The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

Abstract

The selective autophagy pathways of xenophagy and mitophagy are initiated when the adaptor NDP52 recruits the ULK1 complex to autophagic cargo. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to map the membrane and NDP52 binding sites of the ULK1 complex to unique regions of the coiled coil of the FIP200 subunit. Electron microscopy of the full-length ULK1 complex shows that the FIP200 coiled coil projects away from the crescent-shaped FIP200 N-terminal domain dimer. NDP52 allosterically stimulates membrane-binding by FIP200 and the ULK1 complex by promoting a more dynamic conformation of the membrane-binding portion of the FIP200 coiled coil. Giant unilamellar vesicle (GUV) reconstitution confirmed that membrane recruitment by the ULK1 complex is triggered by NDP52 engagement. These data reveal how the allosteric linkage between NDP52 and the ULK1 complex could drive the first membrane recruitment event of phagophore biogenesis in xenophagy and mitophagy.

Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

“…The addition of NDP52 or TAX1BP1-Ub, with GST-Ub4, dramatically enhanced the membrane binding of ULK1 complex (Fig. 4A, second and third columns, B, and C), consistent with the previous observations that NDP52 directly recruited ULK1 complex in mitophagy or xenophagy (28)(29)(30). We noticed that only a little LC3 lipidation occurred even though the ULK1 complex was enriched on the membrane upon the addition of GST-Ub4 with either NDP52 or TAX1BP1 (Fig.…”

“…Negative stain electron microscopy (NSEM) images showed that FIP200 Δ641-779 had essentially the same structure as wild-type, while losing its propensity to aggregate (fig. S2C) ( 29 ). Contour lengths and end-to-end distances of FIP200 Δ641-779 as analyzed by NSEM were comparable as the full-length (fig.…”

“…NDP52 directly binds to and recruits the ULK1 complex to damaged mitochondria and intracellular bacteria by binding to the coiled-coil (CC) of the FIP200 subunit of the ULK1 complex (28)(29)(30). p62 is also recruited to FIP200, but to its CLAW domain instead of the CC (31).…”

“…As part of a long-term effort, we recently reconstituted the events from PI(3)P production by the PI3KC3-C1 to LC3 lipidation in mammalian autophagy in a giant unilamellar vesicle (GUV) model system (37). Separately, we showed that in the presence of clustered ubiquitin chains, NDP52 promotes recruitment of the ULK1 complex to membranes (29).…”

“…As part of a long-term effort, we recently reconstituted the events from PI(3)P production by the PI3KC3-C1 to LC3 lipidation in mammalian autophagy in a giant unilamellar vesicle (GUV) model system ( 37 ). Separately, we showed that in the presence of clustered ubiquitin chains, NDP52 promotes recruitment of the ULK1 complex to membranes ( 29 ). Here, using the GUV model system, and focusing on autophagy receptors involved in mitophagy, we established a start-to-finish reconstitution of selective autophagy initiation from autophagy receptor engagement through LC3 lipidation.…”