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• W2912641855 abstract "•ATL3 is an ER-phagy receptor and promotes tubular ER degradation•ATL3 specifically binds to GABARAP via 2 GABARAP interaction motifs (GIMs)•Sensory neuropathy-associated ATL3 mutations reduce ATL3-GABARAP binding•Sensory neuropathy-associated ATL3 mutations impair ATL3’s function in ER-phagy The endoplasmic reticulum (ER) consists of the nuclear envelope and both peripheral ER sheets and a peripheral tubular network [1Chen S. Novick P. Ferro-Novick S. ER structure and function.Curr. Opin. Cell Biol. 2013; 25: 428-433Crossref PubMed Scopus (120) Google Scholar, 2Nixon-Abell J. Obara C.J. Weigel A.V. Li D. Legant W.R. Xu C.S. Pasolli H.A. Harvey K. Hess H.F. Betzig E. et al.Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.Science. 2016; 354: aaf3928Crossref PubMed Scopus (260) Google Scholar]. In response to physiological or pathological conditions, receptor-mediated selective ER-phagy, engulfing specific ER subdomains or components, is essential for ER turnover and homeostasis [3Dikic I. Open questions: why should we care about ER-phagy and ER remodelling?.BMC Biol. 2018; 16: 131Crossref PubMed Scopus (31) Google Scholar, 4Grumati P. Dikic I. Stolz A. ER-phagy at a glance.J. Cell Sci. 2018; 131: jcs217364Crossref PubMed Scopus (110) Google Scholar, 5Loi M. Fregno I. Guerra C. Molinari M. Eat it right: ER-phagy and recovER-phagy.Biochem. Soc. Trans. 2018; 46: 699-706Crossref PubMed Scopus (33) Google Scholar, 6Fregno I. Molinari M. Endoplasmic reticulum turnover: ER-phagy and other flavors in selective and non-selective ER clearance.F1000Res. 2018; 7: 454Crossref PubMed Scopus (43) Google Scholar]. Four mammalian receptors for ER-phagy have been reported: FAM134B [7Khaminets A. Heinrich T. Mari M. Grumati P. Huebner A.K. Akutsu M. Liebmann L. Stolz A. Nietzsche S. Koch N. et al.Regulation of endoplasmic reticulum turnover by selective autophagy.Nature. 2015; 522: 354-358Crossref PubMed Scopus (538) Google Scholar], reticulon 3 (RTN3) [8Grumati P. Morozzi G. Hölper S. Mari M. Harwardt M.I. Yan R. Müller S. Reggiori F. Heilemann M. Dikic I. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy.eLife. 2017; 6: e25555Crossref PubMed Scopus (219) Google Scholar], SEC62 [9Fumagalli F. Noack J. Bergmann T.J. Cebollero E. Pisoni G.B. Fasana E. Fregno I. Galli C. Loi M. Soldà T. et al.Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery.Nat. Cell Biol. 2016; 18: 1173-1184Crossref PubMed Scopus (245) Google Scholar], and CCPG1 [10Smith M.D. Harley M.E. Kemp A.J. Wills J. Lee M. Arends M. von Kriegsheim A. Behrends C. Wilkinson S. CCPG1 is a non-canonical autophagy cargo receptor essential for ER-phagy and pancreatic ER proteostasis.Dev. Cell. 2018; 44: 217-232.e11Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar]. However, these ER-phagy receptors function in subcellular- and tissue- or physiological- and pathological-condition-specific manners, so the diversity of ER-phagy receptors and underlying mechanisms remain largely unknown [3Dikic I. Open questions: why should we care about ER-phagy and ER remodelling?.BMC Biol. 2018; 16: 131Crossref PubMed Scopus (31) Google Scholar, 4Grumati P. Dikic I. Stolz A. ER-phagy at a glance.J. Cell Sci. 2018; 131: jcs217364Crossref PubMed Scopus (110) Google Scholar]. Atlastins (ATL1, ATL2, and ATL3), in mammals, are a class of membrane-bound, dynamin-like GTPases that function in ER fusion [11Hu J. Shibata Y. Zhu P.P. Voss C. Rismanchi N. Prinz W.A. Rapoport T.A. Blackstone C. A class of dynamin-like GTPases involved in the generation of the tubular ER network.Cell. 2009; 138: 549-561Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 12Orso G. Pendin D. Liu S. Tosetto J. Moss T.J. Faust J.E. Micaroni M. Egorova A. Martinuzzi A. McNew J.A. Daga A. Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin.Nature. 2009; 460: 978-983Crossref PubMed Scopus (340) Google Scholar]. ATL1 is expressed mainly in the central nervous system, while ATL2 and ATL3 are more ubiquitously distributed [13Rismanchi N. Soderblom C. Stadler J. Zhu P.P. Blackstone C. Atlastin GTPases are required for Golgi apparatus and ER morphogenesis.Hum. Mol. Genet. 2008; 17: 1591-1604Crossref PubMed Scopus (157) Google Scholar]. Recent studies showed that ATL2 mainly affects ER morphology by promoting ER fusion, whereas alterations in ER morphology are hardly detectable after ATL3 depletion [14Hu X. Wu F. Sun S. Yu W. Hu J. Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.Protein Cell. 2015; 6: 307-311Crossref PubMed Scopus (21) Google Scholar, 15Pawar S. Ungricht R. Tiefenboeck P. Leroux J.C. Kutay U. Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.eLife. 2017; 6: e28202Crossref PubMed Scopus (22) Google Scholar]. Here, we show that ATL3 functions as a receptor for ER-phagy, promoting tubular ER degradation upon starvation. ATL3 specifically binds to GABARAP, but not LC3, subfamily proteins via 2 GABARAP interaction motifs (GIMs). ATL3-GABARAP interaction is essential for ATL3 to function in ER-phagy. Moreover, hereditary sensory and autonomic neuropathy type I (HSAN I)-associated ATL3 mutations (Y192C and P338R) disrupt ATL3’s association with GABARAP and impair ATL3’s function in ER-phagy, suggesting that defective ER-phagy is involved in HSAN I. Therefore, we reveal a new ATL3 function for GABARAP-mediated ER-phagy in the degradation of tubular ER. The endoplasmic reticulum (ER) consists of the nuclear envelope and both peripheral ER sheets and a peripheral tubular network [1Chen S. Novick P. Ferro-Novick S. ER structure and function.Curr. Opin. Cell Biol. 2013; 25: 428-433Crossref PubMed Scopus (120) Google Scholar, 2Nixon-Abell J. Obara C.J. Weigel A.V. Li D. Legant W.R. Xu C.S. Pasolli H.A. Harvey K. Hess H.F. Betzig E. et al.Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.Science. 2016; 354: aaf3928Crossref PubMed Scopus (260) Google Scholar]. In response to physiological or pathological conditions, receptor-mediated selective ER-phagy, engulfing specific ER subdomains or components, is essential for ER turnover and homeostasis [3Dikic I. Open questions: why should we care about ER-phagy and ER remodelling?.BMC Biol. 2018; 16: 131Crossref PubMed Scopus (31) Google Scholar, 4Grumati P. Dikic I. Stolz A. ER-phagy at a glance.J. Cell Sci. 2018; 131: jcs217364Crossref PubMed Scopus (110) Google Scholar, 5Loi M. Fregno I. Guerra C. Molinari M. Eat it right: ER-phagy and recovER-phagy.Biochem. Soc. Trans. 2018; 46: 699-706Crossref PubMed Scopus (33) Google Scholar, 6Fregno I. Molinari M. Endoplasmic reticulum turnover: ER-phagy and other flavors in selective and non-selective ER clearance.F1000Res. 2018; 7: 454Crossref PubMed Scopus (43) Google Scholar]. Four mammalian receptors for ER-phagy have been reported: FAM134B [7Khaminets A. Heinrich T. Mari M. Grumati P. Huebner A.K. Akutsu M. Liebmann L. Stolz A. Nietzsche S. Koch N. et al.Regulation of endoplasmic reticulum turnover by selective autophagy.Nature. 2015; 522: 354-358Crossref PubMed Scopus (538) Google Scholar], reticulon 3 (RTN3) [8Grumati P. Morozzi G. Hölper S. Mari M. Harwardt M.I. Yan R. Müller S. Reggiori F. Heilemann M. Dikic I. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy.eLife. 2017; 6: e25555Crossref PubMed Scopus (219) Google Scholar], SEC62 [9Fumagalli F. Noack J. Bergmann T.J. Cebollero E. Pisoni G.B. Fasana E. Fregno I. Galli C. Loi M. Soldà T. et al.Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery.Nat. Cell Biol. 2016; 18: 1173-1184Crossref PubMed Scopus (245) Google Scholar], and CCPG1 [10Smith M.D. Harley M.E. Kemp A.J. Wills J. Lee M. Arends M. von Kriegsheim A. Behrends C. Wilkinson S. CCPG1 is a non-canonical autophagy cargo receptor essential for ER-phagy and pancreatic ER proteostasis.Dev. Cell. 2018; 44: 217-232.e11Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar]. However, these ER-phagy receptors function in subcellular- and tissue- or physiological- and pathological-condition-specific manners, so the diversity of ER-phagy receptors and underlying mechanisms remain largely unknown [3Dikic I. Open questions: why should we care about ER-phagy and ER remodelling?.BMC Biol. 2018; 16: 131Crossref PubMed Scopus (31) Google Scholar, 4Grumati P. Dikic I. Stolz A. ER-phagy at a glance.J. Cell Sci. 2018; 131: jcs217364Crossref PubMed Scopus (110) Google Scholar]. Atlastins (ATL1, ATL2, and ATL3), in mammals, are a class of membrane-bound, dynamin-like GTPases that function in ER fusion [11Hu J. Shibata Y. Zhu P.P. Voss C. Rismanchi N. Prinz W.A. Rapoport T.A. Blackstone C. A class of dynamin-like GTPases involved in the generation of the tubular ER network.Cell. 2009; 138: 549-561Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 12Orso G. Pendin D. Liu S. Tosetto J. Moss T.J. Faust J.E. Micaroni M. Egorova A. Martinuzzi A. McNew J.A. Daga A. Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin.Nature. 2009; 460: 978-983Crossref PubMed Scopus (340) Google Scholar]. ATL1 is expressed mainly in the central nervous system, while ATL2 and ATL3 are more ubiquitously distributed [13Rismanchi N. Soderblom C. Stadler J. Zhu P.P. Blackstone C. Atlastin GTPases are required for Golgi apparatus and ER morphogenesis.Hum. Mol. Genet. 2008; 17: 1591-1604Crossref PubMed Scopus (157) Google Scholar]. Recent studies showed that ATL2 mainly affects ER morphology by promoting ER fusion, whereas alterations in ER morphology are hardly detectable after ATL3 depletion [14Hu X. Wu F. Sun S. Yu W. Hu J. Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.Protein Cell. 2015; 6: 307-311Crossref PubMed Scopus (21) Google Scholar, 15Pawar S. Ungricht R. Tiefenboeck P. Leroux J.C. Kutay U. Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.eLife. 2017; 6: e28202Crossref PubMed Scopus (22) Google Scholar]. Here, we show that ATL3 functions as a receptor for ER-phagy, promoting tubular ER degradation upon starvation. ATL3 specifically binds to GABARAP, but not LC3, subfamily proteins via 2 GABARAP interaction motifs (GIMs). ATL3-GABARAP interaction is essential for ATL3 to function in ER-phagy. Moreover, hereditary sensory and autonomic neuropathy type I (HSAN I)-associated ATL3 mutations (Y192C and P338R) disrupt ATL3’s association with GABARAP and impair ATL3’s function in ER-phagy, suggesting that defective ER-phagy is involved in HSAN I. Therefore, we reveal a new ATL3 function for GABARAP-mediated ER-phagy in the degradation of tubular ER. To reveal whether ATL2 and ATL3, the atlastins (ATLs) in COS-7 cells [14Hu X. Wu F. Sun S. Yu W. Hu J. Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.Protein Cell. 2015; 6: 307-311Crossref PubMed Scopus (21) Google Scholar, 16Wu F. Hu X. Bian X. Liu X. Hu J. Comparison of human and Drosophila atlastin GTPases.Protein Cell. 2015; 6: 139-146Crossref PubMed Scopus (22) Google Scholar], function in endoplasmic reticulum (ER) turnover, we first generated 2 single-knockout (KO) COS-7 cell lines for ATL2 and ATL3 using CRISPR/Cas9 approaches [17Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (6406) Google Scholar] (Figure S1A). Consistent with previous reports [14Hu X. Wu F. Sun S. Yu W. Hu J. Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.Protein Cell. 2015; 6: 307-311Crossref PubMed Scopus (21) Google Scholar, 15Pawar S. Ungricht R. Tiefenboeck P. Leroux J.C. Kutay U. Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.eLife. 2017; 6: e28202Crossref PubMed Scopus (22) Google Scholar], ATL3 deficiency caused no obvious changes in the ER network, while the KO of ATL2 resulted in unbranched ER (Figure S1B). Notably, neither ATL2 nor ATL3 deficiency affected LC3B lipidation and p62 degradation upon Earle’s balanced salt solution (EBSS) starvation (Figures S1C–S1E), suggesting that autophagy flux is normal. However, upon starvation induced by EBSS, depletion of ATL3, but not ATL2, significantly inhibited the degradation of tubular ER membrane proteins, such as REEP5, REEP2, reticulon 4 (RTN4), and Calnexin (Figures 1A, 1C–1F, and S1F). In contrast, lack of ATL2, but not ATL3, inhibited the degradation of sheet ER membrane proteins (e.g., Kinectin [KTN1], TRAP-α, and Climp-63) upon starvation (Figures S1G–S1K), a result consistent with its role downstream of sheet ER-phagy receptor FAM134B [18Liang J.R. Lingeman E. Ahmed S. Corn J.E. Atlastins remodel the endoplasmic reticulum for selective autophagy.J. Cell Biol. 2018; 217: 3354-3367Crossref PubMed Scopus (24) Google Scholar]. ATL3 reconstitution in KO cells rescued the turnover of tubular ER membrane proteins (Figure 1G). ATL2 and ATL3 double KO did not exacerbate the inhibition of tubular ER turnover induced by ATL3 single KO (Figures 1B–1F). Also, when we labeled Climp-63 and REEP5 to represent ER sheets and tubular ER, respectively, the fluorescence intensity of Climp-63, but not REEP5, decreased in ATL3 KO COS-7 cells upon starvation (Figures S1L–S1N), suggesting that degradation of tubular ER, but not ER sheets, is inhibited in ATL3 KO COS-7 cells. These results indicate that ATL3 promotes tubular ER turnover. Next, we adopted a fluorescence reporter that fused REEP5, a tubular ER protein, with mCherry and GFP to monitor lysosomal delivery of the ER. When the ER was delivered into lysosomes, mCherry-GFP-REEP5 only emitted red fluorescence (mCherry-positive and GFP-negative puncta). mCherry-positive and GFP-negative puncta of REEP5 significantly increased upon starvation in wild-type (WT) COS-7 cells, and the KO of ATL3 inhibited this phenomenon (Figures 1H and 1I), suggesting that lysosomal delivery of the ER is inhibited in ATL3 KO COS-7 cells. Consistently, upon EBSS treatment in the presence of Bafilomycin A1, Calnexin was localized at Lamp1-positive autolysosomes (Figure S2A), and the KO of ATL3, but not of ATL2, decreased the localization of Calnexin at Lamp1-positive autolysosomes (Figure S2A). The localization of ATL3 at autolysosomes was further confirmed by immuno-electron microscopy (Figures 1J, S2B, and S2C). Taken altogether, these data suggest that ATL3 is a positive regulator of ER-phagy for tubular ER. A previous study showed that full-length RTN3 (RTN3L), but not short RTN3 isoforms (RTN3S/RTN3A), also functioned as a tubular ER-phagy receptor [8Grumati P. Morozzi G. Hölper S. Mari M. Harwardt M.I. Yan R. Müller S. Reggiori F. Heilemann M. Dikic I. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy.eLife. 2017; 6: e25555Crossref PubMed Scopus (219) Google Scholar], prompting us to examine the relationship between ATL3 and RTN3L. The protein level of RTN3S was very low, and RTN3L was barely detectable in COS-7 cells (Figure S2D), a result consistent with previous studies showing that RTN3L expresses only in some tissues and organs [19Di Scala F. Dupuis L. Gaiddon C. De Tapia M. Jokic N. Gonzalez de Aguilar J.L. Raul J.S. Ludes B. Loeffler J.P. Tissue specificity and regulation of the N-terminal diversity of reticulon 3.Biochem. J. 2005; 385: 125-134Crossref PubMed Scopus (37) Google Scholar, 20Shi Q. Ge Y. Sharoar M.G. He W. Xiang R. Zhang Z. Hu X. Yan R. Impact of RTN3 deficiency on expression of BACE1 and amyloid deposition.J. Neurosci. 2014; 34: 13954-13962Crossref PubMed Scopus (33) Google Scholar]. Coincidentally, overexpression of RTN3L, but not RTN3S, could rescue defective ER-phagy in ATL3 KO COS-7 cells (Figures S2E and S2F); ATL3 could interact with RTN3L, but not with the sheet ER-phagy receptor FAM134B (Figures S2G and S2H), suggesting that ATL3 and RTN3L are both tubular ER-phagy receptors [8Grumati P. Morozzi G. Hölper S. Mari M. Harwardt M.I. Yan R. Müller S. Reggiori F. Heilemann M. Dikic I. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy.eLife. 2017; 6: e25555Crossref PubMed Scopus (219) Google Scholar], and ATL3 is the main tubular ER-phagy receptor in cells lacking of RTN3L. To investigate how ATL3 regulates ER-phagy, we examined whether ATL3 interacts with the ATG8 family, a protein family that binds to autophagy receptors to mediate selective autophagy [21Ktistakis N.T. Tooze S.A. Digesting the expanding mechanisms of autophagy.Trends Cell Biol. 2016; 26: 624-635Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 22Deng Z. Purtell K. Lachance V. Wold M.S. Chen S. Yue Z. Autophagy receptors and neurodegenerative diseases.Trends Cell Biol. 2017; 27: 491-504Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. Immunoprecipitation assays showed that ATL3 interacted specifically with the ATG8 family’s GABARAP protein subfamily (GABARAP, GABARAPL1, and GABARAPL2), but not its LC3 subfamily (LC3A, LC3B, and LC3C), both endogenously and exogenously (Figures 2A and S3A–S3D), while ATL2 showed very weak interaction with GABARAP (Figure S3E). GST pull-down assays confirmed the interaction of ATL3 with GABARAPs (Figure 2B). We further purified GST-tagged GABARAPs and the His-tagged cytosolic domain of ATL3 (6×His-cytoATL3, residues 1–445) to perform pull-down assays in vitro. GST-GABARAPs bound to 6×His-cytoATL3 (Figure 2C), suggesting that ATL3 binds directly to GABARAPs. Moreover, upon starvation, the foci of GABARAP were colocalized with ATL3 and REEP5, a tubular ER marker (Figure 2D). We noticed that among the GABARAPs, ATL3 preferred to bind to GABARAP (Figures 2A–2C); therefore, we focused on GABARAP in subsequent studies. Bioinformatic analysis of the ATL3 protein sequence revealed the presence of 2 conserved GABARAP interaction motifs (GIMs) [23Rogov V.V. Stolz A. Ravichandran A.C. Rios-Szwed D.O. Suzuki H. Kniss A. Löhr F. Wakatsuki S. Dötsch V. Dikic I. et al.Structural and functional analysis of the GABARAP interaction motif (GIM).EMBO Rep. 2017; 18: 1382-1396Crossref PubMed Scopus (97) Google Scholar] in the N-terminal cytosolic domain: EYGRL (residues 191–195) and EFKQL (residues 389–393) (Figure 2E). Both GIMs were required for binding, as both immunoprecipitation and GST pull-down assays showed that binding to GABARAP was remarkably reduced with the mutation of either GIM: EYGRL191-195AAAAA (ATL3-GIM1m) or EFKQL389-393AAAAA (ATL3-GIM2m) (Figures 2F, 2G, and S3F). Moreover, just a single ATL3 residue substitution (E389A or K391A) at the second GIM obviously weakened ATL3’s association with GABARAP (Figures S3G and S3H). Mutation of GIMs also weakened the colocalization of ATL3 with GABARAP (Figure S3I). Furthermore, we quantified their colocalization by proximity ligation assay (PLA). The red PLA signal dots were acquired by probing for ATL-3×Flag and GFP-GABARAP, and they represented the colocalization between these 2 proteins. The number of red PLA signal dots in COS-7 cells expressing ATL3-GIM1/2 mutants was significantly less than that in COS-7 cells expressing WT ATL3 (Figures S3J and S3K). When engineering the second GIM of ATL3, a specific GIM, into ATL2, we found that ATL2 showed strong interaction with GABARAP (Figure S3L). These results suggest the importance of GIMs for ATL3-GABARAP interaction. In addition, a GTPase-defective ATL3 mutant, lacking all the GTP-binding motifs [13Rismanchi N. Soderblom C. Stadler J. Zhu P.P. Blackstone C. Atlastin GTPases are required for Golgi apparatus and ER morphogenesis.Hum. Mol. Genet. 2008; 17: 1591-1604Crossref PubMed Scopus (157) Google Scholar], was still able to interact with GABARAP (Figure S3M). Moreover, GABARAP mutations Y25A, K46A, and LL55/63AA apparently disrupted GABARAP’s association with substrates [23Rogov V.V. Stolz A. Ravichandran A.C. Rios-Szwed D.O. Suzuki H. Kniss A. Löhr F. Wakatsuki S. Dötsch V. Dikic I. et al.Structural and functional analysis of the GABARAP interaction motif (GIM).EMBO Rep. 2017; 18: 1382-1396Crossref PubMed Scopus (97) Google Scholar, 24Lystad A.H. Ichimura Y. Takagi K. Yang Y. Pankiv S. Kanegae Y. Kageyama S. Suzuki M. Saito I. Mizushima T. et al.Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures.EMBO Rep. 2014; 15: 557-565Crossref PubMed Scopus (76) Google Scholar, 25Genau H.M. Huber J. Baschieri F. Akutsu M. Dötsch V. Farhan H. Rogov V. Behrends C. CUL3-KBTBD6/KBTBD7 ubiquitin ligase cooperates with GABARAP proteins to spatially restrict TIAM1-RAC1 signaling.Mol. Cell. 2015; 57: 995-1010Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. Y25A severely impaired the interaction of GABARAP with ATL3 (Figures 2H and 2I), implying that Y25 is crucial for the interaction. To reveal the importance of the interaction between ATL3 and GABARAP in ER-phagy, we first examined whether the delivery of ATL3 into lysosomes is dependent on the interaction. We determined ATL3 localization at lysosomes in COS-7 cells expressing ATL3 fused with mCherry and GFP (mCherry-GFP-ATL3) after nutrient starvation in EBSS, as indicated by mCherry-positive and GFP-negative puncta (Figures 3A–3C) [7Khaminets A. Heinrich T. Mari M. Grumati P. Huebner A.K. Akutsu M. Liebmann L. Stolz A. Nietzsche S. Koch N. et al.Regulation of endoplasmic reticulum turnover by selective autophagy.Nature. 2015; 522: 354-358Crossref PubMed Scopus (538) Google Scholar]. Far fewer mCherry-positive and GFP-negative puncta were observed in cells expressing mCherry-GFP-ATL3 GIM mutants, which lacked GABARAP binding activity, than were observed in cells expressing WT ATL3 (Figures 3A–3C). Consistently, there were fewer puncta of ATL3 GIM mutants at Lamp1-positive autolysosomes than there were of WT ATL3 (Figure S4A). Furthermore, ATL3 reconstitution in KO cells rescued EBSS-induced ER-phagy, but ectopically expressed GABARAP-binding-deficient ATL3 GIM mutants failed to re-establish ER-phagy, as determined by degradation of REEP5, REEP2, RTN4, and Calnexin (Figures 3D–3H) and by lysosomal delivery of REEP5 and Calnexin (Figures 3I, S4B, and S4C). Consistent with the result above (Figure S3M), the GTPase-defective ATL3 mutant could rescue defective ER-phagy in ATL3 KO COS-7 cells (Figure S4D). These results demonstrate that ATL3 must bind to GABARAP through its GIMs for it to function in ER-phagy. GABARAP depletion in COS-7 cells also inhibited ER-phagy (Figures 3J and S4E–4J), and it could be rescued by WT GABARAP, but not by the GABARAP-Y25A mutant (Figures 3K and S4K–4N), which didn’t interact with ATL3. Evidently, for it to function in ER-phagy, ATL3 must associate with GABARAP. Recently, 2 mutations of ATL3 (Y192C and P338R) were identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN I) [26Fischer D. Schabhüttl M. Wieland T. Windhager R. Strom T.M. Auer-Grumbach M. A novel missense mutation confirms ATL3 as a gene for hereditary sensory neuropathy type 1.Brain. 2014; 137: e286Crossref PubMed Scopus (37) Google Scholar, 27Kornak U. Mademan I. Schinke M. Voigt M. Krawitz P. Hecht J. Barvencik F. Schinke T. Gießelmann S. Beil F.T. et al.Sensory neuropathy with bone destruction due to a mutation in the membrane-shaping atlastin GTPase 3.Brain. 2014; 137: 683-692Crossref PubMed Scopus (66) Google Scholar]. Coincidentally, the Y192C mutation was found in the first GIM (Figure 2E), prompting us to examine the function of these mutants in ER-phagy. We found that the interaction between ATL3 mutants (Y192C and P338R) and GABARAP was remarkably reduced, as shown by both immunoprecipitation and GST pull-down assays (Figures 4A and 4B ). Consistently, these ATL3 mutants would not reestablish EBSS-induced degradation of REEP5, REEP2, RTN4, and Calnexin in ATL3 KO COS-7 cells (Figures 4C–4G). Therefore, ATL3 mutation-induced dysfunction of ER-phagy may be responsible for sensory neuropathy. Finally, we aligned ATL1 and ATL3 protein sequences and revealed that the first GIM (EYGRL) was conserved between ATL1 and ATL3 (Figure 4H). The tyrosine residue at position 196 of ATL1 matched the tyrosine residue at position 192 of ATL3 (Figure 4H) and was also reported to be mutated to cysteine in a patient with hereditary spastic paraplegia (HSP) [28McCorquodale 3rd, D.S. Ozomaro U. Huang J. Montenegro G. Kushman A. Citrigno L. Price J. Speziani F. Pericak-Vance M.A. Züchner S. Mutation screening of spastin, atlastin, and REEP1 in hereditary spastic paraplegia.Clin. Genet. 2011; 79: 523-530Crossref PubMed Scopus (35) Google Scholar]. As expected, ATL1 also interacted with GABARAP via its GIM as determined by co-immunoprecipitation and GST pull-down assays (Figures 4I–4J). Importantly, the Y196C mutant of ATL1 (HSP-related mutation) also displayed a weakened association with GABARAP (Figures 4I–4J), suggesting that dysfunction of ER-phagy may also be involved in HSP pathogenesis. Here we report that ATL3, as a newly described ER-phagy cargo receptor, regulates tubular ER turnover through specific binding to GABARAP via its 2 GIMs. In mammals, 3 atlastin proteins (ATL1, ATL2, and ATL3) are all believed to mediate ER fusion by GTP-dependent dimerization and conformational changes [29Bian X. Klemm R.W. Liu T.Y. Zhang M. Sun S. Sui X. Liu X. Rapoport T.A. Hu J. Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes.Proc. Natl. Acad. Sci. USA. 2011; 108: 3976-3981Crossref PubMed Scopus (174) Google Scholar, 30Byrnes L.J. Sondermann H. Structural basis for the nucleotide-dependent dimerization of the large G protein atlastin-1/SPG3A.Proc. Natl. Acad. Sci. USA. 2011; 108: 2216-2221Crossref PubMed Scopus (130) Google Scholar, 31Byrnes L.J. Singh A. Szeto K. Benvin N.M. O’Donnell J.P. Zipfel W.R. Sondermann H. Structural basis for conformational switching and GTP loading of the large G protein atlastin.EMBO J. 2013; 32: 369-384Crossref PubMed Scopus (64) Google Scholar, 32Saini S.G. Liu C. Zhang P. Lee T.H. Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration.Mol. Biol. Cell. 2014; 25: 3942-3953Crossref PubMed Scopus (25) Google Scholar, 33Liu T.Y. Bian X. Romano F.B. Shemesh T. Rapoport T.A. Hu J. Cis and trans interactions between atlastin molecules during membrane fusion.Proc. Natl. Acad. Sci. USA. 2015; 112: E1851-E1860Crossref PubMed Scopus (50) Google Scholar, 34O’Donnell J.P. Cooley R.B. Kelly C.M. Miller K. Andersen O.S. Rusinova R. Sondermann H. Timing and reset mechanism of GTP hydrolysis-driven conformational changes of atlastin.Structure. 2017; 25: 997-1010.e4Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. However, recent studies have indicated that ATL3 has a lower dimerization ability than do ATL1 and ATL2 in vitro [14Hu X. Wu F. Sun S. Yu W. Hu J. Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.Protein Cell. 2015; 6: 307-311Crossref PubMed Scopus (21) Google Scholar, 34O’Donnell J.P. Cooley R.B. Kelly C.M. Miller K. Andersen O.S. Rusinova R. Sondermann H. Timing and reset mechanism of GTP hydrolysis-driven conformational changes of atlastin.Structure. 2017; 25: 997-1010.e4Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. The previous reports and our results showed no obvious morphological changes in the ER network in ATL3-depleted cells [14Hu X. Wu F. Sun S. Yu W. Hu J. Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.Protein Cell. 2015; 6: 307-311Crossref PubMed Scopus (21) Google Scholar, 15Pawar S. Ungricht R. Tiefenboeck P. Leroux J.C. Kutay U. Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.eLife. 2017; 6: e28202Crossref PubMed Scopus (22) Google Scholar]. Here, we propose that ATL3 mainly functions as a receptor for ER-phagy, but not as an ER fusogen. Interestingly, the protein level of endogenous ATL3 is significantly upregulated in the early stage of starvation, an observation that needs further investigation. Besides, homo-dimerization of RTN3L, the other tubular ER-phagy receptor, induces ER tubules fragmentation [8Grumati P. Morozzi G. Hölper S. Mari M. Harwardt M.I. Yan R. Müller S. Reggiori F. Heilemann M. Dikic I. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy.eLife. 2017; 6: e25555Crossref PubMed Scopus (219) Google Scholar], and ATL3 can also form homomeric protein complexes [13Rismanchi N. Soderblom C. Stadler J. Zhu P.P. Blackstone C. Atlastin GTPases are required for Golgi apparatus and ER morphogenesis.Hum. Mol. Genet. 2008; 17: 1591-1604Crossref PubMed Scopus (157) Google Scholar]; thus, whether homo-oligomerization of ATL3 is involved in ER-phagy remains to be investigated. During preparation of this manuscript, ATL2 was reported to colocalize with the ER-phagy receptor FAM134B and act downstream of FAM134B-mediated ER-phagy in HCT116 cells [18Liang J.R. Lingeman E. Ahmed S. Corn J.E. Atlastins remodel the endoplasmic reticulum for selective autophagy.J. Cell Biol. 2018; 217: 3354-3367Crossref PubMed Scopus (24) Google Scholar]. Since FAM134B is an ER-phagy receptor that regulates ER sheets turnover [7Khaminets A. Heinrich T. Mari M. Grumati P. Huebner A.K. Akutsu M. Liebmann L. Stolz A. Nietzsche S. Koch N. et al.Regulation of endoplasmic reticulum turnover by selective autophagy.Nat" @default.
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