1. Biochemistry and Chemical Biology
  2. Cell Biology
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The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

  1. Xiaoshan Shi
  2. Chunmei Chang
  3. Adam L Yokom
  4. Liv E Jensen
  5. James H Hurley  Is a corresponding author
  1. Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, United States
  2. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, United States
Research Article
Cite this article as: eLife 2020;9:e59099 doi: 10.7554/eLife.59099
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 2 supplements
EM and HDX-MS of full-length FIP200.

(A) Negative stain EM single particles of full length FIP200 alone. Scale bar 50 nm. (B) Histogram of FIP200 path length and end-to-end distances. (C) Difference of Hydrogen Deuterium Exchange percentages of the FIP200 alone vs FIP200:ATG13:ATG101:ULK1 at 60 s time point. All values are mean (Blue) ± SD (Grey). N = 3 replicates.

Figure 1—figure supplement 1
EM of full-length FIP200.

(A) Negative Stain EM micrograph of full-length GST-FIP200-MBP. 2D class averages of the NTD dimer (B) and CTD Claw domain (C) of GST-FIP200-MBP. Green arrows indicate the GST tags and yellow arrows indicate the MBP tags. Scale bars are 10 nm.

Figure 1—figure supplement 2
Purified ULK1 complex is functional.

(A) ADP-Glo Kinase assay of ULK1 complex with ULKtide as substrate. N = 5 biological replicates. All values are mean ± SD. (B) Hydrogen Deuterium Exchange percentages of the FIP200 alone (Purple) and in FIP200:ATG13:ATG101:ULK1 (Blue) at 60 s time point. (C) Microscopy-based bead protein interaction assay with glutathione sepharose beads coated with GST-NDP52 as baits and incubated with GFP-tagged wild type ULK1 complex. Representative confocal micrographs are shown. Scale bars, 50 µm. (D) Pull-down efficiency of GFP-tagged wild type ULK1 complex by glutathione sepharose beads coated with GST-NDP52 or GST-4xUb as baits. N = 3 biological replicates. All values are mean ± SD.

Figure 2 with 2 supplements
HDX-MS mapping of NDP52 interactions with the ULK1 complex.

(A–D) Difference of Hydrogen Deuterium Exchange percentages of the ATG13 (A), ATG101 (B), ULK1 (C) and FIP200 (D) in ULK1 complex vs in ULK1 complex with NDP52 at the 60 s time point. All values are mean (Blue) ± SD (Grey). N = 3 replicates. (E) Pull-down efficiency of GFP-tagged wild type ULK1 complex or GFP-FIP200 by glutathione sepharose beads coated with different concentrations of GST-NDP52 as baits. All values are mean ± SD. N = 4 biological replicates. (F) Pull-down assays of mutant FIP200 constructs (M1–M8) and wild type with NDP52. Both GSH and Amylose resin were used to pull down GST-FIP200(1274 C):MBP-NDP52 complex from lysate of overexpressing HEK cells. The pull-down results were visualized by SDS-PAGE and Coomassie blue staining.

Figure 2—figure supplement 1
HDX-MS analysis of the interaction of the ULK1 complex with NDP52.

(A–D) Difference of Hydrogen Deuterium Exchange percentages of the ATG13 (A), ATG101 (B), ULK1 (C) and FIP200 (D) in ULK1 complex vs in ULK1 complex with NDP52 at the 6 s and 600 s time point. All values are mean (Blue) ± SD (Grey).

Figure 2—figure supplement 2
The coiled-coil domain of FIP200 is involved in binding with NDP52 and membranes, but not its stability.

(A) Hydrogen Deuterium Exchange percentages of FIP200 in ULK1 complex (Purple) and in ULK1 complex with NDP52 (Blue) at the 60 s time point. (B) Hydrogen Deuterium Exchange percentages of FIP200 in ULK1 complex (Purple) and in ULK1 complex with POPS/POPI SUV (Blue) at the 60 s time point. (C) GSH resin was used to pull down GST-FIP200-MBP, GST-FIP200ΔMR-MBP and GST-FIP200ΔNDP52-MBP from lysate of overexpressing HEK cells. The pull-down results were visualized by SDS-PAGE and Coomassie blue staining.

HDX-MS mapping of membrane interactions with the ULK1 complex.

(A–C) Difference of Hydrogen Deuterium Exchange percentages of the ATG13 (A), ATG101 (B) and FIP200 (C) in ULK1 complex vs in ULK1 complex with POPS/POPI SUV at the 60 s time point. All values are mean (Blue) ± SD (Grey). N = 3 replicates. (D) Liposome sedimentation assay of FIP200 truncations alone with POPS/POPI and POPC/POPE SUVs. Results were visualized by SDS-PAGE and Coomassie blue staining with the supernatant fractions (S) and pellet fractions (P).

Reconstitution of NDP52-stimulated membrane binding of the ULK1 complex.

The schematic drawing illustrates the reaction setting. Colors indicate fluorescent protein fused components. Components in gray are not labeled but are present in the reaction mix. (A) Representative confocal micrographs showing the membrane recruitment of GFP-FIP200. GFP-FIP200 mixed with His-NDP52 or GST-NDP52 was incubated with GUVs, either in the absence or presence of GST-4xUb at room temperature. GFP-FIP200 alone or mixed with GST-4xUb was also incubated with GUVs at room temperature as controls. Images taken at indicated time points were shown. Scale bars, 10 µm. (B) Quantitation of the kinetics of FIP200 recruitment to the membrane from individual GUV tracing in A (means ± SDs; N = 51 (-); 51 (His-NDP52); 48 (GST-NDP52); 53 (GST-4xUb); 72 (GST-4xUb+ His-NDP52); 57 (GST-4xUb+ GST-NDP52)). (C) Representative confocal micrographs showing the membrane recruitment of GFP-ULK1 complex. GFP-ULK1 complex alone or mixed with His-NDP52 or GST-NDP52 in the presence of GST-4xUb was incubated with GUVs at room temperature. Images taken at indicated time points were shown. Scale bars, 10 µm. (D) Quantitation of the kinetics of ULK1 complex recruitment to the membrane from individual GUV tracing in C (means ± SDs; N = 54 (-); 47 (GST-4xUb+ His-NDP52); 49 (GST-4xUb+ GST-NDP52)).

Figure 5 with 1 supplement
NDP52 allosterically activates membrane association of the ULK1 complex.

(A) Microscopy-based bead protein interaction assay with glutathione sepharose beads coated with GST-NDP52 as baits and incubated with GFP-tagged wild type ULK1 complex or mutant as prey. Representative confocal micrographs are shown. Scale bars, 50 µm. (B) Quantification of the GFP-ULK1 complex signal intensity measured on glutathione sepharose beads coated with GST-NDP52 (means ± SDs; N = 20). (C) Representative confocal micrographs showing the membrane recruitment of GFP-ULK1 complex. GFP-tagged wild type ULK1 complex or mutant was mixed with GUVs in the presence of GST-NDP52 and GST-4xUb at room temperature. Images taken at indicated time points were shown. Scale bars, 10 µm. (D) Quantitation of the kinetics of ULK1 complex recruitment to the membrane from individual GUV tracing in A (means ± SDs; N = 22 (WT); 25 (ΔMR); 22 (ΔNDP52)).

Figure 5—figure supplement 1
Membrane binding of NDP52.

Representative confocal micrographs showing the membrane recruitment of NDP52. GST-NDP52-mCherry (upper) or TSF-NDP52-mCherry (lower) was mixed with GUVs in the presence or absence of GST-4xUb at room temperature. Images taken at 30 min were shown. Scale bars, 10 µm.

Model for ULK1 complex membrane recruitment.

Before engagement with NDP52, the FIP200 middle region (790–1050) forms a stable coiled-coil in a low membrane affinity state and the CTD and ‘Claw’ domains are unbound. Initially, NDP52 is recruited by interaction with ubiquitin to autophagic cargo in both xenophagy and mitophagy. FIP200 CTD binds directly to NDP52, driving clustering of FIP200 along with an allosteric conformational change in the MR region. These clusters form a hub for ULK1 auto-transphosphorylation and a site for initial recruitment of phagophore membranes.

Author response image 1
NDP52 binds weakly to PA-containing GUVs.

Binding is unaffected by mutation of the putative PA binding sequence of Yu et al.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line (Homo sapiens)HEK GnTiATCCCRL-3022
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200-MBPThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-Atg13This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-GST-TEVcs-ATG101This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-
MBP-TSF-TEVcs-ULK1
This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-
EGFP-ATG13
This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-
GST-TEVcs-EGFP-ATG101
This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-
GST-TEVcs-EGFP-FIP200-MBP
This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpGST2-NDP52This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-MBP-NDP52This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpGEX5-4xUbZaffagnini et al., 2018From Sascha Martens group (Vienna)
Recombinant DNA reagentpGST2-NDP52-mCherryThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-TSF-NDP52-mCherryThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpET-6xHis-TEVcs-NDP52This paperFrom Sascha Martens group (Vienna)
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200(1274 C)This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200(N-640)-MBPThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200(636-C)-MBPThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200(delta790-1050, ΔMR)-MBPThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200(1363-1370Mut, ΔNDP52)-MBPThis paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Recombinant DNA reagentpCAG-GST-TEVcs-FIP200(1274 C) M1-M8This paperSee ‘plasmid construction’ section. Can be obtained from the Hurley lab.
Commercial assay or kitADP-Glo Max AssayPromega, Madison, WIV6930
Software, algorithmProteome Discoverer 2.1Thermo Fisher Scientific, Waltham, MAhttps://www.thermofisher.com/order/catalog/product/OPTON-30795
Software, algorithmHDExaminerSierra Analytics, Modesto, CAhttp://massspec.com/hdexaminer/
Software, algorithmNikon Elements microscope imaging software 4.60Nikon Corporation, Tokyo, Japanhttps://www.nikoninstruments.com/Products/Software/NIS-Elements-Advanced-Research/NIS-Elements-Viewer
Software, algorithmCustom Python scripts and Jupyter notebooksThis paperAccess at https://github.com/Hurley-Lab/FIP-NDP52-paper
Software, algorithmRelionSCR_016274
OtherGlutathione Sepharose 4B GST-tagged protein purification resinGE healthcare, Chicago, ILCat#17075605
OtherAmylose ResinNew England Biolabs, Ipswich, MACat#E8021L
OtherStrep-Tactin Superflow high capacity 50% suspensionIBA Lifesciences,
Göttingen,
Germany
Cat# 2-1208-010

Additional files

Supplementary file 1

Table S1.

Statistics of HDX differences for Figure 2D The peptides covering residue 800–1250 of FIP200 are listed, and the number of repeat and the sequence of each peptides are provided. The statistical test of HDX differences employed is paired T-test. P value less than 0.05 is highlighted in red, representing significant difference between ULK1 complex and ULK1 complex with NDP52 samples.

https://cdn.elifesciences.org/articles/59099/elife-59099-supp1-v2.xlsx
Supplementary file 2

HDX-MS Data Sets The summary tables of HDX data for each protein in ULK1 complex are provided, and the original peptide pool results are included.

https://cdn.elifesciences.org/articles/59099/elife-59099-supp2-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/59099/elife-59099-transrepform-v2.docx

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