LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy

Abstract

Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. Autophagy receptors target cargo to autophagy by binding ATG8 on autophagosomal membranes. The expansion of the ATG8 family in higher eukaryotes suggests that specific interactions with autophagy receptors facilitate differential cargo handling. However, selective interactors of ATG8 orthologs are unknown. Here we show that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity since cells lacking either protein cannot protect their cytoplasm against Salmonella. LC3C is required for antibacterial autophagy because in its absence the remaining ATG8 orthologs do not support efficient antibacterial autophagy. Structural analysis revealed that the selectivity of NDP52 for LC3C is conferred by a noncanonical LIR, in which lack of an aromatic residue is balanced by LC3C-specific interactions. Our report illustrates that specificity in the interaction between autophagy receptors and autophagy machinery is of functional importance to execute selective autophagy.

Graphical abstract

Figure 1

Selective Binding of LC3C by NDP52 and Requirement for LC3C in Antibacterial Autophagy (A) Lysates of 293ET cells expressing the indicated FLAG-tagged proteins were immunoprecipitated with anti-FLAG beads. Precipitates and lysates were probed with the indicated antibodies. (B) LUMIER binding assay. Normalized ratio between luciferase activity bound to beads and present in lysates. Upper: Lysates of 293ET cells expressing NDP52, p62, or NBR1 each fused to luciferase, incubated with purified GST-tagged ATG8 orthologs bound to beads. Lower: GST-fusion proteins stained with Coomassie and lysates of 293ET cells probed with anti-luciferase antibody. (C) Kinetics of S. Typhimurium replication in HeLa cells transfected with the indicated siRNAs. Bacteria counted on the basis of their ability to form colonies on agar plates. SiRNAs are further characterized in Figure S1. (D and E) Hyperproliferation of S. Typhimurium in a LAMP1-negative compartment despite normal infectivity in cells depleted of LC3C. HeLa cells transfected with the indicated siRNAs and infected with S. Typhimurium. (D) Percentage of cells containing the indicated number of bacteria at the indicated time points. (E) Percentage of LAMP1-positive and -negative bacteria, identified by antibody staining at 6 hr post infection (p.i.). Cells binned according to the number of bacteria they contained. (F) Kinetics of S. flexneri replication in HeLa cells transfected with the indicated siRNAs. Bacteria counted on the basis of their ability to form colonies on agar plates. (All panels) Mean and standard deviation (SD) of triplicate HeLa cultures and duplicate colony counts. Mean and SD of triplicate cultures. ^∗p < 0.05, Student's t test.

Figure 2

LC3C Is Required to Recruit Other ATG8 Orthologs to S. Typhimurium (A–D) Analysis of HeLa cells stably expressing the indicated GFP-tagged ATG8 orthologs and infected with S. Typhimurium. Percentage of bacteria coated at 1 hr p.i. with the indicated ATG8 orthologs (A) and the indicated LC3C alleles (B). Percentage of GFP-positive bacteria stained by an antibody for NDP52 at 1 hr p.i. (C) and percentage of mCherry-LC3C positive bacteria (D). (E and F) Analysis of HeLa cells stably expressing the indicated GFP-tagged ATG8 orthologs, treated with the indicated siRNAs, and infected with S. Typhimurium. (E) Percentage of bacteria coated by the indicated ATG8 orthologs at 1 hr p.i. (F) Confocal microphotographs of HeLa cells expressing GFP-tagged LC3B stained with DAPI and an antibody against ubiquitin. siRNAs are further characterized in Figure S1. (All panels) Mean and SD of triplicate cultures. ^∗p < 0.05, Student's t test.

Figure 3

Mapping the NDP52 LC3C Interacting Region (A) Upper: Domain structure of NDP52. Zn, zinc finger ubiquitin-binding domain. Lower: LUMIER binding assay. Normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing the indicated NDP52 alleles fused to luciferase incubated with the indicated purified GST proteins bound to beads. Blot: Lysates of 293ET cells probed with anti-luciferase antibody. (B) Section of fully assigned ¹⁵N,¹H-HSQC spectra of ¹³C,¹⁵N-labeled NDP52 (aa 21–141, 150 μM) domain with (red) and without (blue) 150 μM unlabeled LC3C (aa 1–126) present. Resonances labeled in bold are exchange-broadened upon addition of LC3C (see Figure S4 for full spectra). (C) Indication of exchange broadening in NDP52 (aa 21–141, 150 μM) upon addition of 60 μM LC3C, derived from the peak intensity ratios. See Figure S5 for further titration points obtained over LC3C concentrations ranging from 5 to 150 μM. (D) LUMIER binding assay. Normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing the indicated NDP52 alleles fused to luciferase were incubated with the indicated purified GST proteins bound to beads. Blot: Lysates of 293ET cells probed with anti-luciferase antibody. (E) Percentage of GFP-positive S. Typhimurium at 1 hr p.i. in HeLa cells stably expressing siRNA-resistant NDP52 alleles and treated with the indicated siRNAs. Lower: Lysates of HeLa cells probed with the indicated antibodies. (F) Alignment of the NDP52 CLIR sequence and the canonical LIR consensus motif. Numbers correspond to human NDP52. (All panels) Mean and SD of triplicate cultures. ^∗p < 0.05, Student's t test.

Figure 4

Origin of LC3C Specificity in NDP52 (A) Crystal structure of the NDP52 SKICH domain (aa 21-141) in cartoon representation and as topology diagram. N and C termini are indicated and β strands are numbered. Inset: The first β strand in ball-and-stick representation with a 2|F_o|-|F_c| electron density (blue mesh) contoured at 1σ. (B) Structure of NDP52 21–141 (cyan) in complex with LC3C (yellow). NDP52 in the same orientation as in (A). Inset: Residues of the CLIR with electron density as in (A). Right: Topology diagram. (C) Interaction of NDP52 CLIR and LC3C. Hydrophobic residues (Phe, Leu, Val, Ile, Pro) of LC3 in green. Inset: NDP52 (cyan) and LC3C (yellow, red labels). (D) Schematic diagram of interactions between LC3C and the NDP52 CLIR. Residues in red boxes differ between LC3C and LC3B (compare Figure 4F). (E) Interaction of p62 LIR and LC3B (pdb-id 2zjd, (Ichimura et al., 2008)), shown as in (C) with a purple p62 LIR peptide. (F) Schematic diagram of interactions between LC3B and the canonical LIR of p62. Residues in red boxes differ between LC3C and LC3B (compare Figure 4D). (G) NDP52 CLIR and p62 LIR peptides under a semitransparent surface (colored as in C, in an orientation as seen by LC3. (H) Close-up of the CLIR (left) and LIR (right) interactions with LC3 under a surface as in (D) and (F), respectively. (I) CLIR and LIR peptides upon superposition of LC3 molecules in NDP52-LC3C and p62-LC3B complexes. Dotted lines distances in Å. (J–K) Hydrogen bonds of the NDP52 CLIR (J) and the p62 LIR (K). Backbone hydrogen bonds boxed.

Figure 5

Manipulation of NDP52 CLIR Affinity and Specificity (A) Detail of the CLIR/LC3C (top) and LIR/LC3B (bottom) interactions with the aromatic pocket in a different orientation than in Figure 4H. (B–C) Fluorescence anisotropy assay of FITC-labeled NDP52 CLIR peptides (aa 128–141) against LC3C (B) and LC3A (C). Errors in K_D represent standard deviation. (D) LUMIER binding assay. Normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing the indicated NDP52 alleles fused to luciferase, incubated with purified GST-tagged ATG8 orthologs bound to beads. Blot: Lysates probed with anti-luciferase antibody. (E) Analysis of HeLa cells stably expressing GFP-tagged LC3B, treated with the indicated siRNAs, transduced with the indicated NDP52 alleles, and infected with S. Typhimurium for 1 hr. Percentage of bacteria coated by LC3B. Blot: Lysates of cells expressing the indicated siRNA-resistant NDP52 alleles and treated with siLC3C#17 were probed with anti-NDP52 antibody. (All panels) Mean and SD of triplicate cultures. ^∗p < 0.05, Student's t test.

Figure 6

Residues in LC3C Providing Specificity for NDP52 (A) Superposition of LC3C (yellow) and LC3B (green), with residues lining the CLIR interaction groove in ball-and-stick representation. (B) LUMIER binding assay. Normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing the indicated luciferase fusion constructs, incubated with purified GST-tagged proteins bound to beads. (C–D) Superposition of NDP52 (blue), LC3C (yellow), LC3B (green) and GABARAP (red) with residues mediating the interaction in ball-and-stick representation. Enlarged: movement of the α2-helix in LC3B and GABARAP compared to LC3C. (E) Percentage of bacteria coated by LC3B in HeLa cells stably expressing GFP-tagged LC3B. Cells transduced with mock, LC3A or the indicated LC3C alleles and treated with siRNA against LC3C were infected with S. Typhimurium for 1 hr. LC3A and LC3C mRNA levels are further characterized in Figure S6C. (All panels) Mean and s.d. of triplicate cultures. ^∗p < 0.05, Student's t test.