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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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.
References
Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132 (2011).
Bento, C. F. et al. Mammalian autophagy: how does it work? Annu. Rev. Biochem. 85, 685–713 (2016).
Yu, L., Chen, Y. & Tooze, S. A. Autophagy pathway: cellular and molecular mechanisms. Autophagy 14, 207–215 (2018).
Dikic, I. & Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349–364 (2018).
Zhao, Y. G. & Zhang, H. Core autophagy genes and human diseases. Curr. Opin. Cell Biol. 61, 117–125 (2019).
Mizushima, N. The ATG conjugation systems in autophagy. Curr. Opin. Cell Biol. 63, 1–10 (2019).
Levine, B. & Kroemer, G. Biological functions of autophagy genes: a disease perspective. Cell 176, 11–42 (2019).
Poillet-Perez, L. & White, E. Role of tumor and host autophagy in cancer metabolism. Genes Dev. 33, 610–619 (2019).
Yang, Y. & Klionsky, D. J. Autophagy and disease: unanswered questions. Cell Death Differ. 27, 858–871 (2020).
Gomez-Sanchez, R., Tooze, S. A. & Reggiori, F. Membrane supply and remodeling during autophagosome biogenesis. Curr. Opin. Cell Biol. 71, 112–119 (2021).
Axe, E. L. et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701 (2008).
Hayashi-Nishino, M. et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat. Cell Biol. 11, 1433–1437 (2009).
Yla-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5, 1180–1185 (2009).
Zoppino, F. C., Militello, R. D., Slavin, I., Alvarez, C. & Colombo, M. I. Autophagosome formation depends on the small GTPase Rab1 and functional ER exit sites. Traffic 11, 1246–1261 (2010).
Graef, M., Friedman, J. R., Graham, C., Babu, M. & Nunnari, J. ER exit sites are physical and functional core autophagosome biogenesis components. Mol. Biol. Cell 24, 2918–2931 (2013).
Hailey, D. W. et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 141, 656–667 (2010).
Ravikumar, B., Moreau, K., Jahreiss, L., Puri, C. & Rubinsztein, D. C. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 12, 747–757 (2010).
Moreau, K., Ravikumar, B., Renna, M., Puri, C. & Rubinsztein, D. C. Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303–317 (2011).
Young, A. R. et al. Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes. J. Cell Sci. 119, 3888–3900 (2006).
Geng, J., Nair, U., Yasumura-Yorimitsu, K. & Klionsky, D. J. Post-Golgi Sec proteins are required for autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 21, 2257–2269 (2010).
Guo, Y. et al. AP1 is essential for generation of autophagosomes from the trans-Golgi network. J. Cell Sci. 125, 1706–1715 (2012).
Longatti, A. et al. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J. Cell Biol. 197, 659–675 (2012).
Ge, L., Melville, D., Zhang, M. & Schekman, R. The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. eLife 2, e00947 (2013).
Ge, L., Zhang, M. & Schekman, R. Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER–Golgi intermediate compartment. eLife 3, e04135 (2014).
Ge, L. et al. Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis. EMBO Rep. 18, 1586–1603 (2017).
Yu, L. et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946 (2010).
Rong, Y. et al. Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation. Proc. Natl Acad. Sci. USA 108, 7826–7831 (2011).
Rong, Y. et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat. Cell Biol. 14, 924–934 (2012).
Du, W. et al. Kinesin 1 drives autolysosome tubulation. Dev. Cell 37, 326–336 (2016).
Itakura, E., Kishi-Itakura, C. & Mizushima, N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151, 1256–1269 (2012).
Takats, S. et al. Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. J. Cell Biol. 201, 531–539 (2013).
Tsuboyama, K. et al. The ATG conjugation systems are important for degradation of the inner autophagosomal membrane. Science 354, 1036–1041 (2016).
Uematsu, M., Nishimura, T., Sakamaki, Y., Yamamoto, H. & Mizushima, N. Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants. Autophagy 13, 1452–1464 (2017).
Traer, C. J. et al. SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment. Nat. Cell Biol. 9, 1370–1380 (2007).
van Weering, J. R. et al. Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules. EMBO J. 31, 4466–4480 (2012).
Anton, Z. et al. A heterodimeric SNX4–SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly. J. Cell Sci. 133, jcs246306 (2020).
Arighi, C. N., Hartnell, L. M., Aguilar, R. C., Haft, C. R. & Bonifacino, J. S. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J. Cell Biol. 165, 123–133 (2004).
Wassmer, T. et al. A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer. J. Cell Sci. 120, 45–54 (2007).
Cullen, P. J. Endosomal sorting and signalling: an emerging role for sorting nexins. Nat. Rev. Mol. Cell Biol. 9, 574–582 (2008).
Wassmer, T. et al. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Dev. Cell 17, 110–122 (2009).
Cullen, P. J. & Steinberg, F. To degrade or not to degrade: mechanisms and significance of endocytic recycling. Nat. Rev. Mol. Cell Biol. 19, 679–696 (2018).
Chen, K. E., Healy, M. D. & Collins, B. M. Towards a molecular understanding of endosomal trafficking by Retromer and Retriever. Traffic 20, 465–478 (2019).
Peter, B. J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).
McMahon, H. T. & Gallop, J. L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).
Suetsugu, S., Toyooka, K. & Senju, Y. Subcellular membrane curvature mediated by the BAR domain superfamily proteins. Semin. Cell Dev. Biol. 21, 340–349 (2010).
van Kerkhof, P. et al. Sorting nexin 17 facilitates LRP recycling in the early endosome. EMBO J. 24, 2851–2861 (2005).
Steinberg, F., Heesom, K. J., Bass, M. D. & Cullen, P. J. SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways. J. Cell Biol. 197, 219–230 (2012).
McNally, K. E. et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat. Cell Biol. 19, 1214–1225 (2017).
Engelender, S. et al. Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin. Hum. Mol. Genet. 6, 2205–2212 (1997).
Hoogenraad, C. C. et al. Mammalian Golgi-associated Bicaudal-D2 functions in the dynein–dynactin pathway by interacting with these complexes. EMBO J. 20, 4041–4054 (2001).
Holleran, E. A. et al. beta III spectrin binds to the Arp1 subunit of dynactin. J. Biol. Chem. 276, 36598–36605 (2001).
Jordens, I. et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein–dynactin motors. Curr. Biol. 11, 1680–1685 (2001).
Matanis, T. et al. Bicaudal-D regulates COPI-independent Golgi–ER transport by recruiting the dynein–dynactin motor complex. Nat. Cell Biol. 4, 986–992 (2002).
Hoogenraad, C. C. et al. Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport. EMBO J. 22, 6004–6015 (2003).
Gauthier, L. R. et al. Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell 118, 127–138 (2004).
Johansson, M. et al. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin. J. Cell Biol. 176, 459–471 (2007).
Hong, Z. et al. The retromer component SNX6 interacts with dynactin p150Glued and mediates endosome-to-TGN transport. Cell Res. 19, 1334–1349 (2009).
Matsui, T. et al. Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17. J. Cell Biol. 217, 2633–2645 (2018).
Yamamoto, H. et al. Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J. Cell Biol. 198, 219–233 (2012).
Orsi, A. et al. Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol. Biol. Cell 23, 1860–1873 (2012).
Reggiori, F. & Tooze, S. A. Autophagy regulation through Atg9 traffic. J. Cell Biol. 198, 151–153 (2012).
Klionsky, D. J., Elazar, Z., Seglen, P. O. & Rubinsztein, D. C. Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4, 849–850 (2008).
Rojas, R., Kametaka, S., Haft, C. R. & Bonifacino, J. S. Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol. Cell. Biol. 27, 1112–1124 (2007).
Weeratunga, S., Paul, B. & Collins, B. M. Recognising the signals for endosomal trafficking. Curr. Opin. Cell Biol. 65, 17–27 (2020).
Kvainickas, A. et al. Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport. J. Cell Biol. 216, 3677–3693 (2017).
Simonetti, B., Danson, C. M., Heesom, K. J. & Cullen, P. J. Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR. J. Cell Biol. 216, 3695–3712 (2017).
Elwell, C. A. et al. Chlamydia interfere with an interaction between the mannose-6-phosphate receptor and sorting nexins to counteract host restriction. eLife 6, e22709 (2017).
Simonetti, B. et al. Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins. Nat. Cell Biol. 21, 1219–1233 (2019).
Griffin, C. T., Trejo, J. & Magnuson, T. Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2. Proc. Natl Acad. Sci. USA 102, 15173–15177 (2005).
Ma, M., Burd, C. G. & Chi, R. J. Distinct complexes of yeast Snx4 family SNX-BARs mediate retrograde trafficking of Snc1 and Atg27. Traffic 18, 134–144 (2017).
Suzuki, S. W. & Emr, S. D. Membrane protein recycling from the vacuole/lysosome membrane. J. Cell Biol. 217, 1623–1632 (2018).
Ravussin, A., Brech, A., Tooze, S. A. & Stenmark, H. The phosphatidylinositol 3-phosphate-binding protein SNX4 controls ATG9A recycling and autophagy. J. Cell Sci. 134, jcs250670 (2021).
Hamasaki, M. et al. Autophagosomes form at ER–mitochondria contact sites. Nature 495, 389–393 (2013).
Kumar, S. et al. Phosphorylation of syntaxin 17 by TBK1 controls autophagy initiation. Dev. Cell 49, 130–144 e136 (2019).
Kumar, S. et al. Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins. J. Cell Biol. 217, 997–1013 (2018).
De Tito, S., Hervas, J. H., van Vliet, A. R. & Tooze, S. A. The Golgi as an assembly line to the autophagosome. Trends Biochem. Sci 45, 484–496 (2020).
Merino-Trigo, A. et al. Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation. J. Cell Sci. 117, 6413–6424 (2004).
Leprince, C. et al. Sorting nexin 4 and amphiphysin 2, a new partnership between endocytosis and intracellular trafficking. J. Cell Sci. 116, 1937–1948 (2003).
Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat. Protoc. 11, 456–475 (2016).
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.).
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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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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.
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.
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.
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.
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.
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.
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.
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.
Supplementary information
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STX17 is retrieved from autolysosomes.
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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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DOI: https://doi.org/10.1038/s41556-022-00861-8
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