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Recycling of autophagosomal components from autolysosomes by the recycler complex

Nature Cell Biology volume 24, pages 497–512 (2022)Cite this article

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Abstract

Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed ‘recycler’. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein–dynactin complex and the SNX4–SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.

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Fig. 1: STX17 is retrieved from autolysosomes.
Fig. 2: SNX4 is required for STX17 recycling from autolysosomes.
Fig. 3: SNX5 is required for STX17 recycling from autolysosomes.
Fig. 4: SNX5 cooperates with SNX4 to mediate STX17 sorting from autolysosomes.
Fig. 5: SNX17 interacts with both the STX17–SNX4–SNX5 and dynein–dynactin complexes to facilitate STX17 retrieval.
Fig. 6: SNX4–SNX5–SNX17 forms the recycler complex.
Fig. 7: ATG9A is retrieved from autolysosomes by recycler.
Fig. 8: Inhibited autophagic flux in recycler-deficient cells is rescued by SNX4, but not SNX4 Y252A, cells.

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Data availability

Source data are provided with this paper. The authors declare that all relevant data supporting the findings of this study are available within the paper and its Supplementary Information files. The proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031183.

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Acknowledgements

We are deeply grateful to L. Yu (Tsinghua University), Q. Zhong (Shanghai Jiao Tong University), W. Liu (Zhejiang University), Q. Sun (Zhejiang University) and L. Ge (Tsinghua University) for helpful suggestions on this study. We thank J. Liu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for gifting plasmids. The work was supported by grants from NSFC 91854116 and 31771529 (to Y.R.) and the Junior Thousand Talents Program of China (to Y.R.). The work was partially supported by the Fundamental Research Funds for the Central Universities 5003510089 (to Y.R.).

Author information

Author notes

  1. These authors contributed equally: Chuchu Zhou, Zhe Wu, Wanqing Du.

Authors and Affiliations

  1. School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Chuchu Zhou, Zhe Wu, Huilin Que, Yufen Wang, Fenglei Jian, Weigang Yuan, Yuan Zhao, Rui Tian & Yueguang Rong

  2. The State Key Laboratory of Membrane Biology, Tsinghua University–Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Wanqing Du & Ying Li

  3. College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China

    Qinqin Ouyang

  4. Center for Precision Medicine Multi-Omics Research, Peking University Health Science Center, Peking University, Beijing, China

    Yang Chen, Shuaixin Gao & Catherine C. L. Wong

  5. School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China

    Yang Chen, Shuaixin Gao & Catherine C. L. Wong

  6. Cell Architecture Research Center, Huazhong University of Science and Technology, Wuhan, China

    Yueguang Rong

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  1. Chuchu Zhou
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Contributions

Y.R., C.Z. and Z.W. conceived and designed the experiments. C.Z., Z.W., H.Q. and Y.W. performed the biological and biochemical experiments. W.D. carried out the in vitro experiments. C.Z., Z.W., W.D., H.Q., Y.W. and Y.R. analysed the data and wrote the manuscript with the help of all authors.

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Correspondence to Yueguang Rong.

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Extended data

Extended Data Fig. 1 GFP-STX17-TM fails to get to lysosomes in FIP200 knock-out cells and ATG9A knock-out cells.

a and c, The delivery of STX17 to autolysosomes was blocked in FIP200-KO cells. Wild-type or FIP200-KO MEF cells stably expressing GFP-STX17-TM were starved with EBSS for 2 hours in a or treated with bafilomycin A1 (100 nM) for another 3 hours after 2 hours EBSS starvation in c. Cells were stained with antibodies against GFP and LAMP1. Scale bar, 5 μm. Inset scale bar, 2 μm. b and d, Images from a and c were analyzed, data are means ± s.d. (n = 3, 50 cells from 3 independent experiments were quantified). Unpaired two-tailed t-test. e and g, The delivery of STX17 to autolysosomes was blocked in ATG9-KO cells. Wild-type or ATG9-KO Hela cells stably expressing GFP-STX17-TM were starved with EBSS for 2 hours in e or treated with bafilomycin A1 (100 nM) for another 3 hours after 2 hours EBSS starvation in g. Then cells were stained with antibodies against GFP and LAMP1. Scale bar, 5 μm. Inset scale bar, 2 μm. f and h, Images e and g were analyzed. Data are means ± s.d. (n = 3, 50 cells from 3 independent experiments were quantified). Unpaired two-tailed t-test. Source numerical data are available in source data.

Source data

Extended Data Fig. 2 The full length STX17 fails to get to lysosomes in FIP200 knock-out cells and ATG9A knock-out cells.

a and c, The delivery of STX17 to autolysosomes is blocked in FIP200-KO cells. Wild-type or FIP200-KO MEF cells stably expressing Flag-STX17 were starved with EBSS for 2 hours in a or treated with bafilomycin A1 (100 nM) for another 3 hours after 2 hours EBSS starvation in c. Cells were stained with antibodies against Flag and LAMP1. Scale bar, 5 μm. Inset scale bar, 2 μm. b and d, Images from a and c were analyzed, data are means ± s.d. (n = 3, 50 cells from 3 independent experiments were quantified). Unpaired two-tailed t-test. e and g, The delivery of STX17 to autolysosomes is blocked in ATG9A-KO cells. Wild-type or ATG9-KO Hela cells stably expressing Flag-STX17 were starved with EBSS for 2 hours in e or treated with bafilomycin A1 (100 nM) for another 3 hours after 2 hours EBSS starvation in g. Then cells were stained with antibodies against Flag and LAMP1. Scale bar, 5 μm. Inset scale bar, 2 μm. f and h, Images from e and g are analyzed. Data are means ± s.d. (n = 3, 50 cells from 3 independent experiments were quantified). Unpaired two-tailed t-test. Source numerical data are available in source data.

Source data

Extended Data Fig. 3 STX17 is recycled from autolysosomes.

a, STX17 is recycled from autolysosomes. U2OS cells stably expressing GFP-STX17 TM, BFP-LC3 were starved for 2 h with EBSS and stained with LysoTracker. Time-lapse images were taken. Selected frames were shown as indicated time points. Scale bar, 1 μm. b-c, The recycling events and recycling frequency on autolysosomes were analyzed. Data are presented as mean values ± s.e.m., (n = 41 biologically independent experiments). d-f, Hela cells, HepG2 cells and COS-7 cells were transfected with GFP-STX17-TM. Twenty-four hours after transfection, cells stained with LysoTracker were starved and time-lapse images were taken. Selected frames were shown as indicated time points. Scale bar, 500 nm. Source numerical data are available in source data.

Source data

Extended Data Fig. 4 ALR genes are not required for STX17 recycling from autolysosomes.

a, ALR genes depletion has no effect on STX17 recycling from autolysosomes. U2OS cells stably expressing Flag-STX17 were transfected with indicated siRNAs. Forty-eight hours after transfection, cells were starved with EBSS for indicated hours and stained with antibodies against Flag and LAMP1. Scale bar, 5 μm. Inset scale bar, 2 μm. b, Quantification of STX17 positive autolysosomes in a. Data are means ± s.e.m. (n = 3, 50 cells from 3 independent experiments were quantified). Unpaired two-tailed t-test. Source numerical data are available in source data.

Source data

Extended Data Fig. 5 Bafilomycin A1 leads to STX17 entrance into autolysosomes.

a, The relative fluorescent intensity of STX17 and LysoTracker on autolysosomes and newly generated STX17 vesicles. Selected images from Fig. 1g were analyzed. Inset scale bar, 2 μm. b, U2OS cells stably expressing GFP-STX17-TM and LAMP1-mCherry were starved with EBSS for 2 h, then cells were treated with or without bafilomycin A1 for another 6 h. Scale bar, 5 μm. Inset scale bar, 2 μm. c, Percentage of cells with STX17 inside autolysosomes. Images in b were analyzed. Data are means ± s.d. (n = 3, 50 cells from 3 independent experiments were quantified), unpaired two-tailed t-test. d, The relative fluorescent intensity of STX17 and LAMP1 on autolysosomes. Selected images from b were analyzed. Inset scale bar, 2 μm. Source numerical data are available in source data.

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Extended Data Fig. 6 The localization of sorting nexins.

The localization of sorting nexins. U2OS cells stably expressing GFP-STX17-TM and LAMP1-CFP were transfected with mCherry-SNX fusion proteins. Twenty-four hours after transfection, cells were starved with EBSS for 2 hours and images were taken. Scale bar, 5 μm. Inset scale bar, 2 μm.

Extended Data Fig. 7 The effect of sorting nexins on STX17 recycling from autolysosomes.

The effect of SNXs on STX17 recycling from autolysosomes. U2OS cells stably expressing Flag-STX17 were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were starved with EBSS for 5 hours and stained with antibodies against Flag and LAMP1. Scale bar, 5 μm. Inset scale bar, 2 μm.

Extended Data Fig. 8 KIBRA, SNX7, and SNX30 are not required for STX17 recycling from autolysosomes.

a-c, U2OS cells stably expressing Flag-STX17 were transfected with indicated siRNAs. Forty-eight hours after transfection, cells were subjected to immunoblot with antibodies against SNX4, KIBRA and SNX30. d, Representative mRNA level for the knockdown efficiency of SNX7. Data are presented as mean values ± s.d. e, The depletion of KIBRA, SNX7, and SNX30 has no effect on STX17 recycling from autolysosomes. U2OS cells stably expressing Flag-STX17 were transfected with non-targeting siRNA (NC) or siRNAs against KIBRA, SNX7, and SNX30. Forty-eight hours after transfection, cells were starved with EBSS for the indicated hours. Scale bar, 5 μm. Inset scale bar, 2 μm. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 9 Retromer is not required for STX17 recycling from autolysosomes.

a-c, U2OS cells stably expressing Flag-STX17 were transfected with indicated siRNAs. Forty-eight hours after transfection, cells were subjected to immunoblot with antibodies against SNX5, VPS35 and SNX6. d, Depletion of VPS35 and SNX6 has no effect on STX17 recycling from autolysosomes. U2OS cells stably expressing Flag-STX17 were transfected with non-targeting siRNA (NC) or siRNAs against VPS35 and SNX6. Forty-eight hours after transfection, cells were starved with EBSS for the indicated hours. Scale bar, 5 μm. Inset scale bar, 2 μm. Source unprocessed blots are available in source data.

Source data

Extended Data Fig. 10 SNX4, SNX5 and SNX17 are required for STX17 recycling from autolysosomes.

a, Depletion of SNX4, SNX5 and SNX17 causes STX17 recycling defect. U2OS cells stably expressing Flag-STX17 were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were starved with or without EBSS for indicated duration and stained with antibodies against Flag, LAMP1 and LC3. Scale bar, 5 μm. Inset scale bar, 2 μm. b, Quantification of the number of STX17-positive autolysosomes. Images in a were analyzed. Data are means ± s.d. (n = 3, 50 cells from 3 independent experiments were quantified). Unpaired two-tailed t-test. c, U2OS cells stably expressing Flag-STX17 were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were subjected to immunoblot with antibodies against SNX4, SNX5 and SNX17. * indicates a non-specific band. Source numerical data and unprocessed blots are available in source data.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–21 and figure legend.

Reporting Summary

Supplementary Video 1

STX17 is retrieved from autolysosomes.

Supplementary Tables

Supplementary Tables 1–3.

Supplementary Data

Statistical source data for Additional Supplementary Figures.

Supplementary Data

Unprocessed western blots for additional supplementary figures.

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Zhou, C., Wu, Z., Du, W. et al. Recycling of autophagosomal components from autolysosomes by the recycler complex. Nat Cell Biol 24, 497–512 (2022). https://doi.org/10.1038/s41556-022-00861-8

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