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ARF GTPases activate Salmonella effector SopF to ADP-ribosylate host V-ATPase and inhibit endomembrane damage-induced autophagy

Nature Structural & Molecular Biology volume 29, pages 67–77 (2022)Cite this article

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Abstract

Selective autophagy helps eukaryotes to cope with endogenous dangers or foreign invaders; its initiation often involves membrane damage. By studying a Salmonella effector SopF, we recently identified the vacuolar ATPase (V-ATPase)-ATG16L1 axis that initiates bacteria-induced autophagy. Here we show that SopF is an ADP-ribosyltransferase specifically modifying Gln124 of ATP6V0C in V-ATPase. We identify GTP-bound ADP-ribosylation factor (ARF) GTPases as a cofactor required for SopF functioning. Crystal structures of SopF–ARF1 complexes not only reveal structural basis of SopF ADP-ribosyltransferase activity but also a unique effector-binding mode adopted by ARF GTPases. Further, the N terminus of ARF1, although dispensable for high-affinity binding to SopF, is critical for activating SopF to modify ATP6V0C. Moreover, lysosome or Golgi damage-induced autophagic LC3 activation is inhibited by SopF or Q124A mutation of ATP6V0C, thus also mediated by the V-ATPase-ATG16L1 axis. In this process, the V-ATPase functions to sense membrane damages, which can be uncoupled from its proton-pumping activity.

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Fig. 1: ARF GTPases are specific binding partners of SopF.
Fig. 2: Crystal structure of the SopF–ARF1 complex.
Fig. 3: Structural basis for SopF binding to NAD+.
Fig. 4: ARF1 binding stimulates the ART activity of SopF for inhibiting bacterial autophagy.
Fig. 5: SopF is a universal inhibitor of membrane damage-induced autophagy.
Fig. 6: Gln124 of ATP6V0C discriminates V-ATPase function in selective autophagy from its proton-pumping activity.

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Data availability

The atomic coordinates and structure factors generated in this study have been deposited in the Protein Data Bank with PDB accession codes 7DN8 and 7DN9 for SopF ∆N72–ARF1Q71L ∆N16 and SopF ∆N62E325D–ARF1Q71L ∆N16, respectively. Source data are provided with this paper.

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Acknowledgements

We thank M. Hottiger (University of Zurich) for proving the antibody for ADP-ribosylated conjugates, M. Zhang (Institute of Biophysics, CAS) for pHluorin reporter and the staff of BL18U1 and BL19U1 beamlines at National Center for Protein Science Shanghai and Shanghai Synchrotron Radiation Facility for assistance during data collection. We also thank members of the Shao laboratory for technical assistance and stimulating discussions. This work was supported by the National Key Research and Development Program of China (grant nos. 2017YFA0505900, 2017YFA0504000 and 2016YFA0501500), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant nos. XDB29020202 and XDB37030202), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant no. 2019-I2M-5-084), Basic Science Center Project (grant no. 81788104) and Excellent Young Scholar Program (grant no. 81922043) of the National Natural Science Foundation of China (NSFC), and a grant from the CAS Youth Innovation Promotion Association (grant no. 2017127).

Author information

Authors and Affiliations

  1. National Institute of Biological Sciences, Beijing, China

    Yue Xu, Huan Zeng, Ping Zhou, Yan Ma, Lin Li, Feng Shao & Jingjin Ding

  2. Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yue Xu

  3. Institute of Analytical Chemistry and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Sen Cheng & Xiaoyun Liu

  4. Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Sen Cheng & Xiaoyun Liu

  5. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Huan Zeng, Feng Shao & Jingjin Ding

  6. Research Unit of Pyroptosis and Immunity, Chinese Academy of Medical Sciences and National Institute of Biological Sciences, Beijing, China

    Feng Shao

  7. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

    Feng Shao

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Contributions

F.S. conceived the study. Y.X., assisted by S.C., identified ARFs as SopF-binding proteins and performed biochemical and cellular experiments. H.Z. contributed to biochemical analyses of SopF-ARFs interaction and SopF NAD+ hydrolysis assay. S.C. was supervised by X.L. J.D. determined and analyzed the crystal structures. P.Z., Y.M. and L.L. provided technical assistance. Y.X., J.D. and F.S. analyzed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Feng Shao or Jingjin Ding.

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Nature Structural & Molecular Biology thanks Herwig Schüler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann, Sara Osman and Florian Ullrich were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 SopF interactions with ARF proteins.

a, Yeast two-hybrid analysis of SopF–ARF interaction. The assay was performed similarly as that in Fig. 1d except that different ARFs and their indicated mutants were used. b, Myristoylation of ARF1 is not required for binding to SopF. 293T cells co-transfected with Flag-SopF (or Flag-RFP) and the G2A mutant of an indicated ARF. The assay was performed similarly as that in Fig. 1b. c, Co-expression of SopF and the GTP-bound mutant of different ARFs results in formation of stable SopF–ARF complexes, assessed by gel filtration chromatography and Coomassie-stained SDS-PAGE gel. All data are representative of three independent experiments.

Source data

Extended Data Fig. 2 Structural homology search reveals that SopF is a member of the ART family.

a, Dali search results of SopF structure. b, Cartoon schemes of structurally characterized ART prototypes, including SopF (this study) and the ART domains of RNA 2'-phosphotransferase (PDB code: 1WFX), diphtheria toxin (PDB code: 1TOX), and cholera toxin (PDB code: 2A5F). The conserved six-stranded β-sheets are colored in magenta and the catalytic triads are shown as yellow sticks.

Extended Data Fig. 3 NAD+-hydrolysis activity of SopF.

a, Stereo-view of the Sigma-A weighted 2Fo − Fc electron density map (contoured at 1.0 σ) for the NAD+ in the SopF–ARF1 complex structure (Fig. 3a). b, LC-MS analyses of NAD+, Nam (nicotinamide), and ADP-ribose following incubation of NAD+ with WT, E325A, or D261A mutant version of SopF ∆N62–ARF1 complex. Data in b are representatives of three independent experiments.

Extended Data Fig. 4 The activity of SopF catalytic mutants in inhibiting bacterial autophagy.

a, Secretion of SopF mutants from S. Typhimurium into infected HeLa cells was determined by the SunTag translocation assay. b, c, Indicated amounts of WT or indicated SopF mutant protein (in complex with ARF1) were electroporated into HeLa GFP-LC3 cells (~ 2×105 cells in 100 µL). 12 h later, cells were infected with S. Typhimurium ΔsopF. Representative fluorescence images are in a (scale bar, 5 µm) and b (scale bar, 10 µm), in which the intracellular bacteria were stained with anti-Salmonella antibody (blue). c, percentages of GFP-LC3-positive bacteria (mean values ± SD from three measurements).

Source data

Extended Data Fig. 5 Characterization of SopF interaction with ARF1 and modification of the V-ATPase.

a, Analyses of the ability of ARF1 effectors to disrupt SopF–ARF1 complex formation. SopF ∆N62 in complex with His-tagged ARF1 Q71L was immobilized on Ni-NTA beads, and then incubated with indicated molar ratio of the ARF-binding domains of GGA1 or ARHGAP21. b, The V-ATPase complex used for in vitro modification by SopF. 293T cells stably expressing ATP6AP2-Flag were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. c, d, Optimization and analyses of the reconstituted V-ATPase ADP-ribosylation by SopF. The assay was performed similarly as in Fig. 4e with omission of an indicated component (c) or in the presence of indicated molar ratio of the ARF-binding domains of GGA1, GGA3 or ARHGAP21 (d). Proteins in the reaction in d were shown by a Coomassie-stained SDS-PAGE gel. e, Mass spectrometry determination of molecular weight of His-ARF1 expressed alone or co-expressed with SopF in E. coli. Theoretical mass for the methionine-removed form of ARF1 is 24221.26 Da. f, 293T cells were transfected with ARF1-3×Flag alone or together with IpaJ or SopF. ARF1-3×Flag was immunopurified and its total molecular mass was determined by mass spectrometry. As a control, co-expression with IpaJ resulted in a 267.45-Da mass loss in ARF1 due to cleavage of the N-myristoylated glycine. Data are representative of two independent experiments.

Source data

Extended Data Fig. 6 The N-terminal region of ARF1 is required for SopF catalytic activation.

a, Multiple sequence alignment of the ARF family. Secondary structures of ARF1 are marked and labeled along the sequence. The alignment was performed using the Clustal Omega algorithm. Identical residues are highlighted by red background and conserved residues are in red. The N-terminal and switch I/II regions of ARFs are highlighted by pink and green boxes, respectively. Residues in ARF1 that make contacts with SopF in are marked by blue background. The residue number is indicated on the left of the sequence. b–d, NAD+-hydrolysis activity of SopF ∆N62 in complex with different ARFs. The experiments were performed similarly as in Fig. 3d. Kinetic parameters of NAD+ hydrolysis by the SopF–ARF complexes were determined. d, ARF1N16-ARF5Q71L ΔN16 was generated by replacing the N-terminal 16 residues in ARF5 (Q71L) with that of ARF1. e, ADP-ribosylation of ATP6V0C by indicated SopF–ARF complexes in the reconstitution system. Data in b, d are mean values ± SD from three determinations. f. Structural comparison of NAD+-bound SopF–ARF1 complex and cholera toxin subunit A1 (CTA1) in complex with ARF6 (PDB code: 2A5F). The structure of ARF6 in CTA1–ARF6 complex was superimposed with that of ARF1 in the SopF–ARF1 complex. The switch I/II of ARF1/6 are highlighted with different colors as indicated. The bound NAD+ is shown as ball and sticks models. The N-terminus of ARFs are marked. Data (b–e) are representative of two independent experiments.

Source data

Extended Data Fig. 7 Inhibition of endogenous LC3B activation by SopF and activation of LC3B paralogues by lysosome/Golgi-damaging agents.

a, HeLa cells were infected with S. Typhimurium sopF deletion or complementation strain, and infected cells were stained by anti-Salmonella antibody (blue) and anti-LC3B antibody (green) (scale bar, 5 µm). b, HeLa cells, in which endogenous LC3B was stained by the anti-LC3B antibody (green), or HeLa cells expressing GFP-LC3 paralogs were treated with indicated lysosome or Golgi-damaging drugs. The cells were subjected to fluorescence imaging (scale bar, 5 µm). c, Time-course analyses of LC3-II formation, analyzed by anti-LC3 immunoblotting, in nigericin or NH4Cl-treated normal or SopF-expressing HeLa cells. Data are representative of two independent experiments.

Source data

Extended Data Fig. 8 AMDE-1 treatment and STING activation stimulate LC3 recruitment to the Golgi.

a, HeLa GFP-LC3 cells stably expressing STING alone or together with GalT-mRuby3 were treated with AMDE-1 or transfected with poly(dA:dT); cells without GalT-mRuby3 expression were stained with anti-TGN46 antibody (red). Fluorescence imaging of the cells are shown (scale bar, 5 µm). b, Stable expression of SopF does not disrupt the Golgi structure. The Golgi in HeLa cells or HeLa cells expressing mCherry-SopF was stained with anti-TGN46 or anti-GM130 antibody (green). Representative images are shown (scale bar, 5 µm). Data are representative of two independent experiments.

Extended Data Fig. 9 Evidences supporting that the V-ATPase-ATG16L1 axis senses pH alteration in endomembrane organelles to induce LC3 activation.

a, b, LC3 recruitment to bacteria-containing vacuoles or damaged lysosomes or Golgi is independent of labeling by Gal3 but correlates well with being resistant to lysotracker staining. a, HeLa GFP-LC3 cells (stained by lysotracker after bacterial infections) or HeLa cells expressing Gal3-mCherry were infected with S. Typhimurium ΔsopF or S. flexneri ΔvirAΔicsB. Bacteria were stained by their respective antibodies (blue) (scale bar, 5 µm). b, HeLa cells were treated with indicated lysosome or Golgi-damaging drugs and assayed similarly as in a. c, AMDE-1-induced pH changes in the Golgi measured by super-ecliptic pHluorin (SEP). HeLa cells expressing GalT (the first 61 residues of B4GALT1)-tagged mRuby3 (pH-insensitive, red) and the SEP reporter (pH-sensitive, green) were treated with AMDE-1 and subjected to fluorescence imaging (scale bar, 5 µm). BafA treatment was included as a control. All data are representative of three independent experiments.

Supplementary information

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Xu, Y., Cheng, S., Zeng, H. et al. ARF GTPases activate Salmonella effector SopF to ADP-ribosylate host V-ATPase and inhibit endomembrane damage-induced autophagy. Nat Struct Mol Biol 29, 67–77 (2022). https://doi.org/10.1038/s41594-021-00710-6

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