ER ‐to‐lysosome‐associated degradation of proteasome‐resistant ATZ polymers occurs via receptor‐mediated vesicular transport

Article3 August 2018free access Source DataTransparent process ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport Ilaria Fregno Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Elisa Fasana Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Timothy J Bergmann Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Andrea Raimondi Experimental Imaging Center, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Marisa Loi Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Tatiana Soldà Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Carmela Galli Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Rocco D'Antuono Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Diego Morone Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Alberto Danieli Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Paolo Paganetti Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, Taverne-Torricella, Switzerland Search for more papers by this author Eelco van Anken Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Maurizio Molinari Corresponding Author [email protected] orcid.org/0000-0002-7636-5829 Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Ilaria Fregno Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Elisa Fasana Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Timothy J Bergmann Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Andrea Raimondi Experimental Imaging Center, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Marisa Loi Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Tatiana Soldà Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Carmela Galli Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Rocco D'Antuono Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Diego Morone Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Alberto Danieli Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Paolo Paganetti Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, Taverne-Torricella, Switzerland Search for more papers by this author Eelco van Anken Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Maurizio Molinari Corresponding Author [email protected] orcid.org/0000-0002-7636-5829 Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Author Information Ilaria Fregno1,2,‡, Elisa Fasana1,‡, Timothy J Bergmann1,2, Andrea Raimondi3, Marisa Loi1,2, Tatiana Soldà1, Carmela Galli1, Rocco D'Antuono1, Diego Morone1, Alberto Danieli4, Paolo Paganetti5, Eelco Anken4 and Maurizio Molinari *,1,6 1Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland 2Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland 3Experimental Imaging Center, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy 4Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy 5Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, Taverne-Torricella, Switzerland 6School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 918200319; E-mail: [email protected] EMBO J (2018)37:e99259https://doi.org/10.15252/embj.201899259 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies. Synopsis Misfolded proteins in the endoplasmic reticulum (ER) are dislocated across the ER membrane and degraded by the ubiquitin-proteasome-system. Proteasome-resistant alpha1-antitrypsin Z (ATZ) misfolded polymers undergo a novel ER-to-lysosome clearance pathway that requires ER-phagy components, vesicular traffic and endolysosome fusion. ATZ polymers are delivered from the ER to endolysosomal degradative compartments via receptor-mediated vesicular traffic. The ER-chaperone Calnexin segregates ATZ polymers in ER subdomains and in ER-derived vesicles under the control of ER-phagy receptor FAM134B. ATZ-loaded vesicles recruit LC3 to dock and fuse with endolysosomes, leading to degradation of the ATZ polymers. ER-resident Syntaxin-17 and lysosomal SNARE VAMP8 mediate membrane fusion events to guide allow degradation of misfolded polymers. Introduction About 40% of the eukaryotic cell's proteome is synthesized in the ER (Uhlen et al, 2015). Protein folding is rather inefficient (Schubert et al, 2000; Vabulas & Hartl, 2005) and maintenance of cellular proteostasis, i.e., the capacity to produce the proteome in appropriate quality and quantity requires continuous removal from biosynthetic compartments of polypeptides that fail to attain the native structure. Paradoxically, the ER does not contain catabolic devices in its lumen. Misfolded ER clients are dislocated in the cytosol for clearance by the ubiquitin-proteasome system (UPS; Guerriero & Brodsky, 2012; Pisoni & Molinari, 2016). Yet, misfolding may in certain cases cause polypeptide aggregation that impairs dislocation and UPS intervention. The disease-causing polymerogenic E342K (ATZ) variant of alpha1 antitrypsin (AT) (Teckman & Perlmutter, 2000; Kamimoto et al, 2006; Kroeger et al, 2009), various serpin mutants (Kroeger et al, 2009), the E90K mutant of the gonadotropin-releasing hormone receptor (GnRHR; Houck et al, 2014), the β subunits of thyrotrophic hormone (Noda & Farquhar, 1992), proalpha1(I) chains of type I collagen (Ishida et al, 2009), and the L1341P mutant of dysferlin (Fujita et al, 2007) are reported cases of proteasome-resistant misfolded proteins generated in the ER and degraded with the contribution of the lysosomal system. Core components of the macroautophagy machinery required for membrane-tethering of the cytosolic ubiquitin-like LC3 and/or GABARAP proteins (e.g., ATG5, ATG7, or VPS34/BCN1) are involved in lysosomal clearance of these proteasome-resistant polypeptides (Teckman & Perlmutter, 2000; Kamimoto et al, 2006; Fujita et al, 2007; Ishida et al, 2009; Kroeger et al, 2009; Gelling et al, 2012; Houck et al, 2014). Originally, LC3 lipidation was reported to occur at the phagophore membrane, as an early event in biogenesis of double-membrane autophagosomes regulating macroautophagy (Suzuki et al, 2007; Mizushima et al, 2011). It has therefore been inferred that macroautophagy regulates clearance of proteasome-resistant aggregates from the ER, as it does for cytosolic aggregates (Lamark & Johansen, 2012; Bento et al, 2016; Hurley & Young, 2017; Menzies et al, 2017). However, luminal aggregates are shielded by the ER membrane from the macroautophagy machinery that operates in the cell cytosol. Thus, an intervention of macroautophagy in clearance of luminal aggregates would imply their dislocation across the ER membrane, or the capture of ER portions containing them by autophagosomes (Marciniak et al, 2016). This latter option is particularly intriguing in view of the recent identification of ER-resident, LC3-binding proteins proposed to selectively label ER subdomains for lysosomal clearance via processes collectively defined as ER-phagy (Khaminets et al, 2015; Fumagalli et al, 2016; Grumati et al, 2017; Fregno & Molinari, 2018; Loi et al, 2018; Smith et al, 2018). Conventional macroautophagy inducers such as rapamycin and starvation enhance clearance of cytosolic inclusions (Lamark & Johansen, 2012; Bento et al, 2016; Hurley & Young, 2017; Menzies et al, 2017) and of some proteasome-resistant, large polypeptides misfolding in the ER lumen such as proalpha1(I) chains of type I collagen (Ishida et al, 2009) and mutant dysferlin (Fujita et al, 2007). For ATZ and other serpin polymers and for the E90K GnRHR, however, lysosomal clearance in cellular and animal disease models is not affected, or it is actually impaired, on conventional macroautophagy activation upon mTOR inhibition (Teckman et al, 2002; Kroeger et al, 2009; Hidvegi et al, 2010; Houck et al, 2014). Thus, it can be speculated that macroautophagic (rapamycin-responsive) and non-macroautophagic (rapamycin-unresponsive) catabolic pathways operate in mammalian cells to back-up the UPS for clearance of compartmentalized aggregates. In such a scenario, the LC3 lipidation machinery that intervenes in clearance of rapamycin-insensitive substrates could do so in a non-canonical fashion (Bestebroer et al, 2013; Ktistakis & Tooze, 2016). To explore this notion, we monitored the fate of proteasome-resistant ATZ polymers, whose lysosomal clearance is not enhanced by rapamycin or nutrient deprivation. Results ATZ polymers are delivered to LAMP1/RAB7-positive endolysosomes for clearance To understand lysosomal clearance of proteasome-resistant ATZ polymers, we first identified the degradative organelle by transiently inhibiting lysosomal activity with bafilomycin A1 (BafA1; Klionsky et al, 2008). Confocal laser scanning microscopy (CLSM) of mouse embryonic fibroblasts (MEF) expressing HA-tagged ATZ shows that in cells exposed to BafA1 ATZ accumulates in endolysosomes (EL, as defined in Huotari & Helenius, 2011) that display LAMP1 [Fig 1A (mock-treated cells) vs. B (cells exposed to BafA1) and D] and RAB7 (Fig EV1) at the limiting membrane. The ATZ accumulating in inactive endolysosomes is decorated both with the anti-HA (Fig 1B, Inset HA) and with the 2C1 antibody (Inset 2C1), which is specific for the polymeric form of ATZ (Fig EV2A; Miranda et al, 2010). The wash-out of BafA1 readily re-establishes the clearance of polymeric ATZ as shown by the disappearance of the ATZ-specific signal in the LAMP1-positive organelles (Fig 1C, Insets, D). Biochemical analyses where the intracellular level of ATZ polymers immunoisolated with the 2C1 antibody from cell lysates is checked in Western blots (WB) confirm the increase in the polymer level on cell exposure to BafA1 (Fig 1E, lane 1 vs. 2, F) and its return to steady-state level 4 h after BafA1 wash-out (Fig 1E, lane 3, F). All in all, LAMP1-positive endolysosomes are the degradative organelles, where polymeric ATZ is delivered from the ER for clearance. These organelles are also visible in non-transfected cells [e.g., cells in the upper left and right corners, Fig 1B, panels LAMP1, Merge (HA), and Merge (2C1)]. This shows that their formation is not induced by the luminal expression of proteasome-resistant misfolded polypeptides such as ATZ. Figure 1. Reversible accumulation of polymeric ATZ within LAMP1-positive endolysosomes on acute lysosomal inhibition A. Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, confocal laser scanning microscopy (CLSM). B. Same as (A) for MEF exposed to 50 nM BafA1 for 12 h. C. Same as (B), 4 h after BafA1 wash-out. D. Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n = 13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, nsP > 0.05, ****P < 0.0001. E. ATZ polymers immunoisolated from lysates of WT MEF mock-treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on Western blot (WB). F. Quantification of (E), n = 3, mean ± SEM. Unpaired two-tailed t-test, nsP > 0.05, *P < 0.05. G–K. Same as (B) in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt-, and ERp57-KO MEF. L. Quantification of ATZ-positive EL (n = 10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, nsP > 0.05, ****P < 0.0001. Data information: Scale bars: 10 μm. Source data are available online for this figure. Source Data for Figure 1E [embj201899259-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Endolysosomes display LAMP1 and GFP-RAB7 at the limiting membraneCLSM analysis showing LAMP1 (cyan) and GFP-RAB7 (green) co-localization at the limiting membrane of endolysosomes containing ATZ-HA (red). Scale bar: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. ATZ, but not NHK, forms 2C1-positive polymers Flow cytofluorimetric analyses to assess HA (x-axis) and 2C1 (y-axis) immunoreactivity in MEF mock-transfected (first panel), expressing ATZ-HA (second panel) or NHK-HA (third panel). HA/2C1 double staining is revealed only in ATZ-HA-expressing cells. Same as panel 2 in CST-treated and in CnxKO MEFs (fourth and fifth panels, respectively). Control of ATZ-HA (lane 2) and NHK-HA (lane 3) expression by WB with anti-HA antibodies (upper panel). Loading control (lower panel). CLSM analyses showing distribution of the ERAD substrate NHK in WT MEF (upper panels). On BafA1 treatment (lower panels), NHK does not accumulate in LAMP1-positive endolysosomes. Scale bars: 10 μm. Source data are available online for this figure. Download figure Download PowerPoint Another disease-causing variant of the SERPINA1 gene, the folding-defective AT variant NHK, is also retained in the ER lumen. At steady state, NHK and ATZ are present at similar intraluminal levels based on the intensities of the HA signal determined in cytofluorimetry and by WB of total cell lysates (Fig EV2A and B). However, in contrast to ATZ (Fig EV2A, second panel), NHK does not form polymers as revealed by the absence of 2C1 immunoreactivity (third panel). NHK also does not accumulate within LAMP1-positive endolysosomes on lysosomal inactivation (Fig EV2C, BafA1). In fact, NHK is eventually dislocated across the ER membrane to be degraded by cytosolic proteasomes (Liu et al, 1999). Thus, delivery to endolysosomes for clearance is not a common fate of misfolded proteins generated in the ER, most of which are degraded by ER-associated degradation (ERAD; Guerriero & Brodsky, 2012; Pisoni & Molinari, 2016). It rather occurs to remove proteasome-resistant species (Teckman & Perlmutter, 2000; Kamimoto et al, 2006; Fujita et al, 2007; Ishida et al, 2009; Kroeger et al, 2009; Gelling et al, 2012; Houck et al, 2014), polymeric ATZ being a paradigmatic example. Delivery of polymeric ATZ to endolysosomes requires the ER lectin CNX The involvement of ER-resident chaperones in selection of folding-defective polypeptides for ERAD is well understood (Guerriero & Brodsky, 2012; Pisoni & Molinari, 2016). In contrast, their involvement in selection of proteasome-resistant aberrant proteins for lysosomal clearance is not studied. The only exception, to the best of our knowledge, shows that pharmacologic inactivation of the calnexin (CNX)/calreticulin (CRT)/ERp57 chaperone system with the plant alkaloid castanospermine (CST; Elbein, 1991; Hebert et al, 1995) prevents lysosomal delivery and disposal of GnRHRE90K (Houck et al, 2014). The membrane-bound lectin chaperone CNX, its soluble and functional homolog CRT, and the associated oxidoreductase ERp57 catalyze oxidative and reductive reactions during polypeptide folding and unfolding in the ER (Caramelo & Parodi, 2007). It has therefore been speculated that their intervention in lysosomal clearance is required to attain a GnRHRE90K structure competent for lysosomal delivery (Houck et al, 2014). As reported for GnRHRE90K, CST also abolishes delivery of ATZ to LAMP1-positive endolysosomes (Fig 1G vs. H and L). Our tests in cells lacking CNX reveal defective delivery of ATZ to the endolysosomes (Fig 1I and L). Ablation of CRT (Fig 1J and L) or ERp57 (Fig 1K and L) has in contrast no consequence. We have extensively studied ER-chaperone-assisted protein biogenesis and clearance. The folding machinery is highly redundant and pharmacologic inactivation of the CNX/CRT/ERp57 chaperone system on cell exposure to CST modestly impact on folding efficiency for most cellular proteins (Denzel et al, 2002; Molinari et al, 2004, 2005; Pieren et al, 2005; Soldà et al, 2006, 2007). On the same line, less invasive approaches, such as the ablation of either CNX or CRT or ERp57, are efficiently compensated in culture cells by the activation of surrogate pathways (Soldà et al, 2006). For that reason, we consider unlikely that the block of ATZ delivery to the endolysosomes on CST treatment, which is recapitulated in cells lacking CNX, results from a specific involvement of CNX in attainment of a delivery-competent ATZ structure. Consistently, the generation of ATZ polymers is unperturbed in cells exposed to CST or lacking CNX (Fig EV2A, fourth and fifth panels, respectively). Rather, we hypothesize a role of CNX as a membrane-bound receptor that segregates ATZ polymers in ER subdomains to be delivered to endolysosomes for clearance. ATZ expression favors formation of a CNX:FAM134B:LC3II complex Our hypothesis that CNX participates in a membrane receptor to segregate ATZ polymers from the ER lumen is corroborated by recent literature indicating CNX as an interactor of FAM134B (Grumati et al, 2017). FAM134B is a recently characterized ER-phagy receptor (i.e., an ER-resident, LC3-binding protein involved in ER fragmentation and delivery of ER subdomains to LAMP1-positive endolysosomes for clearance; Fregno & Molinari, 2018; Loi et al, 2018). The biological significance of CNX association with FAM134B is not known, as this interaction is not required for FAM134B-regulated, ER stress-induced, or starvation-induced ER-phagy (unpublished and Fumagalli et al, 2016; Khaminets et al, 2015). Immunoisolation of epitope-tagged FAM134B from lysates of HEK293 cells confirms the association of endogenous CNX and of endogenous LC3II (Fig 2A, lane 8). Significantly, the co-expression of ATZ-HA substantially enhances the co-precipitation of endogenous CNX and LC3II with V5-tagged FAM134B (Fig 2A, lane 9, B). FAM134BLIR, where the LC3-binding function has been inactivated on replacement of six residues in the cytosolic LC3-interacting region (LIR) of FAM134B (-453DDFELL458- to -453AAAAAA458-), also associates with ATZ and CNX but, as expected, it does not engage LC3 (Fig 2A, lane 10). Under these experimental conditions, i.e., ectopic expression of FAM134BLIR, endolysosomal delivery of ATZ is blocked (Fig 2C vs. D). Our analyses by immuno-electron microscopy (IEM, immunogold-labeled ATZ, Fig 2E–G) and by CLSM (Fig 2H) reveal that ATZ is released in ER-derived vesicles (red arrowheads in Fig 2F, EV in the insets of Fig 2H and see below). However, the ATZ-containing EV remain dispersed in the cytosol, thus failing to release their content within endolysosomes (Fig 2C vs. D, E vs. F and G). As a consequence, clearance of polymeric ATZ is substantially delayed as determined by a cycloheximide (CHX) chase (Fig 2I and J), thus resulting in intracellular accumulation of ATZ polymers at levels comparable to those obtained by cell exposure to BafA1 as quantified by cytofluorimetry (Fig 2K). All in all, the LC3-binding function of FAM134B is dispensable for formation of a complex with CNX and ATZ and for the generation of ATZ-containing EV, but it is required to target EV to endolysosomes where their content must be released for clearance. We postulate, and show below, that FAM134B association with LC3 promotes EV docking to the endolysosomal membrane that precedes membrane:membrane fusion events eventually leading to delivery of polymeric ATZ within endolysosomes for clearance. Figure 2. Disposal of polymeric ATZ requires a functional LIR domain of FAM134B A. HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 6 h with 100 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Materials and Methods. Lanes 1–5, WB of the total cell extract (TCE); lanes 6–10, WB of anti-V5 immunoprecipitates to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with anti-V5 (upper panels), anti-CNX, anti-HA, and anti-LC3 antibodies. B. Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n = 3, unpaired two-tailed t-test, *P < 0.05. C. Same as Fig 1B, WT MEF. Scale bar: 10 μm. D. Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes. Scale bar: 10 μm. E, F. Distribution of gold-labeled ATZ-HA by IEM in BafA1-treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome. G. Quantification of ATZ-gold density of (E, F) (n = 75 and 79 EL, respectively). Unpaired two-tailed t-test, ****P < 0.0001. H. Max projection of the same cell as in (D) after deconvolution. Insets show orthogonal section of select regions. Scale bar: 10 μm. I. Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with anti-HA in WB) in HEK293 cells mock-transfected (lanes 1–4) or expressing FAM134BLIR-V5 (lanes 5–8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control. J. Quantification of (I) (Mean ± SEM, n = 3, unpaired two-tailed t-test, *P < 0.05). K. Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock-treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n = 5, unpaired two-tailed t-test, nsP > 0.05, **P < 0.01, ***P < 0.001). Source data are available online for this figure. Source Data for Figure 2A and I [embj201899259-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint FAM134B is required for delivery of polymeric ATZ to endolysosomes To confirm the role of FAM134B in clearance of proteasome-resistant polymeric ATZ from the ER lumen, delivery of ATZ polymers within endolysosomes was analyzed in WT MEF and in MEF ablated of FAM134B by use of CRISPR/Cas9 genome editing technology (Fig 3A). CLSM analyses confirm delivery of ATZ in LAMP1-positive endolysosomes in WT MEF (Fig 3C and F), which is compromised in FAM134B-ablated cells (Fig 3D and F). Deletion of SEC62 (Fig 3B), another ER-resident, LC3-binding protein that regulates ER-phagy after termination of an ER stress (Fumagalli et al, 2016; Fregno & Molinari, 2018; Loi et al, 2018) does not perturb endolysosomal delivery of ATZ polymers for clearance (Fig 3E and F). Figure 3. Delivery of polymeric ATZ to LAMP1-positive endolysosomes and clearance of ATZ polymers requires FAM134B A. WB analysis showing KO efficiency for CRISPR134B MEF. Asterisks, cross-reacting bands. B. Same as (A) for CRISPR62 MEF. C–E. Same as Fig 1B in CRISPR WT MEF, CRISPR134B MEF, CRISPR62 MEF, respectively. Scale bars: 10 μm. F. Quantification of (C–E) (n = 10, 8, 7 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, nsP > 0.05, ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 3A and B [embj201899259-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint Delivery of polymeric ATZ to the endolysosomes requires a functional LC3 lipidation machinery FAM134B associates with lipidated LC3II. The LC3-binding function of FAM134B is required to deliver ER fragments within autophagolysosomes during ER-phagy (Khaminets et al, 2015) and for delivery of ATZ polymers to endolysosomes for clearance (Fig 2). To assess whether the LC3 lipidation machinery participates in clearance of ATZ polymers, we monitored their delivery to LAMP1-positive endolysosomes in MEF with defective LC3 lipidation due to ablation of ATG4B (Marino et al, 2010) or ATG7 (Komatsu et al, 2005; Fig EV3A and B). Indeed, lysosomal delivery of ATZ such as occurs in WT MEF (Fig 4A, Insets, I) is defective in cells lacking ATG4B (Fig 4B and I) or ATG7 (Fig 4C and I). On the same line, SAR405, a specific inhibitor of VPS34 that prevents LC3 lipidation and autophagosome biogenesis (Ronan et al, 2014; Bento et al, 2016), fully blocks delivery of proteasome-resistant misfolded ATZ from the ER lumen to the endolysosomes (Fig EV4). Click here to expand this figure. Figure EV3. Macroautophagy activity in AtgKO cell lines used in the study KO efficiencies were controlled for ATG4B, ATG7, ULK1, ATG13, ATG9A, and RUBICON (arrows). Macroautophagy activity on nutrient deprivation is assessed by monitoring variation of the levels of the macroautophagy substrate p62 by WB in WT, Atg4BKO, Atg7KO, Fip200KO Ulk1/2DKO, Atg13KO, Atg9KO MEFs (the values for two independent experiments are given). For each cell line, also LC3 levels are shown. All the controls performed in this figure confirm the results published by the groups sharing these cell lines (cited in the text and in the Acknowledgements section). Source data are availa