Abstract
Autophagy-related (Atg) proteins catalyze autophagosome formation at the phagophore assembly site (PAS). The assembly of Atg proteins at the PAS follows a semihierarchical order, in which Atg8 is thought to be quite downstream but still able to control the size of autophagosomes. Yet, how Atg8 coordinates multiple branches of autophagy machinery to regulate autophagosomal size is not clear. Here, we show that, in yeast, Atg8 positively regulates the autophagy-specific phosphatidylinositol 3-OH kinase complex and the retrograde trafficking of Atg9 vesicles through interaction with Atg1. Mechanistically, Atg8 does not enhance the kinase activity of Atg1; instead, it recruits Atg1 to the surface of the phagophore likely to orient Atg1’s activity toward select substrates, leading to efficient phagophore expansion. Artificial tethering of Atg1 kinase domains to Atg8s enhanced autophagy in yeast, human and plant cells and improved muscle performance in worms. We propose that Atg8-mediated relocation of Atg1 from the PAS scaffold to the phagophore is a critical step in positive autophagy regulation.
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Data availability
All data supporting the findings of this study are available within the article and Supplementary Information. Source data are provided with this paper.
Change history
13 October 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41594-025-01700-8
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Acknowledgements
We would like to thank T.-B. Liu (Southwest University), J.-H. Lu (University of Macau), Y. Shen (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences), L. Weisman (University of Michigan), H. Zhang (Institute of Biophysics, Chinese Academy of Sciences) and X.-Q. Zhao (Shanghai Jiao Tong University (SJTU)) for gifts of experimental materials, T. Shi (SJTU), Q. Xu (SJTU) and J.-T. Zheng (SJTU) for research advice and Y. Ma (SJTU), F. Wei (SJTU), X.-M. Yang (SJTU), Y.-C. Zhang (SJTU) and Z. Zhou (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) for technical assistance. We would also like to thank past lab members B. Hong, F.-J. Jiang, Z.-Q. Yu, T. Ni and H.-Y. Wang for their assistance in preparing the paper. This work was supported by the National Key R&D Program of China (2020YFA0907700, to Z.X.), National Natural Science Foundation of China (32270796, to Z.X.), Shanghai Rising-Star Program (19QA1409700, to Z.Q.), Tongji Hospital Start-Up Funding for Scientific Research (RCQD2301, to Z.Q.), and Shanghai Municipal Science and Technology Commission (22ZR1433800, to Z.X.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.
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Conceptualization, J.-Z.S., H.L., Q.G., J.W., Z.Q. and Z.X. Investigation, J.-Z.S., H.L., H.Y., R.L., W.Z., T.H., M.-X.X., C.C., L.C., S.W., Y.R., L.-F.P. and J.Z. Writing—original draft, J.-Z.S., Q.G., J.W., Z.Q. and Z.X. Supervision, Q.G., J.W., Z.Q. and Z.X.
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Extended data
Extended Data Fig. 1 Atg8 enhances the retrograde traffic of Atg9 vesicles.
(A-C) Atg9 is concentrated at the PAS in atg8Δ and atg7Δ cells. (A) Representative snapshots (max intensity projection for fluorescent channel) of cells expressing Atg9-2GFP after 1 h of starvation. (B) Fractions of cells containing bright Atg9-2GFP puncta. Mean ± standard deviation, n = 3 independent repeats (C) Fluorescent intensities of bright Atg9-2GFP puncta. a. u., arbitrary units. Sina plot data summed from 3 independent repeats, with mean ± standard deviation marked. (D-F) Retrograde Atg9 trafficking is retarded in atg8Δ and atg7Δ cells. (D) Schematic depiction of the assay. Kinetic of Atg9 vesicle trafficking was evaluated in cells expressing atg1-ts. PAS accumulation (23 → 37 °C) and release (37 → 23 °C) of Atg9 were triggered by temperature shifts that inactivate and re-activate Atg1-ts protein. (E) Representative snapshots at different time points (presented as in A). (F) Fractions of cells containing bright Atg9-2GFP puncta at different time points (presented as in B). Scale bar, 5 um.
Extended Data Fig. 2 Evaluation of Atg8 and Atg1 mutants by multiple assays.
(A-B) Interaction of Atg8 variants with Atg1 (A), Atg3, Atg4, Atg7, or Atg19 (B) by conventional yeast two-hybrid assay. Mutants in red displayed abnormal interaction. (C-H) Co-immunoprecipitation assay evaluating the interaction between Atg8 variants and Atg3 (C-D), Atg4 (E-F), or Atg7 (G-H). Constructs were expressed under ADH1 promoter in wild-type cells. (C, E, G) Representative immunoblots. (D, F, H) Levels of co-precipitated Atg proteins. Mean ± standard deviation, n = 3 independent repeats. Normalized against sample containing wild-type proteins. Mutants in red displayed abnormal interaction. (I-K) Status of Atg8 lipidation evaluated by immunoblotting. (I) Representative immunoblots. (J, K) Amounts of Atg8-PE, and ratios of Atg8-PE/Atg8. Mean ± standard deviation, n = 3 independent repeats. (L-M) Interaction of Atg1 variants with Atg8 (L), Atg13, Atg17, or Atg11 (M) by conventional yeast two-hybrid assay. (N-S) Co-immunoprecipitation assay evaluating the interaction between Atg1 variants and Atg4 (N-O), Atg13 (P-Q), or Atg17 (R-S). Presented as in (C-H).
Extended Data Fig. 3 The retrograde traffic of Atg9 vesicles does not depend on Vam3.
(A-C) Steady state distribution of Atg9. (A) Representative snapshots (max intensity projection for fluorescent channel) of cells expressing Atg9-2GFP after 1 h of starvation. (B) Fractions of cells containing bright Atg9-2GFP puncta. Mean ± standard deviation, n = 3 independent repeats (C) Fluorescent intensities of bright Atg9-2GFP puncta. a. u., arbitrary units. Sina plot data summed from 3 independent repeats, with mean ± standard deviation marked. Note that data on wild type and atg8L55A cells are the same as in Fig. 4U and X-Y. (D-E) Rate of retrograde Atg9 trafficking. (E) Representative snapshots at different time points (presented as in A). (F) Fractions of cells containing bright Atg9-2GFP puncta at different time points (presented as in B). Scale bar, 5 um.
Extended Data Fig. 4 Atg8-Atg1 interaction has mild impacts on PAS scaffold proteins.
(A-C) Subcellular distribution of PAS scaffold proteins examined by fluorescent microscopy. Cells were starved for 1 h. (A) Representative snapshots (max intensity projection for fluorescent channel). (B) Numbers of puncta per cell. Mean ± standard deviation, n = 3 independent repeats. (C) Brightness of individual puncta. a. u., arbitrary units. Sina plot data summed from 3 independent repeats, with mean ± standard deviation marked. (D-E) Vsp34-Vac8 interaction does not depend on Atg8-Atg1 interaction. The interaction between Vps34 and Vac8 was evaluated by co-immunoprecipitation. Vac8 and Vps34 constructs were expressed under their own promoters. Cells were nitrogen starved for 1 h. (D) Representative immunoblots. (E) Levels of co-precipitated Vac8. Normalized against sample containing wild-type proteins. (F-G) Reduced recruitment of Atg13 was secondary to a decline of PtdIns-3-K activity. Status of Atg13-2GFP in vps34K759D cells was evaluated as in (A-B). Scale bar, 5 um.
Extended Data Fig. 5 Only Atg1, but not the PAS scaffold proteins, are transported to the vacuole for degradation.
(A-B) Subcellular distribution of 2xGFP tagged Atg1, Atg13, Atg17, Atg29, and Atg31 at 1 h (S1) and 4 h (S4) after starvation, in wild type (A) or pep4Δ (B) background cells. Only Atg1-2GFP showed prominent translocation into vacuoles at 4 h. Representative image slices are shown. Scale bar, 5 um. (C) The processing of GFP tagged proteins examined by immuno-blotting. Only Atg1-GFP showed prominent processing into free GFP at 4 h.
Extended Data Fig. 6 Phosphorylation status of Atg1 substrates in Atg8-Atg1 interaction mutant cells.
(A-D) Auto-phosphorylation of Atg1 occurred independent of Atg8-Atg1 interaction. Cells were starved for 1 h. Band shifts in immunoblots were used to indicate the status of Atg1 auto-phosphorylation. Autophosphorylation was reduced in atg13Δ, atg1T226A, and atg1K54A M102A (KM) cells, but normal in atg1V432F, atg8L55A, and atg8Δ cells. (A, C) Representative immunoblots. (B, D) Ratios of phosphorylated Atg1 over total Atg1. Mean ± standard deviation, n = 3 independent repeats. (E) Several known Atg1 substrates did not display substantial Atg1-dependent phosphorylation under our experimental condition. Phosphorylation status of Atg1 substrate proteins was examined by phos-tag gel. Cells were nitrogen-starved for 2 h. Representative immunoblots from phos-tag gel (top row) and regular gel (bottom row) are shown. Proteins in bold font displayed band-shifts independent of Atg1 activity. (F-G) Phosphorylation of Ykt6 was reduced in both atg1V432F and atg8L55A mutant cells. (F) Representative immunoblots presented as in (E). Positions of phosphorylated bands to be quantified were marked by bars or arrows. (G) Quantified ratios of phosphorylated proteins over total proteins (for Atg13, Atg29, Atg38, and Atg2) or relative amount of phosphorylated proteins (for Ykt6, normalized against wild-type sample). Mean ± standard deviation, n = 3 independent repeats.
Extended Data Fig. 7 Autophagy is enhanced via targeting of Atg1 kinase domain to Atg8, but not to others.
(A) Fusion of 3xAIM motif to the kinase domain of Atg1 was sufficient for interaction with Atg8. Proteins interactions evaluated by conventional yeast two-hybrid assay. (B) Increased autophagic flux was also observed with construct containing 4xMyc tag. (C) Fusion of Atg1 kinase domain to PAS scaffold proteins did not enhance autophagy. (D) Fusion of Atg1 kinase domain to other proteins functioning in autophagy did not enhance autophagy. (B-D) Autophagic flux examined by the pho8Δ60 assay. Mean ± standard deviation, n = 3 independent repeats.
Extended Data Fig. 8 Impact of KD-Atg8 on various branches of autophagy machinery.
(A-D) Subcellular distribution of 2GFP tagged proteins examined by fluorescent microscopy (A) Representative image snapshots (max intensity projection for fluorescent channel). Scale bar, 5 um. (B) Numbers of Atg protein puncta per cell. Mean ± standard deviation, n = 3 independent repeats. (C) Brightness of Atg protein puncta. In the case of Atg9, only the bright puncta in cells displaying concentrated Atg9 were measured. Sina plot data summed from 3 independent repeats, with mean ± standard deviation marked. (D) Fractions of cells containing bright Atg9 puncta. Mean ± standard deviation, n = 3 independent repeats. (E) Phosphorylation of Atg1 substrate proteins evaluated by phos-tag gel. Cells were nitrogen-starved for 2 h. Representative immunoblots from phos-tag gel (top row) and regular gel (bottom row).
Supplementary information
Supplementary Information
Supplementary Note 1: Additional information on cell lines used in this study. Supplementary Fig. 1: Additional data.
Supplementary Tables 1–5
Supplementary Table 1: Plasmids. Supplementary Table 2: Strains. Supplementary Table 3: Primers. Supplementary Table 4: Antibodies. Supplementary Table 5: Reagents.
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Source Data Figs. 1–8 and Extended Data Figs. 1–4 and 6–8
Statistical source data.
Source Data Figs. 1–4 and 6–8 and Extended Data Figs. 2, 4–6 and 8
Unprocessed western blots.
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Song, JZ., Li, H., Yang, H. et al. Recruitment of Atg1 to the phagophore by Atg8 orchestrates autophagy machineries. Nat Struct Mol Biol 32, 1606–1621 (2025). https://doi.org/10.1038/s41594-025-01546-0
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DOI: https://doi.org/10.1038/s41594-025-01546-0