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, 13 (5), 834-853

ATG4B Contains a C-terminal LIR Motif Important for Binding and Efficient Cleavage of Mammalian Orthologs of Yeast Atg8

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ATG4B Contains a C-terminal LIR Motif Important for Binding and Efficient Cleavage of Mammalian Orthologs of Yeast Atg8

Mads Skytte Rasmussen et al. Autophagy.

Abstract

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.

Keywords: ATG4; GABARAP; GABARAPL1; LC3B; LIR; X-ray structure; autophagy; peptide arrays.

Figures

Figure 1.
Figure 1.
ATG4B contains a C-terminal LIR motif important for a strong interaction with Atg8-family orthologs. (A) Schematic overview of ATG4B indicating disordered regions and LIR motifs predicted by the iLIR and PONDR-FIT servers. (B) Identification of a C-terminal LIR motif. An array of 20-mer peptides covering full-length ATG4B (each peptide shifted 3 amino acids relative to the previous) was mixed with GST-GABARAP (1 µg/ml) and binding detected with GST antibodies. The extension of the most strongly interacting peptides is indicated below in black. (C) The C-terminal LIR in ATG4B interacts with the LC3 and GABARAP subfamilies. The indicated peptides from ULK1, ULK2, ATG4B and FYCO1 (synthesized in duplicates marked 1 and 2) were examined as in B in a peptide array for binding to GST-tagged Atg8-family orthologs. (D) C-terminal sequences of ATG4B constructs carrying mutations affecting the C-terminal LIR motif. (E) The C-terminal LIR motif is important for the interaction of full-length ATG4B with Atg8-family orthologs. Myc-tagged ATG4B constructs were in vitro translated in the presence of [35S]methionine, and tested in GST affinity isolation experiments for binding to the indicated Atg8-family orthologs fused to GST. Bound proteins were detected by autoradiography (AR), and immobilized GST or GST-tagged proteins by Coomassie brilliant blue staining (CBB). (F) Quantification of E, % binding relative to WT ATG4B based on 3 independent experiments. (G) A putative N-terminal LIR is not important for the interaction of ATG4B with LC3B or GABARAPL1. MYC-tagged ATG4B constructs were in vitro translated and tested for binding to GST-LC3B and GST-GABARAPL1 as in (E). (H) Quantification of (G) from 3 independent experiments.
Figure 2.
Figure 2.
Structure of the GABARAPL1-ATG4B LIRC complex. (A) Close-up of structure for wild-type ATG4B LIRC motif bound to GABARAPL1. The LIRC of ATG4B (amino acids 384 to 393) is displayed in green cartoon with the interacting residues shown as sticks. GABARAPL1 is displayed in white cartoon and transparent surface with the hydrophobic pocket 1 and 2 colored in pink and blue surfaces, respectively. (B) Close-up of structure of p-S392 (phosphorylated) ATG4B LIRC motif bound to GABARAPL1. The phosphorylated LIRC peptide (amino acids 384 to 393) is displayed in orange cartoon with the interacting residues shown as sticks. GABARAPL1 is displayed in white cartoon and transparent surface with the hydrophobic pocket 1 and 2 colored in pink and blue surfaces, respectively. (C) Superposition of the wild-type and (S392)-phosphorylated LIRC peptide bound to GABARAPL1. Both structures are colored according to A and B. For clarity, only the surface of GABARAPL1 for the p-S392 (phosphorylated) LIRC is displayed. (D) Same as (C). Close-up view of the hydrophobic pocket 1 in both structures. Some sidechains were removed for clarity. (E) Sequence alignment of human Atg8-family orthologs generated with ESPript 3.0. Identical and similar residues are boxed in red and yellow, respectively. Red asterisks indicate key residues discussed in the text.
Figure 3.
Figure 3.
The C-terminal LIR in ATG4B relies both on electrostatic interactions, as well as aromatic and hydrophobic pocket interactions for efficient binding. (A) Two-dimensional peptide array scan analyzing the effects of single amino acid substitutions at all positions of the indicated 20-mer peptide from the C terminus of ATG4B. Arrays were probed with GST-GABARAPL1 or GST-LC3B. (B) E386 and E389 in the LIRC of ATG4B form important electrostatic interactions with both LC3B and GABARAPL1. A C-terminal ATG4B peptide (amino acids 374 to 393), fused to GFP and carrying the indicated point mutations, were in vitro translated and tested in GST affinity isolation assays for binding to LC3B and GABARAPL1. The ΔLIRC construct is indicated with an asterisk because it comprises amino acids 365 to 384 of ATG4B (LNLSLDSSDVERLERFFDSE) and 2mLIR harbors the F388A and L391A double mutation of the canonical aromatic and hydrophobic core LIRC residues. (C) Quantification of the experiment shown in B, based on 3 independent experiments. The binding to the WT peptide is set to 100%. (D) H9 and R47 are crucial for binding of GABARAP or GABARAPL1 to the ATG4B LIRC. The C-terminal ATG4B peptide (amino acids 374 to 393), fused to GFP was in vitro translated and tested in GST affinity isolation assays for binding to specific point mutants of GABARAPL1 fused to GST and bound to GST beads. ((E)and G) Both LIRC-LDS interactions and catalytic site-substrate interactions contribute to ATG4B-LC3B binding. MYC-ATG4B (E) and MYC-ATG4B 2mLIR (G) were in vitro translated and tested in GST affinity isolation assays for binding to GST-LC3B with or without mutations affecting the LIRC-LDS interaction (F52A and L53A, R10A and R11A, as well as R70A) or the catalytic site-substrate interaction (F80A and L82A). (F) Quantification of (E), the % binding relative to the binding of WT GST-LC3B to WT ATG4B based on 3 independent experiments. (H) Quantification of (G), % binding relative to the binding of WT GST-LC3B to ATG4B 2mLIR based on 3 independent experiments.
Figure 4.
Figure 4.
ATG4A and ATG4B harbor a C-terminal LIR, binding efficiently to LC3B and GABARAP, while ATG4C and ATG4D do not. (A) Model of full-length ATG4B bound to LC3B. The structure of the phosphorylated ATG4B LIRC motif bound to GABARAPL1 was superposed to the ATG4B (1 to 354)-LC3B complex (PDB ID: 2ZZP) to generate a model of the full-length ATG4B bound to LC3B. ATG4B is displayed in blue cartoons and LC3B is displayed in white cartoons and transparent surface. The 32 missing residues connecting ATG4B C-terminal LIR motif to Q354 are represented by a dashed line. (B) Schematic illustration of human ATG4B and yeast Atg4 indicating the protease domain and the functional LIRs (red bar) with the location of 2 candidate LIRs in yeast Atg4 tested in F indicated as gray bars. (C) A phylogenetic tree (left), and alignment of the far C-terminal sequence of the 4 different mammalian ATG4s and yeast Atg4 LIR (referred to in yeast as the Atg8-interacting motif). (D) MYC-tagged human ATG4 orthologs (WT and LIRC-deleted) were in vitro translated and tested in GST affinity isolation assays for binding to GST-LC3B and GST-GABARAP. The region deleted in the LIRC-deleted constructs is indicated by an open arrowhead in (C). The affinity isolation with the respective GST controls is shown below. (E) Quantification of the experiment shown in (D), based on 3 independent experiments. Bars indicate relative binding, and the interaction with ATG4B is set to 100%. For LIRC-deleted constructs, % binding relative to the corresponding full-length construct is indicated. (F) Yeast Atg4 has predicted LIR motifs binding to yeast Atg8 at a similar C-terminal distance to the protease domain as human ATG4B. Yeast Atg4 (WT and candidate LIR mutants) were in vitro translated and analyzed by GST affinity isolation assays for binding to GST-Atg8 (yeast). (G) Quantification of the experiment shown in (F), based on 3 independent experiments. WT was set to 100% binding. (H) Yeast MYC-tagged Atg4 was in vitro translated and analyzed by GST affinity isolation assays for binding to GST-Atg8 (WT and LDS mutated).
Figure 5.
Figure 5.
The C-terminal LIR motif of ATG4B is important for efficient cleavage of Atg8-family proteins. (A) GST-ATG4B ΔLIRC displays reduced ability to cleave off a C-terminal GST tag on both LC3B and GABARAP. The GST-tagged substrates were incubated with recombinant ATG4B (WT or mutated) for 0, 5, 15 or 60 min. The rate of cleavage was measured after SDS-PAGE as a loss of the band corresponding to the GST-tagged substrate. The % of uncleaved substrate remaining is indicated. (B) Lipidation of LC3 and GABARAP is functional in atg4b knockout cells rescued with WT or LIRC mutated GFP-ATG4B. Cells rescued with GFP-ATG4B constructs were treated or not with doxycycline (10 µg/ml) for 24 h to induce expression and cell lysates analyzed by western blotting. (C) Reduced ability of LIRC mutated ATG4B constructs to cleave transiently transfected LC3B or GABARAPL1 fused to the Gaussia luciferase. Cells reconstituted with the indicated ATG4B constructs were transiently transfected with the indicated LC3B or GABARAPL1 Gaussia luciferase constructs or vector control. Cleavage was measured by release of Gaussia luciferase over a period of 18 to 24 h for cells expressing the indicated LC3B or GABARAPL1 Gaussia luciferase constructs or vector control. Mean +/− SD of 4 independent experiments, wild type set to 100%, n ≥ 6, NS P > 0.05 *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANNOVA followed by the Tukey multiple comparison test. (D) Deletion of LIRN in ATG4B has no effect on lipidation of LC3B, GABARAP, GABARAPL1 or GABARAPL2 in atg4b knockout cells rescued with GFP-ATG4B. Cells rescued with WT or mutated GFP-ATG4B were treated or not with doxycycline (1 µg/ml) for 24 h to induce expression and cell lysates analyzed by western blotting. (E) Deletion of LIRN has a slightly positive effect on cleavage of transiently transfected LC3B or GABARAPL1 fused to the Gaussia luciferase. Cells reconstituted with the indicated ATG4B constructs were transiently transfected with the indicated constructs and in vivo cleavage measured as in (C). Mean +/− SD of 3 independent experiments, wild type set to 100%, n ≥ 6, NS P > 0.05 *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANNOVA followed by the Tukey multiple comparison test.
Figure 6.
Figure 6.
LC3B puncta formation is similar in atg4b KO MEFs expressing WT or LIRC-deleted GFP-ATG4B. (A) Cells were stained with LC3B antibodies and analyzed by confocal imaging. (B) Quantification of fractions of cells containing LC3 puncta, SQSTM1 puncta, or puncta positive for both LC3B and SQSTM1. (C) atg4b KO MEFs reconstituted with WT or LIRC-deleted GFP-ATG4B were transiently transfected with mCherry-GFP-LC3B, and analyzed by confocal imaging 16 h after transfection. There was no difference in yellow or red only structures between the cell lines, but cells expressing the LIRC-deleted construct contained large ring structures rarely seen in cells expressing WT ATG4B. Note that cells were not treated with doxycycline, and expression of GFP-ATG4B was therefore below detection level in these studies.
Figure 7.
Figure 7.
The LIRC motif of ATG4B is required for stabilization of GABARAP and GABARAPL1. (A) The endogenous level of GABARAP is severely diminished in cells that do not express ATG4B. Extracts of atg4b KO cells reconstituted with the indicated GFP-ATG4 constructs were analyzed by western blotting. (B) GABARAP is degraded by the proteasome in cells that do not express ATG4B. Cells reconstituted with GFP or indicated GFP-ATG4B constructs were treated or not with the proteasomal inhibitor MG132 (10 µM) for 4 h and analyzed by western blotting. The FK2 anti-ubiquitin antibody was used as a positive control for proteasomal inhibition. (C) In cells that do not express ATG4B, or inactive ATG4B, there is very little, if any, accumulation of GABARAP in response to lysosomal inhibition by BafA1. Cells were treated with or without BafA1 (0.2 µM) for 8 h, and cell lysates analyzed by western blotting. (D) GABARAP, but not LC3B, is stabilized by an elevated expression of WT ATG4B, and stabilization is LIRC dependent. Cell lysates from cells expressing different amounts of GFP-ATG4B or GFP-ATG4B ΔLIRC were analyzed by western blotting. To increase or reduce the expression of GFP-ATG4B, cells were treated as indicated with doxycycline (0.1 or 1 µM) or Atg4b siRNA for 48 h, respectively. (E) Endogenous GABARAP is efficiently immunoprecipitated by ATG4B in a LIRC-dependent manner. Lysates of atg4b KO cells reconstituted with the indicated GFP-ATG4B constructs were immunoprecipitated with GFP antibodies and analyzed by western blotting.
Figure 8.
Figure 8.
ATG4B stabilizes GABARAP in a LIR-dependent manner. (A) GABARAP stabilized by catalytic inactive ATG4B has a diffuse localization pattern in cells. atg4b KO cells reconstituted with GFP-ATG4B WT, C74S or ΔLIRC mutant were transiently transfected with mCherry-GABARAP and analyzed by confocal microscopy 24 h post transfection. (B) High-level expression of ATG4B WT and catalytic inactive, but not ATG4B ΔLIRC, stabilizes and redistributes ectopically expressed mCherry-GABARAP into a diffuse localization pattern. atg4b KO cells reconstituted with GFP-ATG4B WT, C74S or ΔLIRC mutant were transiently transfected with mCherry-GABARAP, induced with doxycycline (1 µg/ml), and analyzed by confocal microscopy 24 h post transfection. (C) GFP-ATG4B (C74S) and ectopically expressed mCherry-GABARAP display an almost completely overlapping localization pattern, both in the cytoplasm and in the cell nucleus. atg4b KO cells reconstituted with GFP-ATG4B (C74S) were transiently transfected with mCherry-GABARAP, induced by doxycycline (1 µg/ml), and analyzed by confocal microscopy 24 h post transfection. Representative images are shown. Bars: 10 μm. (D) Illustration of functional activities of ATG4B, indicating how the C-terminal LIR stabilizes the interaction between ATG4B and GABARAP and thereby contributes to the maintenance of a pool of unlipidated GABARAP. In (E) a potential trans-mediated interaction of the C-terminal LIR with an adjacent Atg8-family molecule conjugated to the same membrane is illustrated.

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