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First published online 24 July 2008
doi: 10.1242/jcs.026005

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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

View larger version (54K): [in this window] [in a new window]   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). LC3N28 contains the ubiquitin core alone. (B) CHO cells stably expressing GFP-LC3, GFP-LC3N10, GFP-LC3N28 or GFP-LC3N40 were cultured for 2 hours in MEM (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 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-LC3N28 expressing cells were subjected to western blot analysis using anti-GFP or anti-LC3 antibodies. Middle panels, cells expressing GFP-LC3N28 were incubated in MEM 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-LC3N28 or GFP-LC3N28 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-LC3N28 or GFP-LC3N40 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-LC3N28 or GFP-LC340 with LAMP-1-labeled lysosomes (right) was performed as described in the Materials and Methods. Scale bars: 5 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 2. LC3 specifically interacts with p62 in an N-terminus-dependent manner. (A) CHO cells stably expressing GFP-LC3 or GFP-LC3N28 were pulsed with [35S]methionine (40 mCi/ml), cultured for 2 hours in MEM (control) or EBSS medium (starvation) in the presence of 0.1 µM Baf A and lysed using RIPA extraction buffer. GFP-LC3 or GFP-LC3N28 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.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Two sites in LC3 are essential for p62 binding. HeLa cells were transfected with the gene of interest and after 24 hours were cultured for 2 hours in EBSS medium in the presence of 0.1 µM Baf A. Cells were then lysed using RIPA extraction buffer. GFP or GFP fusion constructs of LC3 and its mutants (A) or LC3 family proteins and their mutants (B) were immunoprecipitated (IP) from total cellular extract (500 µg) and subjected to SDS-PAGE. Copurified p62 was detected by immunoblotting with anti-p62 antibody (upper panel). WB, western blot. Immunoprecipitated GFP fusion proteins were detected with anti-GFP antibody (lower panel). Amount of total p62 or GFP fused proteins detected in 50 µg total protein extract (extracts) is indicated.

View larger version (55K): [in this window] [in a new window]   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 MEM (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.

View larger version (74K): [in this window] [in a new window]   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.

View larger version (59K): [in this window] [in a new window]   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, 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.

View larger version (45K): [in this window] [in a new window]   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 MEM 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.

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