) 3D structure of LC3 and its different N-terminal truncations (illustrated in red). PyMol molecular visualization system was used for presentation of 1UGM:A file from RCSB PDB (Sugawara et al., 2004). LC3⌬N28 contains the ubiquitin core alone. (B) CHO cells stably expressing GFP-LC3, GFP-LC3⌬N10, GFP-LC3⌬N28 or GFP-LC3⌬N40 were cultured for 2 hours in αMEM (control) or EBSS (starvation) medium, fixed and analyzed by fluorescence microscopy. (C) Stably expressing CHO cells were starved in the presence of 100 nM bafilomycin A (Baf A), lysed with RIPA extraction buffer and cell lysates were analyzed by immunoblotting using anti-GFP antibodies (right). Asterisk labels the

Journal of Cell Science lipidated form (LC3-II). Quantification of GFP-LC3 lipidation (left) was performed as described in Materials and Methods. Data are means ± s.d. (D) Left, extracts of GFP-LC3 and GFP-LC3⌬N28 expressing cells were subjected to western blot analysis using anti-GFP or anti-LC3 antibodies. Middle panels, cells expressing GFP-LC3⌬N28 were incubated in αMEM or EBSS medium in the presence or absence of Baf A, fixed and immunostained with anti-LC3 antibodies. Colocalization was analyzed by confocal microscopy. The boxed areas shown are enlarged on the right. Right, colocalization analysis of GFP-LC3⌬N28 or GFP-LC3⌬N28 G120A with endogenous LC3 in starved cells was performed using the JACoP plugin in ImageJ (NIH Image) as described in the Materials and Methods. (E) Cells expressing GFP-LC3, GFP-LC3⌬N28 or GFP-LC3⌬N40 were incubated for 2 hours under starvation conditions in the presence of 100 nM Baf A. Lysosomes were labeled with monoclonal antibodies against LAMP-1 and colocalization was analyzed by confocal microscopy (left panels). The areas shown in white boxes are enlarged on the right. Colocalization analysis of GFP-LC3WT, GFP-LC3⌬N28 or GFP-LC3⌬40 with LAMP-1-labeled lysosomes (right) was performed as described in the Materials and Methods. Scale bars: 5 μm.

of the protein are involved in the interaction with p62 for the core decorated by two N-terminal α-helices (Coyle et al., 2002; consequential delivery of this protein to lysosomes for degradation. Knight et al., 2002; Paz et al., 2000; Sugawara et al., 2003). To However, p62 is not required for either LC3 lipidation or for determine the specific contribution of these protein regions to the autophagosome formation and targeting to the lysosomes. Moreover, autophagic process, Chinese hamster ovary (CHO) cells, previously total autophagy-mediated protein degradation was not affected by characterized for their autophagic activity (Fass et al., 2006), were p62 knockdown. This study provides molecular information stably transfected with GFP fused to either wild-type LC3, LC3⌬N10 regarding the cargo-recruiting activity of LC3, which involves the (lacking the first α-helix), LC3⌬N28 (lacking two α-helices), or conserved residue Phe52, together with the first ten amino acids of LC3⌬N40 (lacking two α-helices and first β-sheet) (Fig. 1A; the the protein. truncated regions are marked in red). The cells were incubated in αMEM (control) or EBSS (starvation) medium to induce autophagy Results and the subcellular distribution of transfected proteins was The ubiquitin core of LC3 is sufficient for its lipidation and microscopically examined. As shown in Fig. 1B, GFP-LC3WT, GFP- targeting to autophagosomes LC3⌬N10 or GFP-LC3⌬N28 were translocated to autophagosomes in A key step in the autophagic process is the conjugation of Atg8s, starved cells, whereas the subcellular distribution of GFP-LC3⌬N40 a novel UBL protein family, to the autophagosomal membrane. The remained unchanged. Next, we examined whether these truncations mammalian homologues of Atg8 include GATE-16, GABARAP affected the processing of the protein by the conjugation machinery and LC3, all sharing a similar structure composed of a ubiquitin (converted to the cytosolic form, LC3-I, or to the lipidated form, LC3 recruits p62 into autophagosomes 2687

Fig. 2. LC3 specifically interacts with p62 in an N-terminus-dependent manner. (A) CHO cells stably expressing GFP-LC3 or GFP-LC3⌬N28 were pulsed with [35S]methionine (40 mCi/ml), cultured for 2 hours in αMEM (control) or EBSS medium (starvation) in the presence of 0.1 μM Baf A and lysed using RIPA extraction buffer. GFP-LC3 or GFP-LC3⌬N28 proteins were immunoprecipitated from 500 μg total cell extracts using 0.5 μl anti-GFP antibodies, and coprecipitated proteins were determined by autoradiography. (B) Cell extracts (3.5 mg) prepared from GFP-LC3-expressing cells were precipitated using 3.5 μl anti-GFP antibodies. Samples were separated on 12% SDS-PAGE and stained using silver staining technique. M, Marker; E, eluate; T, total; F, flow through. The interacting protein (boxed band) was identified by mass spectrometry (MS). (C) Amino acid sequence of rat p62/SQSTM1. Amino acids identified by mass spectrometry are in red. (D) p62 copurified with GFP-LC3 was detected with specific monoclonal anti-p62 antibodies.

LC3-II). To this end, the formation of the lipidated form was induced protein (GFP-LC3), whereas no effect was observed in cells in cells by amino acid starvation, and its degradation was blocked expressing GFP-LC3⌬N40. These findings indicate that GFP- by bafilomycin A, a known lysosomal inhibitor. This agent was LC3⌬N28 localizes to LC3-labeled autophagosomes that are properly previously shown (Yoshimori et al., 1991) to block lysosomal targeted and fuse with lysosomes. acidification, leading to the accumulation of autolysosomes (Fass et al., 2006; Mousavi et al., 2001) and autophagosomes-associated LC3 specifically interacts with p62 in an N-terminus- and LC3 (LC3-II). The cells were lysed and cell extracts were subjected Phe52-dependent manner to western blot analysis with anti-GFP antibodies. While wild-type To study the role of LC3 N-terminus we searched for proteins that GFP-LC3, GFP-LC3⌬N10 and GFP-LC3⌬N28 were effectively interact specifically with the full-length LC3 but not with the protein ⌬N40 α ⌬N28 Journal of Cell Science processed to LC3-II, GFP-LC3 remained unprocessed (Fig. 1C lacking the two N-terminal -helices (LC3 ). For that purpose, and LC3-II marked by an asterisk). Based on these results we cells expressing GFP-LC3 or GFP-LC3⌬N28 were radiolabeled with conclude that the ubiquitin core of the protein (LC3⌬N28) is sufficient [35S]methionine, incubated under starvation or control conditions and essential for its conjugation to PE. in the presence of Baf A (to prevent the degradation of LC3 in the To assure the sufficiency of the ubiquitin core for the appropriate lysosomes), and the GFP fused proteins were then targeting of the protein to bona fide autophagosomes, we utilized immunoprecipitated using anti-GFP antibodies. As shown in Fig. anti-peptide antibodies raised against LC3 N-terminus, which 2A, a ~60 kDa band was predominantly precipitated in cells recognize the full-length protein but not the truncated protein (Fig. expressing the full-length LC3 but not the truncated protein 1D, left panel). Next, cells stably expressing GFP-LC3⌬N28 were LC3⌬N28. To identify this protein we scaled up the either incubated under normal growth conditions, or starved in the immunoprecipitation experiment eightfold and the 62 kDa band, absence or presence of Baf A, and stained with these specific anti- visualized by silver staining, was subjected to mass spectrometry LC3 antibodies. Confocal microscopy analysis revealed that amino analysis (Fig. 2B). This analysis identified three different peptides acid starvation induced the appearance of vesicles labeled with both (Fig. 2C) corresponding exclusively to p62/SQSTM (no other truncated GFP-LC3⌬N28 and endogenous LC3, which jointly peptides were found in this sample), polyubiquitin-binding protein accumulated in the presence of Baf A (Fig. 1D, middle panel). As and a common component of protein aggregates that has been shown by quantitative colocalization analysis, the colocalization of recently reported to directly interact with different LC3 family GFP-LC3⌬N28 and endogenous LC3 (Fig. 1D, right panel) was members (Bjorkoy et al., 2005; Pankiv et al., 2007). The identity blocked by introducing a point mutation into the LC3 C-terminal of p62/SQSTM (hereafter termed p62) was then confirmed by glycine (GFP-LC3⌬N28 G120A), which is essential for its processing western blot with specific anti-p62 monoclonal antibodies (Fig. 2D). and conjugation to autophagosomal membrane (Kabeya et al., 2004). To narrow down the specific region of LC3 that is crucial for Notably, these antibodies also showed some nonspecific nuclear interaction with p62, LC3 lacking the first α-helix (10 amino acids) staining as revealed by knockdown of LC3 protein (Fig. 5). We was fused with GFP and immunoprecipitated from Baf-A-treated further examined whether GFP-LC3⌬N28-labeled autophagosomes starved cells. As shown in Fig. 3A, p62 was co-purified only with are delivered to lysosomes by treating starved cells with vacuolar full-length GFP-LC3, but not with GFP-LC3⌬N10, indicating that H+-ATPase inhibitor Baf A. As shown in Fig. 1E, cells treated with the first 10 amino acids of LC3 are required for this interaction. Baf A accumulated GFP-LC3⌬N28-labeled vesicles within LAMP- We next examined whether the ubiquitin core of the protein also 1-labeled lysosomes (autolysosomes), similarly to the full-length contributes to the interaction with p62. For that purpose, we tested 2688 Journal of Cell Science 121 (16)

ubiquitin core of these proteins, but is not dependent on their C- terminal processing.

LC3 is essential for incorporation of p62 into autophagosomes and its consequential degradation within lysosomes p62 partly localizes to LC3-labeled autophagosomes under both normal growth and starvation conditions and, consistently with a previous report (Bjorkoy et al., 2005), its delivery to the lysosomes is increased under amino acid starvation (data not shown). Blocking lysosomal activity enables the determination of their contribution to p62 degradation and the visualization of p62 within these organelles. To test whether various LC3 mutants affect the incorporation of p62 into autophagosomes and its consequential degradation in the lysosomes, wild-type LC3 and its mutants (LC3F52A, LC3⌬N10, LC3⌬N28, LC3G120A) were transiently expressed at high levels in HeLa cells. We next determined whether these proteins acquired appropriate subcellular localization by utilizing antibodies directed against different markers of the autophagic pathway. As shown in Fig. 4A, all mutant proteins (excluding LC3G120A) were partially colocalized with Atg16, indicating that these proteins are properly localized to the phagophore or isolation membranes. To test whether the LC3-labeled autophagosomes are efficiently delivered to lysosomes, HeLa cells transiently expressing these proteins were starved in the presence of Baf A, stained with LAMP-1 and analyzed by confocal microscopy. Quantitative Fig. 3. Two sites in LC3 are essential for p62 binding. HeLa cells were colocalization analysis revealed that delivery of mutants LC3F52A, transfected with the gene of interest and after 24 hours were cultured for 2 LC3⌬N10 and LC3⌬N28 into the lysosomes is similar to that of the hours in EBSS medium in the presence of 0.1 μM Baf A. Cells were then wild-type protein (Fig. 4B). The various LC3 mutants can be divided lysed using RIPA extraction buffer. GFP or GFP fusion constructs of LC3 and F52A its mutants (A) or LC3 family proteins and their mutants (B) were into two types based on their interaction with p62: mutants LC3 , ⌬N10 ⌬N28 immunoprecipitated (IP) from total cellular extract (500 μg) and subjected to LC3 and LC3 , which do not bind p62 but are efficiently SDS-PAGE. Copurified p62 was detected by immunoblotting with anti-p62 conjugated to the autophagosomal membrane, and LC3G120A, which antibody (upper panel). WB, western blot. Immunoprecipitated GFP fusion binds p62 with similar affinity as the wild-type protein but does proteins were detected with anti-GFP antibody (lower panel). Amount of total not undergo conjugation to autophagosomal membrane. Hence, p62 or GFP fused proteins detected in 50 μg total protein extract (extracts) is indicated. when the effect of these mutants on p62 delivery into lysosomes

Journal of Cell Science was analyzed in cells starved in the presence of Baf A, two patterns of p62 localization were detected. As depicted in Fig. 4C, expression of wild-type GFP-LC3 did not affect the level of p62 accumulation the binding of p62 to LC3 mutated at residues located within the in lysosomes (appears as small vesicles), and most p62 vesicles ubiquitin core (Phe80-Phe82 and Phe52-Leu53), which has been colocalized with GFP-LC3. When LC3⌬N10, LC3⌬N28 or LC3F52A proposed to be part of two distinct protein-protein interaction sites fused to GFP were overexpressed in cells, fewer vesicles with p62 in Atg8 and LC3 (Amar et al., 2006; Fass et al., 2007). As shown were observed and only partial colocalization with different LC3 in Fig. 3A, alanine mutation at residue Phe52 completely prevented proteins was detected. By contrast, when LC3G120A, the mutant that the interaction between p62 and LC3, whereas a mutation at residue actively prevents incorporation of p62 into autophagosomes, was Leu53 had only a partial effect. Similar results were obtained with overexpressed in vivo, p62 accumulated in several large structures, the other LC3 mammalian homologues, GABARAP and GATE-16 often associated with GFP-LC3G120A (Fig. 4C, left panels). Notably, (data not shown). Notably, double mutation at residues Phe80- when LC3G120A was expressed at high levels, it often colocalized Leu82, which are essential for LC3 C-terminal processing (Fass et with p62 to separate large structures, which may represent protein al., 2007), had no effect on the interaction between LC3 and p62 aggregates (data not shown). Consistently, extensive quantitative (Fig. 3A). analysis showed that although colocalization of LC3⌬N10, LC3⌬N28 To test whether interaction between p62 and other LC3 family and LC3F52A with p62 was significantly inhibited compared to wild- members is dependent on the processing of the C-terminus, we type LC3, overexpression of LC3G120A resulted in strong inhibition prepared proteins carrying mutated Gly120 in LC3 and at Gly116 of p62 delivery into lysosomes (Fig. 4C, right). To carry out in GABARAP and GATE-16. Such mutations are known to prevent biochemical analysis on to the effect of these mutants on p62 the C-terminus cleavage and, consequentially, conjugation of these degradation, HeLa cells expressing these proteins were starved in proteins to PE (Kabeya et al., 2004). As depicted in Fig. 3B, similar the absence or presence of Baf A and the level of p62 was amounts of p62 protein coprecipitated with both wild-type and the determined by western blot using specific anti-p62 antibodies. The mutated, uncleaved forms of all LC3 family members, indicating degradation of p62 following overexpression of different LC3 that this interaction does not require the C-terminal processing of mutants was estimated as the difference between its level in the these proteins. Taken together, these results indicate that interaction absence or presence of Baf A and calculated relative to its of p62 with all LC3 family members requires the first N-terminal degradation in cells transfected with the wild-type protein. As α-helix (10 amino acids) and residues Phe52-Leu53 located in the depicted in Fig. 4D, overexpression of all LC3 mutants (LC3F52A, LC3 recruits p62 into autophagosomes 2689 Journal of Cell Science

Fig. 4. Overexpression of LC3 mutants inhibits incorporation of p62 into autophagosomes and its degradation within lysosomes. (A) HeLa cells were transfected with GFP fused to LC3WT or to its mutants and after 24 hours were cultured for 3 hours in EBSS medium, fixed, immunostained with anti-Atg16 antibodies and analyzed by confocal microscopy. The boxed areas are enlarged on the right. (B) Cells transfected as in A were starved in the presence of 0.1 μM Baf A, fixed, immunostained with monoclonal anti-LAMP-1 antibodies and analyzed by confocal microscopy. The areas shown in white boxes are enlarged on the right. Colocalization analysis of GFP fused proteins with LAMP-1 (right panel) was performed as described in the Materials and Methods. (C) Cells transfected as in A were starved in the presence of 0.1 μM Baf A, fixed, immunostained with monoclonal anti-p62 antibodies and analyzed by confocal microscopy. The boxed areas are enlarged on the right. Arrows indicate nontransfected cells; arrowheads indicate cells overexpressing GFP-LC3 proteins. Quantitative colocalization analysis of GFP fused proteins with p62 (right panel) was performed as described in the Materials and Methods. Mean ± s.d. of five independent experiments is presented below. **P<0.001. (D) Cells transfected as in A were starved in the presence or absence of 0.1 μM Baf A, lysed with RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-GFP antibodies (left panel). Quantification of relative p62 degradation (right panel) was calculated as described in Materials and Methods, and mean ± s.d. of four independent experiments is presented below. *P<0.05. (E) The rate of degradation of long-lived proteins was measured in HeLa cells incubated in either αMEM (control) or EBSS (starvation) medium, 24 hours following transfection of GFP-fused proteins. Values expressing the percentage of cellular proteins degraded in 4 hours are represented as the means ± s.d. of six determinations. Scale bars: 5 μm. 2690 Journal of Cell Science 121 (16) Journal of Cell Science

Fig. 5. LC3 is essential for starvation-induced degradation of p62. (A) HeLa cells were transfected with either transfection reagent (mock), nontargeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B and C isoforms) using DharmaFect reagent. After 72 hours, the cells were incubated in EBSS medium in the absence or presence of 0.1 μM Baf A, lysed with RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-LC3 antibodies. The asterisk indicates a nonspecific band. Arrows indicate LC3-I and LC3-II. (B) Quantification of relative p62 degradation (left panel) or relative p62 level (right panel) was performed as described in the Materials and Methods. Data are means ± s.d. of five independent experiments. The cells were transfected as in A, starved for 3 hours in the absence (C) or presence (D) of 0.1 μM Baf A, immunostained with anti-p62 and anti-LC3 antibodies, and analyzed using confocal microscopy. Scale bars: 5 μm.

LC3⌬N10, LC3⌬N28 and LC3G120A) led to a significant inhibition in To directly determine the involvement of LC3 in the delivery of p62 degradation compared with wild-type protein. These results are p62 into autophagosomes, LC3 was efficiently knocked down (Fig. consistent with the notion that LC3 delivers p62 to autophagosomes 5A) with a specific pool of siRNA against all three LC3 isoforms for lysosomal degradation. (for details see Materials and Methods) and the effect on p62 We next examined the effect of these LC3 mutants on autophagy- degradation and localization was examined. Transfected cells were mediated protein degradation. To this end, cells transfected with starved in the absence or presence of Baf A and relative p62 different GFP-fused proteins were prelabeled with [14C]valine for degradation was calculated and compared with control cells (Fig. 16 hours, and starvation-induced protein degradation was measured 5A,B). As shown in Fig. 5, knockdown of LC3 inhibited p62 in these cells. As depicted in Fig. 4E, overexpression of different degradation during the starvation period (Fig. 5B, left). Moreover, LC3 mutants had no effect on protein degradation activity compared the basal level of p62 in the LC3-knockdown cells was elevated with cells expressing the wild-type protein, suggesting that these up to 10-fold in comparison to control cells (Fig. 5B, right). mutants are not dominant-negative with respect to the overall Microscopy analysis of cells starved in the absence (Fig. 5C) or autophagic process. In summary, although overexpression of LC3 presence (Fig. 5D) of Baf A revealed that LC3 knockdown led to mutants resulted in inhibition in p62 degradation (Fig. 4C,D), no the accumulation of large p62 structures even in the absence of the defect was observed in autophagic activity of these cells (Fig. 4E), drug. Taken together, these results indicate that LC3 is essential for implying that association of p62 with autophagosomes is not p62 recruitment into autophagosomes for consequential degradation essential for autophagy. in lysosomes. LC3 recruits p62 into autophagosomes 2691

Fig. 6. Knockdown of LC3 leads to accumulation of detergent-insoluble p62 and polyubiquitylated proteins. (A) HeLa cells were transfected with either nontargeting siRNA (control siRNA), or with a pool of LC3 siRNAs (A, B and C isoforms) using DharmaFect reagent. After 72 hours, the cells were incubated in EBSS medium, lysed with RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-ubiquitin antibodies (left panel). Quantification of relative ubiquitin level (right panel) was performed as described in Materials and Methods. Mean ± s.d. of three independent experiments is presented below. (B) The cells were treated as in A, fixed, immunostained using anti-LC3, anti-ubiquitin (UB) and anti-p62 antibodies, and analyzed using confocal microscopy. (C) Cells were transfected as in A, starved for 3 hours in the presence of Baf A, then treated for 20 minutes with 1% Triton X-100 (right panel), fixed and stained with anti-LC3,

Journal of Cell Science anti-ubiquitin (UB) and anti-p62 antibodies. Scale bars: 5 μm. (D) The cells were transfected as in A, starved for 3 hours, lysed with RIPA extraction buffer, the lysates were centrifuged for 30 minutes at 350,000 g. The supernatant (soluble, left) and pellet (insoluble, right) fractions were analyzed by western blot with anti-p62, anti-actin and anti-ubiquitin antibodies.

Knockdown of LC3 leads to the accumulation of aggregates co-labeled with p62 that were insoluble in Triton X-100 polyubiquitylated aggregates (Fig. 6C). Consistently, western blot analysis of cell lysates separated P62 has been recently reported to serve as an adaptor molecule, into supernatant and pellet fractions revealed that knockdown of mediating degradation of polyubiquitylated protein aggregates LC3 led to accumulation of insoluble p62 and polyubiquitylated through the autophagic machinery (Bjorkoy et al., 2005). We proteins (Fig. 6D). Taken together, these results are consistent with therefore examined whether impaired degradation of p62 affected the notion that LC3 is essential for the delivery of p62 and the level of polyubiquitylated proteins. To this end, LC3-knockdown polyubiquitylated aggregates for lysosomal degradation. cells were starved and polyubiquitylated proteins were detected either by western blot analysis (Fig. 6A) or by confocal microscopy P62 is not essential for starvation-induced autophagy (Fig. 6B) using specific anti-ubiquitin monoclonal antibodies. As To further test the involvement of p62 in the autophagic process, shown in Fig. 6A, the level of polyubiquitylated proteins was the protein was knocked down by RNAi and autophagy was significantly (threefold) elevated compared with that in control cells. examined by several assays: LC3 lipidation, LC3 delivery to the This elevation was accompanied by accumulation of large structures, lysosomes and starvation-induced protein degradation. p62 was associated both with p62 and polyubiquitin proteins (Fig. 6B). It effectively knocked down by the specific siRNA (Fig. 7A, and B should be noted that LC3 and p62 that accumulated in lysosomes lower panel); however, no effect on LC3 lipidation either under of starved cells following Baf A treatment (Fig. 6C, left panels) control or starvation conditions was detected (Fig. 7A, and 7B upper were soluble in Triton X-100 (Fig. 6C, right panels) whereas panel). Next, we show that the formation of LC3-labeled knockdown of LC3 resulted in the accumulation of ubiquitin autophagosomes and their delivery to lysosomes are not affected 2692 Journal of Cell Science 121 (16) Journal of Cell Science

Fig. 7. p62 is not an essential factor in starvation-induced autophagy. (A) HeLa cells were transfected with either transfection reagent (mock), non-targeting siRNA (control siRNA), or p62 siRNA using DharmaFect reagent. After 48 hours, the cells were incubated under control or starvation conditions in the absence or presence of 0.1 μM Baf A, lysed using RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-LC3 antibodies. (B) Quantification of LC3 lipidation and the level of p62 protein were performed as described in Materials and Methods. (C) The cells were transfected as in A, starved for 3 hours in the presence of 0.1 μM Baf A, immunostained with anti-p62 and anti-LC3 antibodies, and analyzed using confocal microscopy. Scale bar: 10 μm. (D) The cells were transfected as in A, starved for 3 hours in the presence of 0.1 μM Baf A, immunostained with anti-LC3 and anti-LAMP-1 antibodies, and analyzed using confocal microscopy (left panels). Scale bar: 10 μm. Quantitative colocalization analysis of endogenous LC3 with LAMP-1 (right) was performed as described in the Materials and Methods. Data are means ± s.d. of three independent experiments. (E) Following 48 hours of transfection, the rate of degradation of long-lived proteins was measured in siRNA-transfected cells incubated in either αMEM or EBSS medium, in the absence or presence of 3-MA (10 mM) or Baf A (0.1 μM). Values express the percentage of cellular proteins degraded in 4 hours represented as the mean ± s.d. of six determinations. LC3 recruits p62 into autophagosomes 2693

by p62 knockdown (Fig. 7C,D). Finally, autophagy-mediated membrane (lipidation) is not sufficient for full function of Atg8 degradation of long-lived proteins was similar in both control and (Amar et al., 2006). Also, Ohsumi and colleagues have recently p62-knockdown cells (Fig. 7E). Notably, this degradation was shown that following conjugation to PE, Atg8 might act in significantly inhibited both by 3-methyladenine and Baf A, agents membrane tethering and hemifusion, two essential processes for known to block induction of autophagy or lysosomal activity, the expansion of the autophagosomal membrane (Nakatogawa et respectively (Fass et al., 2006). Taken together, our results indicate al., 2007). This study also identified multiple residues within the that p62 is not essential for starvation-induced autophagy. ubiquitin core that are essential for these activities of the protein. Although similar in structure, all UBL proteins are involved in Discussion different cellular functions. To better understand the function of Atg8 is a key autophagic factor involved in the early stages of mammalian Atg8, it is important to identify their specific interacting autophagosome biogenesis in yeast. This protein belongs to a UBL partners and the way in which such an interaction takes place. Here protein family, conserved from yeast to mammals, whose members we identified a specific residue within the ubiquitin core of share a similar structure composed of a ubiquitin-like core and two mammalian Atg8, which is essential for binding to p62. It has been unique α-helices attached at their N-termini. Among their other recently demonstrated that Ile44 in ubiquitin is involved in the functions, all Atg8 homologues are involved in autophagy whereas interaction with many different effector molecules (Seet et al., 2006). their active form is conjugated to phosphatidylethanolamine (PE) Based on our findings, we propose that Phe52 of LC3 (and its of the autophagosomal membrane. Atg8 lipidation is a crucial step equivalents in other members of the Atg8 family) has a similar role for dynamic membrane rearrangement during autophagosome in binding effectors. formation and therefore, this process is well controlled by multiple Unlike other UBL molecules, Atg8 family members contain two autophagic factors (Meijer and Codogno, 2006; Ohsumi, 2001; unique α-helices at their N-termini. Here we addressed the role of Scherz-Shouval et al., 2007). Here we determined the minimal this region in LC3 function. Several reports propose that the N- structural requirement for this conjugation process. We found for terminus of both GABARAP (Coyle et al., 2002; Wang and Olsen, LC3 – a member of the UBL protein family – that the ubiquitin 2000) and LC3 (Kouno et al., 2005) contains a tubulin-binding core alone is sufficient and essential for both C-terminal processing domain, which is required for interaction with tubulin or and appropriate targeting to bona fide autophagosomes. The role microtubules. Nevertheless, the implication of this interaction in of the unique N-terminal region flanking the ubiquitin core of Atg8 autophagy remains unclear. Crystal structure analysis of GABARAP and its homologues with respect to autophagy remains unclear. By revealed two possible conformational forms of this protein (‘open’ examining the activity of truncated proteins we show that the first and ‘closed’), which differ mainly in the orientation of the first α- N-terminal α-helix of mammalian Atg8 is involved in the interaction helix (Coyle et al., 2002). It was also suggested that the lipidation with p62, a protein recently reported to mediate the delivery of of Atg8 induces a conformational change in its N-terminal region polyubiquitylated proteins for autophagic degradation. Moreover, (Ichimura et al., 2004), implying that not only conjugation to a lipid specific residues (Phe52-Leu53) located at a distinct site within the but also a consequential conformational change in the N-terminal ubiquitin core of LC3 are also involved in this interaction. region may contribute to the function of Atg8. By utilizing an in Knockdown of LC3 or overexpression of LC3 mutants that fail to vitro system for Atg8 lipidation, it has been shown that Atg8

Journal of Cell Science interact with p62 significantly reduced the incorporation of p62 into conjugated to PE mediates tethering between adjacent membranes autophagosomes, indicating that LC3 is responsible for delivery of and stimulates membrane hemifusion, whereas truncation of the N- this protein into lysosomes for degradation. P62, however, affected terminal region resulted in a significant reduction in these activities neither LC3 lipidation nor autophagosome formation and targeting (Nakatogawa et al., 2007). Here we propose a novel role for this into lysosomes, implying that this protein acts to deliver region by showing that the N-terminal α-helices, particularly the polyubiquitylated aggregates for lysosomal degradation but is not first 10 amino acids of LC3, are essential for interaction with p62. essential for the autophagic process. Most importantly, this interaction also depends on residues Phe52- All Atg8 family members belong to a widespread family of UBL Leu53 located at the ubiquitin core (equivalent to residues 49 and proteins, which includes SUMO, ISG15, Nedd8 and Atg12, all of 50 of the yeast Atg8), implying that these two regions are involved which function as critical regulators of many cellular processes in this interaction. Residue Phe52 appears essential for the (Kerscher et al., 2006; Kirkin and Dikic, 2007). In spite of their interaction of LC3 and P62 whereas mutation of Leu53 led only to low sequence homology, they share the same three-dimensional a partial defect in binding, which may result from an effect of such structure (ubiquitin-like fold) and all use a C-terminal glycine as mutation on its neighboring residues. Since p62 binding was not the site of substrate conjugation. Here we show that the LC3 dependent on LC3 C-terminal processing, we suggest that p62-LC3 ubiquitin core is sufficient for its conjugation to PE, indicating that interaction requires the N-terminal region, but not LC3 this structural element contains all the information necessary for conformational change following lipidation. However, the recognition by a priming enzyme (Atg4) and by the conjugation significance of this interaction in the autophagic process is still machinery. Moreover, this scaffold is also sufficient for the unexplained. By utilizing different ways to examine autophagy, we appropriate targeting to a membrane of bona fide autophagosomes. demonstrated that p62 is not required for total autophagic activity, It has been recently reported that a point mutation in the ubiquitin consistently with a recent report by Komatsu and co-workers who core of both yeast and mammalian Atg8 affected their C-terminal demonstrated that the starvation-induced autophagy was similar in processing and the consequent conjugation to lipid (Amar et al., control and p62-deficient hepatocytes (Komatsu et al., 2007). 2006; Fass et al., 2007). Consistent with this finding, we show here However, by knockdown of LC3 or expression of its mutants that that the truncation of this conserved ubiquitin core affects all these fail to bind p62, we showed that LC3 is essential for p62 recruitment processes, indicating that the intact ubiquitin fold serves as a into autophagosomes and the consequential degradation within structural recognition module for interacting proteins. A recent lysosomes. Notably, when p62 incorporation into autophagosomes report, however, showed that appropriate conjugation to a target was inhibited, this protein accumulated with polyubiquitylated 2694 Journal of Cell Science 121 (16)

proteins in detergent-insoluble structures. These results further rabbit polyclonal anti-GFP antibody (Molecular Probes) and subjected to western support the notion that p62 acts as an adaptor, linking between blot analysis. polyubiquitylated protein aggregates and the autophagic machinery Quantification analysis (Bjorkoy et al., 2005; Pankiv et al., 2007). Thus, autophagy, Results from separate experiments (a minimum of three) were analyzed using essentially a nonselective process operating under starvation ImageQuant image program and quantified as follows: the percentage of p62 degradation was calculated as the difference in total p62 (normalized to actin as loading conditions, mediates targeting of specific cargo molecules for control) in the presence or absence of Baf A. The largest number obtained was set selective degradation. to 100% and the relative degradation in cells undergoing other treatments was calculated accordingly. The relative level of p62 or ubiquitin proteins was calculated Materials and Methods as difference in total p62/ubiquitin (normalized to actin as loading control) in LC3 siRNA transfected cells compared with non-targeting siRNA-transfected cells. The Antibodies and reagents number obtained from non-targeting siRNA transfected cells was set to one and the Minimal essential medium (αMEM), Earle’s balanced salt solution (EBSS), valine- relative protein level in LC3 siRNA-transfected cells was calculated accordingly. For free αMEM medium and fetal calf serum (FCS) were obtained from Biological the LC3 lipidation analysis, the fraction of lipidated LC3 out of the total LC3 was Industries (Beit Haemek Laboratories, Israel). Bafilomycin A1 (Baf A) was provided calculated for each treatment in each experiment. The largest value obtained in each by LC Laboratories, 3-methyladenine (3-MA) was obtained from Sigma-Aldrich and experiment was set to 100% and the relative lipidation in other treatments was L-[U-14C]-valine was obtained from Amersham Pharmacia Biotech. The following calculated accordingly. antibodies were used: mouse monoclonal and rabbit polyclonal anti-p62 antibodies (Santa-Cruz); anti-actin monoclonal antibodies (Sigma); mouse monoclonal anti- ubiquitin antibodies (Covance); mouse monoclonal anti-LAMP-1 (Developmental Fluorescence microscopy HeLa cells were plated on sterile coverslips (13 mm diameter) and cultured under Studies Hybridoma Bank, University of Iowa); mouse monoclonal anti-green fluorescence protein (GFP) (BabCo); rabbit polyclonal anti-GFP (Molecular Probes); the conditions indicated, fixed with cold methanol for 5 minutes at –20°C and rhodamine-conjugated goat anti-mouse IgG; FITC-conjugated donkey anti-rabbit IgG permeabilized by quick washing with cold acetone. Cells were blocked by incubation (Jackson ImmunoResearch Laboratories); and horseradish peroxidase (HRP)-coupled with 10% FCS in PBS for 30 minutes at room temperature, followed by incubation goat antibody against mouse IgG (Bio-Rad). Anti-LC3 antibody was produced as for 1 hour with the primary antibody. Cells were then incubated with the secondary previously described (Fass et al., 2006). antibody for 30 minutes. For fluorescence imaging, a Nikon Eclipse TE300 microscope equipped with a GFP filter (HQ FITC 41001) and Hamamatsu digital DNA construction camera C4742-95 were used. A fluorescence filter was used to observe Rhodamine (excitation 543 nm, mirror, emission 560-600 nm), and GFP/FITC (excitation 488, pEGFP-LC3 vector encoding human LC3 fused with GFP was prepared as previously dichroic 560 nm, emission 505-525 nm). Confocal images were taken by a FV500 described (Fass et al., 2006). The point mutation for glycine to alanine at position laser-scanning confocal microscope equipped with a PLAPO 60ϫ 1.4 NA oil- 120 of LC3 (LC3G120A) was created by PCR-based site-directed mutagenesis using immersion lens and Fluoview software (Olympus). LC3 sense primer (5Ј-CAGGAGACGTTCGCGATGAAATTGTCA-3Ј) and LC3 antisense primer (5Ј-TGACAATTTCATCGCGGACGTCTCCTG-3Ј). The point mutation for phenylalanine to alanine at position 52 of LC3 was created by PCR- Colocalization analysis based site-directed mutagenesis using LC3 sense primer (5Ј-GGATAA - The quantitative colocalization analysis was performed using JACoP (Just Another AACAAAGGCCCTTGTACCTGACC-3Ј) and LC3 antisense primer (5Ј- Co-localization Plugin) plugin in ImageJ program (NIH Image). For analysis, images GGTCAGGTACAAGGGCCTTTGTTTTATCC-3Ј). The point mutation for leucine of approximately 50-60 cells were taken in each experiment and 3-5 experiments to alanine at position 53 of LC3 was created by PCR-based site-directed mutagenesis were analyzed, bringing the total number of cells to 150-300 per determination. The using LC3 sense primer (5Ј-GGATAAAACAAAGTTCGCTGTACCTGACCATGTC- value shown represents Pearson’s coefficient (Manders et al., 1992). A linear 3Ј) and LC3 antisense primer (5Ј-GACATGGTCAGGTACAGCGAACTTTGTT - equation describing the relationship between the intensities in two images is calculated TTATCC-3Ј). The truncation of first 10 amino acids (LC3ΔN10) was created by PCR- by linear regression. The Pearson’s coefficient provides an estimate of the goodness based site-directed mutagenesis using LC3 sense primer (5Ј-CAACCCTT - of this approximation. Its value can range from 1 to –1, with 1 representing complete TCTCGAGCGCACCTTCGAACAAAGAG-3Ј) and LC3 antisense primer (5Ј- positive correlation and –1 a negative correlation; zero represents no correlation.

Journal of Cell Science GTGCCATTGCTCGAGGGATCTGAGTCCGGACTTGTA-3Ј). The truncation of the first 24 amino acids (LC3ΔN24) was created by PCR-based site-directed mutagenesis Degradation of long-lived proteins using LC3 sense primer (5Ј-CAATCTCGAGACCAAAATCCCG GTGATA ATAGA - Cells were transfected as described earlier and grown to 70-80% confluence in 12- ACG-3Ј) and LC3 antisense primer (5Ј-GTGCCATTGCTCGAGGGATCTGAGTC- well plates (35 mm). Cells were then labeled for 16 hours in medium containing 14 CGGACTTGTA-3 Ј). [ C]valine (0.5 mCi/ml) and 5% FCS in valine-free αMEM. After three rinses with PBS, cells were incubated in valine-free αMEM containing 0.1% BSA and 10 mM Cell culture and transfection cold valine. When required, 100 nM bafilomycin A or 10 mM 3-MA was added. α HeLa and CHO cells were grown on αMEM medium supplemented with 10% fetal After the 3 hour incubation, the medium was replaced with either MEM or EBSS medium also containing 0.1% BSA and 10 mM cold valine, and cells were incubated calf serum (FCS) and 1% penicillin-streptomycin (Sigma) at 37°C in 5% CO2. CHO cells were transfected with 5 μg DNA/10 cm plate using LipofectAmine (4 μl/1 μg for an additional 4 hours. The medium was precipitated in 10% TCA, and TCA- soluble radioactivity was measured. Total cell radioactivity was measured after lysis DNA). Stable clones of GFP-LC3 transfected CHO cells were selected in 1 mg/ml 14 geneticin (G418). Subconfluent HeLa cells were transfected with 10 μg DNA/10 cm with 0.1 M NaOH. [ C]-valine release was calculated as a percentage of the plate using standard calcium phosphate technique for overexpression of GFP-LC3 radioactivity in the TCA-soluble supernatant relative to total cell radioactivity. and its mutants, or with DharmaFect 1 (Dharmacon, Lafayette, CO) for siRNA transfection. siRNA SMARTpools (50 nM), consisting of four RNA duplexes We thank Terje Johansen for providing anti-p62 antibodies. We thank targeting p62 (catalogue number M-010230-00), or LC3 and non-targeting siRNAs Y. Avivi for discussions and critical evaluation of the manuscript, and control (catalogue number D-001206-14), were purchased from Dharmacon. For LC3, V. Kiss for technical assistance. Z.E. is an incumbent of the Sholimo 50 nM of each LC3A (M-013579-00), LC3B (M-012846-00) and LC3C (M-032399- 01) RNAs were targeted, bringing the total number of transfected duplexes to 12. and Michla Tomarin Career Development Chair of Membrane Cells were grown to densities of 5ϫ104 per 4 cm dish in 2 ml αMEM without Physiology. This work was supported in part by the Israel Science antibiotics, and transfected with DharmaFect according to the manufacturer’s Foundation, Binational Science Foundation and the Weizmann Institute instructions. Experiments with p62 siRNA were performed 48 hours after transfection Minerva Center. and with LC3 siRNA, 72 hours after transfection. To obtain starvation conditions, cells were washed three times with PBS and incubated in EBSS medium at 37°C for 3 hours. To inhibit lysosomal degradation, cells were starved in the presence of 100 References nM Baf A. Total cell extracts were made using RIPA extraction buffer (10 mM Tris- Abeliovich, H., Dunn, W. A., Jr, Kim, J. and Klionsky, D. J. (2000). Dissection of autophagosome biogenesis into distinct nucleation and expansion steps. J. Cell Biol. HCl pH 7.5, 10 mM NaCl, 1.5 mM MgCl , 1% DOC, 1% Triton X-100) with protease 2 151, 1025-1034. inhibitor mixture (Sigma). Amar, N., Lustig, G., Ichimura, Y., Ohsumi, Y. and Elazar, Z. (2006). Two newly identified sites in the ubiquitin-like protein Atg8 are essential for autophagy. EMBO Co-immunoprecipitation Rep. 7, 635-642. For immunoprecipitation experiments, cells were lysed 24 hours after transfection Babu, J. R., Geetha, T. and Wooten, M. W. (2005). Sequestosome 1/p62 shuttles using RIPA extraction buffer with protease inhibitor mixture. Cells were centrifuged polyubiquitinated tau for proteasomal degradation. J. Neurochem. 94, 192-203. at 350,000 g and supernatant was taken for immunoprecipitation experiment. GFP- Bjorkoy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., Stenmark, fused proteins were immunoprecipitated from total cellular extracts (500 μg) using H. and Johansen, T. (2005). p62/SQSTM1 forms protein aggregates degraded by LC3 recruits p62 into autophagosomes 2695

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