LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy

Summary 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.


Supplemental Figure Legends
A) Alignment of ATG8 family members from S. cerevisiae (Sc) and Homo sapiens (Hs). Mature proteins with C-terminally exposed Gly are shown. -helices andstrands are indicated by waved lines and arrows, respectively. B) LUMIER binding assay. Normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells or E. coli expressing NDP52 fused to luciferase, incubated with purified GST-tagged ATG8 orthologs bound to beads.      Plasmids. M5P or closely related plasmids were used to produce recombinant MLV for the expression of proteins in mammalian cells (Randow and Sale, 2006). pETM plasmids were gifts from A. Geerlof. Open reading frames encoding human ATG8 orthologs, NDP52, p62, NBR1, were amplified by PCR or have been described (Thurston et al., 2009;Bloor et al., 2010;2008). Mutations were generated by PCR and verified by sequencing.
Bacteria. S. Typhimurium (strain 12023), kindly provided by David Holden, was grown overnight in LB and sub-cultured (1:33) in fresh LB for 3.5 h prior to infection.
HeLa cells in 24-well plates were infected with 20 l of such cultures for 15 min.
Following two washes with warm phosphate buffered saline (PBS, pH 7.4) and incubation with 100 g/ml gentamycin for 2 h, cells were cultured in 20 g/ml gentamycin. To measure intracellular bacterial growth, cells from triplicate wells were lysed in 1 ml cold PBS containing 0.1 % Triton X100. Serial dilutions were plated in duplicate on TYE agar.

S. flexneri M90T, kindly provided by Chris Tang, was grown overnight in Tryptic Soy
Broth (TSB) and sub-cultured (1:100) in fresh TSB until an OD of approximately 0.3 was reached. Bacteria were resuspended in warm IMDM and 100 l were added to HeLa cells in 24-well plates. Samples were centrifuged for 10 min at 2000 rpm.
Following an incubation at 37 °C for 30 min, cells were washed with warm PBS and cultured in 100 g/ml gentamycin for 2 h and 20 g/ml thereafter.
Cell culture. Cells were grown in IMDM supplemented with 10% FCS at 37°C in 5% CO2. HeLa cells were obtained from the European Collection of Cell Cultures.
RNAi. 5x10 4 cells per well were seeded in 24-well plates. The following day cells were transfected with 40 pmol of siRNA (Invitrogen) using Lipofectamine 2000 (Invitrogen) in Optimem medium (Invitrogen). Optimem was replaced with complete IMDM medium after 4 h and experiments were performed after 72 h. siRNAs targeted the following sequences: Samples were separated on 4-12% denaturing Bis-Tris gels (Invitrogen).
Visualization following immunoblotting was performed using ECL detection reagents (Amersham Bioscience). NMR Analysis. NDP52 (21-141) was expressed as described above but with the following modification. Bacteria from a 40 mL overnight culture grown in LB medium were used to inoculate 4 L M9 minimum medium supplemented with 8 g of 15 N ammonium chloride, and 16 g of 13 C glucose for double-labeled samples, or 16g of glucose for single-labeled samples. Cells were lysed by sonication in NMR buffer (phosphate buffered saline (PBS), 18 mM Na 2 HPO 4 , 7 mM NaH 2 PO 4 .xH 2 O, 150 mM NaCl, 5 mM DTT, pH 7.2). Purification was performed as described above, with the final gel filtration performed in NMR buffer. All NMR experiments were performed in PBS at neutral pH (pH 7.2) in the presence of 150 mM NaCl to mask non-specific interactions. All proteins were monodisperse in these buffer conditions at all analyzed protein concentrations.
NMR data were acquired using a 700MHz ( 1 H frequency) Bruker Avance2+ spectrometer with a cryogenic triple resonance probe, and a sample temperature of 298 K. All datasets were processed in TopSpin 2.1 (Bruker) and analyzed in Sparky after 5 days. The space group of these crystals was P4 1 2 1 2 and this crystal form was used for phasing. A similar crystallization condition lacking cadmium (24% (w/v) polyethylene glycol (PEG) 4000, 100 mM Tris (pH 8.5)) resulted in crystals in a different space group (P2 1 2 1 2 1 ) that diffracted to high resolution (Supplementary Diffraction data was processed using iMosflm (Leslie, 2006) or XDS (Kabsch, 2010) and the CCP4 software suite (CCP4, 1994). The structure of NDP52 SKICH domain was determined by single anomalous dispersion methods using cadmium anomalous signal in autoSHARP (Bricogne et al., 2003). The structure of the NDP52-LC3C complex was solved by molecular replacement with Phaser (McCoy et al., 2005) using the NDP52 SKICH domain and LC3B (PDB-id 1ugm, (Sugawara et al., 2004)) as search models. Subsequent rounds of model building in Coot (Emsley and Cowtan, 2004) and refinement in Phenix (Adams et al., 2002)  were performed in 384-well format on a PheraStar plate reader with excitation and emission wavelengths at 488 nm and 518 nm, respectively. Anisotropy readings were normalized to a control of 10 µL of Binding Buffer mixed with 10 µL of 100 µM FITClabeled NDP52 peptides. Binding data was analyzed with GraphPad Prism 5.