The Cargo Receptor NDP52 Initiates Selective Autophagy by Recruiting the ULK Complex to Cytosol-Invading Bacteria

Abstract

Summary Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo. Here, we show that, essential for anti-bacterial autophagy, the cargo receptor NDP52 forms a trimeric complex with FIP200 and SINTBAD/NAP1, which are subunits of the autophagy-initiating ULK and the TBK1 kinase complex, respectively. FIP200 and SINTBAD/NAP1 are each recruited independently to bacteria via NDP52, as revealed by selective point mutations in their respective binding sites, but only in their combined presence does xenophagy proceed. Such recruitment of the upstream autophagy machinery by NDP52 reveals how detection of cargo-associated “eat me” signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated in higher eukaryotes.

Ultrastructural insights into pathogen clearance by autophagy

“…It is likely that such membrane nucleation and expansion must depend on tight interactions between selective autophagy adaptors recognising the Salmonella eat‐me signals (eg, ubiquitination) and the autophagy machinery, and this probably explains why the phagophore membranes follow tightly the contour of the bacterial outer membrane. Recent work has discovered interactions between NDP52 and the ULK complex as well as between TBK1 and WIPI2 . Given the tightness of these engulfing membranes with their targets, additional such interactions will undoubtedly be discovered between adaptors and early autophagy proteins.…”

“…For example, it was recently shown that NDP52 forms a complex with FIP200 and SINTBAD/NAP1 leading to the recruitment of the autophagy machinery to Salmonella in the cytosol. The ULK complex localises to the Galectin‐8‐positive Salmonella surface, highlighting the importance of the damaged SCV for phagophore formation . In addition, the WIPI2 PI3P effector promotes the localization of the TBK1 kinase to the invading Salmonella prior to autophagic engulfment (https://www.ncbi.nlm.nih.gov/ pubmed/27370208).…”

Mitophagy: An Emerging Role in Aging and Age-Associated Diseases

“…Parkin-mediated poly-ubiquitination of outer mitochondrial membrane proteins triggers the recruitment of autophagy receptors such as optineurin (OPTN), calcium binding and coiled-coil domain 2 (CALCOCO2, better known as NDP52) and Tax1 binding protein 1 (TAX1BP1), concomitantly with the activation of the TANK binding kinase 1 (TBK1) that phosphorylates OPTN (at serine 177, 473, and 513) further enhancing its ubiquitin chain binding ability (Wild et al, 2011;Wong and Holzbaur, 2014;Heo et al, 2015;Lazarou et al, 2015). Once recruited to the mitochondria, autophagy receptors can employ initiator proteins from the autophagic machinery such as unc-51 like autophagy activating kinase 1 (ULK1), zinc finger FYVE-type containing 1 (ZFYVE1, better known as DFCP1) and WD repeat domain, phosphoinositide interacting 1 (WIPI1, also known as ATG18) to assemble the autophagosome (Wong and Holzbaur, 2014;Lazarou et al, 2015;Ravenhill et al, 2019;Turco et al, 2019;Vargas et al, 2019) and ATG8s, which could further recruit autophagy receptors to amplify mitophagy signals (Padman et al, 2019). The key function of the ULK1-containing complex for selective autophagy has been recently discussed elsewhere (Turco et al, 2020).…”

TBK1‐mediated phosphorylation of LC3C and GABARAP‐L2 controls autophagosome shedding by ATG4 protease

“…We tested the effect of phosphorylation at both these sites (S93 and S96) by modeling phosphate groups onto serine residues in the LC3C-ATG4B complex and performed molecular dynamics (MD) simulations (up to 1.5 ls). TBK1 kinase activity is induced at autophagy initiation [31,32] (e.g., during mitochondrial depolarization), which is when phosphorylation potentially disrupts the LC3-ATG4 interface. The C-terminal tail of LC3C remained bound and strongly anchored to the active site of ATG4B throughout the simulation.…”