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. 2019 May 3;10(1):2055.
doi: 10.1038/s41467-019-10059-6.

Molecular Determinants Regulating Selective Binding of Autophagy Adapters and Receptors to ATG8 Proteins

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Free PMC article

Molecular Determinants Regulating Selective Binding of Autophagy Adapters and Receptors to ATG8 Proteins

Martina Wirth et al. Nat Commun. .
Free PMC article

Abstract

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Pericentriolar material 1 (PCM1) binds to GABARAP via a C terminally extended LC3-interacting region (LIR) motif. a Structure of PCM11951–1964 LIR bound to GABARAP (P212121). The PCM1 LIR sequence is shown in orange cartoon with interacting residues shown as sticks. GABARAP is displayed in white cartoon and transparent surface with hydrophobic pocket 1 and 2 colored in pink and blue surfaces, respectively. b, c Close-up view of Q59GAB interaction with main chain carbonyl residues of P1962PCM1 and K1964PCM1 in two different crystal forms (b: P212121; c: P43). d Sequence alignment of human ATG8-family orthologs using ESPript. Identical (red) and similar residues (yellow) are boxed. Red asterisks and green boxes indicate non-conserved residues between LC3A/B and LC3C/GABARAPs further analyzed in this study. e MYC-TRAP immunoprecipitation (IP) of HEK293A cells expressing MYC-ATG8 constructs and immunoblot with indicated proteins. f Sequence alignment of LIR peptides. The core LIR motif is boxed and aromatic and hydrophobic residues in position Θ0 and Γ3 depicted in blue. Residues in position X6–8 are boxed to highlight proline residues (yellow). Acidic residues are shown in red. g Affinities (Kd values) of LIR peptides to human ATG8 proteins determined by bio-layer interferometry (BLI). Color code indicates fold changes relative to wild-type (WT) GABARAP (data are mean ± s.d., n = 2–4)
Fig. 2
Fig. 2
ATG13 and ULK1 bind GABARAP via C terminally extended LC3-interacting region (LIR) motifs. a Structure of the ATG13441–454 LIR bound to GABARAP in alternate conformation A. The ATG13 LIR sequence is shown in light green cartoon with interacting residues depicted as sticks. GABARAP is displayed in white cartoon and transparent surface with hydrophobic pocket 1 (pink) and 2 (blue). b, c Close-up view of Q59GAB forming hydrogen bonds with main chain carbonyls of ATG13 LIR residues in alternate conformation A (light green) and B (dark green), respectively. d Structure of ULK1354–366 LIR-GABARAP complex. The ULK1 LIR sequence is shown in pink cartoon with interacting residues depicted as sticks. e Close-up view of interactions between GABARAP residues, Q59GAB, F62GAB, and L63GAB, with ULK1 LIR residues in position X7,8 (F364ULK1, P365ULK1). f Schematic overview of non-canonical LIR interactions observed in the structures of GABARAP bound to ULK1 (pink), ATG13 (green), and PCM1 (orange) LIR motifs. Blue lines indicate hydrophobic interactions, green lines hydrogen bonds, and green double arrow salt bridges. Strictly conserved residues are boxed and displayed in black; non-conserved residues are boxed and displayed in red
Fig. 3
Fig. 3
The C-terminal region is critical for ULK1 LC3-interacting region (LIR) binding specificity. a, b Mutational peptide array of 24-mer ULK1 peptide covering LIR motif incubated with GST-GABARAP (a) or GST-LC3B (b) and immunoblotted with anti-GST. Each amino acid position was substituted for every other amino acid. c Twenty four-mer peptide array analysis of ULK1 LIR peptides containing point mutations incubated with indicated GST-ATG8 protein and immunoblotted with anti-GST. Each peptide is spotted in triplicates. Mutated residues are highlighted in red. d GST pulldown of HEK293A cells expressing indicated HA-ULK1 constructs and immunoblot with indicated antibodies
Fig. 4
Fig. 4
Position X2 in the pericentriolar material 1 (PCM1) core LC3-interacting region (LIR) motif inhibits LC3 binding. a Twenty four-mer peptide array analysis of PCM1-FYCO1 LIR chimera sequences incubated with indicated GST-ATG8 protein and immunoblotted with anti-GST. Each peptide is spotted in triplicates. PCM1 and FYCO1 peptide sequence are highlighted in orange and green, respectively. b Twenty four-mer peptide array analysis of PCM1 LIR containing point mutations incubated with GST-ATG8 protein and immunoblotted with anti-GST. Each peptide is represented in triplicates. Mutated residues are highlighted in red. c Affinities (Kd values) of LIR peptides to ATG8 proteins determined by bio-layer interferometry (BLI). Color code indicates fold changes relative to Kd value of PCM1 WT LIR peptide binding to the corresponding ATG8 protein. (data are mean ± s.d., n = 2). d, e Mutational peptide array of 24-mer PCM1 peptide covering LIR motif incubated with GST-GABARAP (d) or GST-LC3B (e) and immunoblot. Each amino acid position was substituted for every other amino acid
Fig. 5
Fig. 5
Increased LC3 binding alters pericentriolar material 1 (PCM1) dynamics in cells. a GST pulldown of HEK293A cells expressing indicated GFP-PCM1 constructs and immunoblot. b Quantification of GFP signal intensities at the centrosome (centriole and pericentriolar material marked by γ-tubulin) normalized to total GFP signal intensities of whole cells. Each measurement represents one cell; n = 4 independent experiments. c, d Quantification of GFP-PCM1 colocalization with LC3 (c) and LAMP1 (d); HEK293A cells expressing indicated GFP-PCM1 constructs starved for 2 h in Earle’s balanced salt solution (EBSS) in the absence (SM) or presence of bafilomycin A1 (BafA1) (SMB), fixed and labeled with anti-LC3 and anti-LAMP1. See also Supplementary Fig. 5c. Pearson’s coefficient was measured in 30 cells per condition per independent experiment; n = 3 independent experiments. e HEK293 cells expressing indicated EYFP-mCherry-PCM1 constructs starved for 6 h in EBSS and fixed for confocal microscopy. Scale bars represent 10 µm. f Quantification of EYFP colocalization with mCherry. HEK293 cells expressing indicated EYFP-mCherry-PCM1 constructs (shown in e) starved for 6 h in EBSS. Pearson’s coefficient was measured in at least 50 cells per condition; n = 3 independent experiments. g HEK293 cells expressing indicated EYFP-mCherry-PCM1 constructs or empty vector (EV) were incubated in full medium (FM) or EBSS (SM) for 6 h in the presence or absence of BafA1 prior to immunoblotting. Statistical analysis using one-way analysis of variance (ANOVA) test; mean ± s.e.m.; ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns not significant
Fig. 6
Fig. 6
Non-conserved GABARAP residues are key to selective LC3-interacting region (LIR) binding. a, c, e Quantification of endogenous pericentriolar material 1 (PCM1) (a), ULK1 (c), and FIP200 (e) binding to indicated GFP-GABARAP constructs expressed and immunoprecipitated by GFP-TRAP from HEK293A cells. Representative immunoblot of GFP-TRAP immunoprecipitation (IP) experiments is shown in Supplementary Fig. 6e. b, d, f Quantification of endogenous PCM1 (b), ULK1 (d), and FIP200 (f) binding to indicated GFP-LC3B constructs expressed and immunoprecipitated by GFP-TRAP from HEK293A cells. Representative immunoblot of GFP-TRAP IP experiments is shown in Supplementary Fig. 6f. Statistical analysis using one-way analysis of variance (ANOVA) test; mean ± s.d.; data from at least three independent experiments. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. g Affinities (Kd values) of LIR peptides to indicated ATG8 proteins measured by bio-layer interferometry (BLI). Color code represents fold changes relative to wild-type (WT) GABARAP and LC3B, respectively (data are mean ± s.d., n = 2–5)
Fig. 7
Fig. 7
Rendering GABARAP more LC3B-like impairs NDP52 degradation. a Control (wild-type) and hexa ATG8 CRISPR knockout (KO) HeLa cell lines stably expressing indicated MYC-ATG8 constructs or empty vector (EV) in full medium, Earle’s balanced salt solution (EBSS) (SM) or EBSS + bafilomycin A1 (BafA1) (SMB) for 7 h prior to lysis and immunoblot with indicated antibodies. Expression of MYC-ATG8 constructs was induced by 1 µg/ml doxycycline for 6 days. Note, immunoblot of non-induced cells showing equal p62 and NDP52 protein levels in the reconstituted hexa ATG8 CRISPR KO HeLa cell lines is included as Supplementary Fig. 7a. b, c Quantification of p62 (b) and NDP52 (c) protein levels (normalized to actin) shown in a. Statistical analysis of (SM) using one-way analysis of variance (ANOVA) test; mean ±s.d.; data from four (p62) and three (NDP52) independent experiments. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. d Structure of FYCO1 LIR motif bound to LC3B (PDB ID 5WRD). Non-conserved LIR docking site (LDS) residues of LC3B are displayed in white sticks. LC3B is displayed in white cartoon and the FYCO1 LIR in green transparent cartoon. Hydrophobic pocket (HP) 1 and HP2 are indicated in pink and blue, respectively. e Structure of PCM1 LIR motif bound to GABARAP (P212121). Non-conserved LDS residues of GABARAP are displayed in white sticks. GABARAP is displayed in white cartoon and the PCM1 LIR motif is shown in orange transparent cartoon. f Surface electrostatic potential of FYCO1:LC3B structure in the same orientation as shown in d. g Surface electrostatic potential of PCM1:GABARAP (P212121) structure with ULK1 LIR superimposed (pink cartoon). f, g Projections shown are −5 (red) and +5 (blue) kT/e using pymol apbs plugin with red and blue representing negative and positive potential, respectively. Red dashed lines encircle basic patch in LC3B (d, f) and extension of HP2 in GABARAP (e, f)

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