Title: TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3
Abstract: Article1 February 2013free access TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3 Emilio Boada-Romero Emilio Boada-Romero Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Michal Letek Michal Letek Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Aarne Fleischer Aarne Fleischer Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Kathrin Pallauf Kathrin Pallauf Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Cristina Ramón-Barros Cristina Ramón-Barros Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Felipe X Pimentel-Muiños Corresponding Author Felipe X Pimentel-Muiños Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Emilio Boada-Romero Emilio Boada-Romero Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Michal Letek Michal Letek Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Aarne Fleischer Aarne Fleischer Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Kathrin Pallauf Kathrin Pallauf Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Cristina Ramón-Barros Cristina Ramón-Barros Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Felipe X Pimentel-Muiños Corresponding Author Felipe X Pimentel-Muiños Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Author Information Emilio Boada-Romero1, Michal Letek1, Aarne Fleischer1, Kathrin Pallauf1, Cristina Ramón-Barros1 and Felipe X Pimentel-Muiños 1 1Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain *Corresponding author. Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca 37007, Spain. Tel.:+34 923294818; Fax:+34 923294795; E-mail: [email protected] The EMBO Journal (2013)32:566-582https://doi.org/10.1038/emboj.2013.8 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 Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection. Introduction Macroautophagy (hereafter referred to as autophagy) is a catabolic process that promotes degradation of bulk cytoplasmic constituents and recycles the resulting basic components as metabolic precursors (Mizushima, 2007; Yang and Klionsky, 2009). This phenomenon involves sequestration of the doomed material into double-membrane vesicles (autophagosomes) that eventually fuse with lysosomes for degradation of their contents (Rubinsztein et al, 2012). Although the autophagic process digests random cytoplasm to maintain nutrient supply during stressful situations (Rabinowitz and White, 2010; Singh and Cuervo, 2011), it can also target specific, superfluous or potentially harmful components, a less characterized phenomenon called selective autophagy (Komatsu and Ichimura, 2010; Kroemer et al, 2010; Mizushima and Komatsu, 2011). The autophagic pathway is highly conserved in all eukaryotic organisms (Nakatogawa et al, 2009; Mizushima et al, 2011). In mammalian cells, two protein complexes (ULK1/ATG1–mTOR–ATG13–FIP200 and ClassIII Pi3K–BECLIN/ATG6–ATG14) act coordinately to initiate autophagosome nucleation (He and Klionsky, 2009). Membrane elongation and autophagosome closure are driven by two ubiquitin-like modification systems that converge in the lipidation of processed LC3/ATG8 (LC3I) to produce a membrane-bound form called LC3II (Noda et al, 2009; Yang and Klionsky, 2009). ATG12 and LC3 are ubiquitin-like modifiers, and ATG7 is the E1 enzyme for both molecules. In an E2-like step, ATG10 promotes the bonding of ATG12 to ATG5, and ATG3 binds LC3. ATG5–ATG12 then interacts with ATG16L1 to assemble a final E3 system for the conjugation of phosphatidylethanolamine (PE) to LC3I (Tanida, 2011). In this complex, ATG5–ATG12 holds the E3-ligase activity (Hanada et al, 2007), whereas ATG16L1 determines the site of LC3 lipidation (Fujita et al, 2008). Thus, ATG16L1 recruits ATG12–ATG5 to defined membrane localizations, and brings LC3 close to a membranous PE source through interaction between ATG12 and ATG3–LC3I (Fujita et al, 2008). LC3 lipidation and its association with autophagosomes are widely used as autophagic reporter systems (Mizushima et al, 2010). Autophagy has important implications in processes like tumour suppression, neurodegeneration or native immunity (Cecconi and Levine, 2008; Levine and Kroemer, 2008; Levine et al, 2011), and selective autophagy plays a role in these activities (Mizushima et al, 2008; Mizushima and Komatsu, 2011). For example, tumour suppression might result from a specific elimination of damaged organelles that produce pro-inflammatory and DNA-damaging reactive oxygen species (Mathew et al, 2009; Dikic et al, 2010; Mathew and White, 2011). Atg deletion in the central nervous system (Hara et al, 2006; Komatsu et al, 2006) causes neurodegeneration through accumulation of insoluble protein aggregates (Komatsu et al, 2007), revealing a critical housekeeping role of autophagy in clearing toxic garbage (Garcia-Arencibia et al, 2010; Mizushima and Komatsu, 2011). Elimination of foreign invaders involves their recognition by the autophagic machinery, whether they are loose in the cytoplasm (Deretic and Levine, 2009; Deretic, 2011; Shahnazari and Brumell, 2011) or enclosed in phagosomes (Gutierrez et al, 2004; Sanjuan et al, 2007). Although the mechanisms that provide cargo specificity in selective autophagy remain poorly understood, some examples are available. Damaged mitochondria (Youle and Narendra, 2011), insoluble protein precipitates (Knaevelsrud and Simonsen, 2010) or cytosolic bacteria (Fujita and Yoshimori, 2011; Randow, 2011) first become ubiquitinated, and adaptor proteins that simultaneously bind ubiquitin and LC3 target them for autophagic degradation (Kirkin et al, 2009b; Johansen and Lamark, 2011). p62 (Pankiv et al, 2007), NBR1 (Kirkin et al, 2009a), NDP52 (Thurston et al, 2009) and Optineurin (Wild et al, 2011) are some of these adaptors. Depolarized mitochondria are also subjected to autophagy by recruiting NIX/bNIP3L, another LC3-binding protein (Novak et al, 2010). Interestingly, all these linker proteins share a common LC3-interacting motif (Noda et al, 2010). In an additional example, phagosomes containing activated TLR2 recruit BECLIN/ATG6 to promote LC3 labelling of this otherwise non-autophagic compartment (Sanjuan et al, 2007). NOD proteins recognize bacteria at the entry site and bind ATG16L1 to cause LC3 activation (Travassos et al, 2010). Thus, a variety of proteins function as adaptor modules that couple the selected substrates directly with the autophagic machinery to promote LC3 decoration of the targeted item. Given the breadth of processes where selective autophagy is involved, one could anticipate the existence of additional linker families that, similar to ubiquitin/LC3 adaptors, might engage specific autophagic effectors through common protein signatures. Here we show that the human transmembrane molecule TMEM59 defines a novel ATG16L1-binding motif through which the protein promotes labelling of its own endocytic compartment with LC3, in a process that links conventional endocytosis to autophagic degradation. Interestingly, the motif is present with a similar ATG16L1-binding activity in other molecules. Results TMEM59 induces LC3 activation During an extended screening to identify cDNAs whose expression causes cell death (Alcalá et al, 2008), we found clone P15 as able to induce a morphologically atypical death modality (not shown). P15 encoded TMEM59 (Supplementary Figure S1A), a predicted type-I transmembrane protein (C terminus intracellular), known to regulate glycosylation of the amyloid precursor protein (APP) (Ullrich et al, 2010). Unconventional death morphologies have been linked to autophagic (type II) cell death (Shimizu et al, 2004), so to explore a possible pro-autophagic property of TMEM59 we confronted this molecule with reporter systems based on ATG8/LC3 (Mizushima et al, 2010). TMEM59 expression induced HA–LC3 lipidation (Figure 1A and C) and GFP–LC3 redistribution to vesicular structures (Figure 1B, D and E), thus confirming its pro-autophagic capacity. Although functional divergence between different LC3-family members may exist (Chen and Klionsky, 2011), TMEM59 activated the LC3A and B isoforms comparably (see Figure 1C–E). Figure 1.TMEM59 induces LC3 activation. (A) Clone P15 induces HA–LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA–LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti-HA for HA–LC3A). The figure shows that expression of P15 induces HA–LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35. (B) P15 induces GFP–LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP–LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal pictures are shown. (C) TMEM59 induces HA–LC3 lipidation in different cell lines lacking the SV40 large T-antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA–LC3A (left) or HA–LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules. (D) TMEM59 induces GFP–LC3 activation. 293 cells were transfected with the indicated plasmids and GFP–LC3A (top) or GFP–LC3B (bottom). Representative confocal pictures are shown. (E) Quantification of the phenotype in D. The number of GFP–LC3-positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page. Source data fig 1 [embj20138-sup-0001-SourceData-S1.jpg] Download figure Download PowerPoint Characterization of endogenous TMEM59 Northern-blot assays revealed two TMEM59-specific mRNAs present in most cell types (Supplementary Figure S1B). Antibodies against the putative extracellular domain (N terminus) recognized a 34–36-kDa band (Supplementary Figure S1C and D) whose diffuse nature is likely due to glycosylation (Supplementary Figure S1E). TMEM59 localized to small cytoplasmic vesicles that were difficult to detect in immunolocalization studies (Supplementary Figure S2A), although the ectopic protein was easily observed (Supplementary Figure S2B). These results suggested low basal levels, perhaps as a consequence of active degradation. Consistently, inhibition of protein synthesis rapidly reduced TMEM59 expression (Supplementary Figure S2C), and lysosome inhibitors induced the protein without altering its mRNA levels (Supplementary Figure S2D–F). Therefore, the low basal expression of TMEM59 is probably due to intense lysosomal degradation. This constitutive degradation is not autophagic, because neither defective autophagy (Supplementary Figure S2G) nor increased autophagic turnover (Supplementary Figure S2H) altered TMEM59 expression levels. The TMEM59-positive vesicles strongly colocalized with LAMP2 and CD63, and partially with EEA1 (Supplementary Figure S3A and B), suggesting a main localization in late endosomes/lysosomes and a transient presence in early endosomes. Although previous studies with a different antibody (against the C terminus) showed Golgi localization (Ullrich et al, 2010), we found no colocalization with Golgi markers (Supplementary Figure S3A and B). Intriguingly, transfected TMEM59 was expressed at the cell surface (Supplementary Figure S3C) but the endogenous molecule was not found in this location (Supplementary Figure S3D). Some lysosomal membrane proteins transit first through the plasma membrane (Janvier and Bonifacino, 2005; Saftig and Klumperman, 2009), an indirect route revealed by agents that promote their accumulation at the cell surface by inhibiting trafficking to the lysosome (e.g., chloroquine) (Lippincott-Schwartz and Fambrough, 1987). Consistent with this possibility, chloroquine treatment provoked surface exposure of TMEM59 (Supplementary Figure S3D–F). Bafilomycin caused the same effect, although less efficiently (not shown). Importantly, as all these staining procedures were done on unpermeabilized cells and the antibody recognizes the N-terminal part of the molecule, these results confirm a type I topology. Therefore, TMEM59 is a glycosylated, type I transmembrane protein that mainly localizes to late endosomes/lysosomes. The protein is probably first exported to the cell surface and then actively endocytosed to transiently localize in early endosomes on its way to the late endosomal/lysosomal compartment. In this final destination, TMEM59 becomes quickly degraded, a phenomenon that results in low expression levels. Since TMEM59 has no sequence features that could provide mechanistic clues about its pro-autophagic activity, we reasoned that the characterization of this function might reveal undescribed mechanisms of autophagic regulation. TMEM59 induces autophagy through a minimal 19-amino-acid subdomain To dissect the autophagic activity of TMEM59, we first determined the signalling region. A deleted version of the molecule lacking the whole intracellular domain (ID) was unable to activate LC3 (Figure 2A–C), thus ascribing a necessary role to this part of the protein. To evaluate whether this domain was also sufficient to induce autophagy, we placed it in a different molecular context that preserves the type I transmembrane configuration. Chimeric molecules containing the extracellular part of CD16 and the transmembrane region of CD7 (CD16:7) fulfil this requirement, and have been used before in functional assays since they can be stimulated by aggregation with anti-CD16 antibodies (Kolanus et al, 1993). Aggregation of the CD16:7 chimera fused to the ID of TMEM59 caused HA–LC3 conversion (Figure 2D–F) and GFP–LC3 redistribution to a perinuclear vesicle cluster (Figure 2G and H). These results indicate that the cytoplasmic region of TMEM59 suffices for autophagy induction, and suggest that an aggregation event can unleash the activity. Figure 2.The ID of TMEM59 is necessary and sufficient for LC3 activation. (A) TMEM59 ID is required for HA–LC3 lipidation. 293 cells were transfected with full-length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263–323), HA–LC3A (left) or HA–LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules. (B) The ID is necessary for GFP–LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP–LC3A or GFP–LC3B (as indicated). Representative confocal pictures are shown. (C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E. (D) TMEM59 ID (amino acids 263–323) suffices for HA–LC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HA–LC3A and GST, subjected to aggregation with the shown amounts of anti-CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection. (E) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti-CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates. (F) The ID suffices for HA–LC3B lipidation. (G) The ID suffices for GFP–LC3 activation. 293 cells were transfected with the indicated chimeras and GFP–LC3A or GFP–LC3B, aggregated and mounted. Representative confocal pictures are shown. Activated GFP–LC3 appears as a collapsed mass of indiscernible vacuoles. (H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFP–LC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page. Source data fig 2 [embj20138-sup-0002-SourceData-S2.jpg] Download figure Download PowerPoint Serial C-terminal deletions of TMEM59 were tested by overexpression to further map the active subdomain. These experiments showed that amino acids 263–281 are necessary for the activity, because deletions beyond Δ282 were no longer functional (Figure 3A and B). When evaluated in the context of the CD16:7 chimera, this subdomain proved sufficient to activate LC3 (Figure 3C, D and F). In fact, this 19-amino-acid stretch retained the full potential of the whole ID to stimulate autophagy when both constructs were evaluated in parallel (Figure 3C, D and F). In contrast, the remaining ID (282–323) was inactive (Figure 3E and F). Therefore, a minimal 19-amino-acid subdomain between amino acids 263 and 281 (Figure 3G) holds the autophagic potential of the molecule. Figure 3.A minimal 19-amino-acid subdomain between amino acids 263–281 holds the autophagic activity of TMEM59. (A, B) Ability of serial C-terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C-terminal deletions, HA–LC3A (A) or HA–LC3B (B) and GST, and lysed for western blotting against the indicated molecules. (C) Amino acids 263–281 retain the full potential of TMEM59 ID to promote HA–LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA–LC3A or HA–LC3B (as indicated) and GST, and subjected to anti-CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples). (D) Amino acids 263–281 suffice for GFP–LC3 activation and retain the full potential of TMEM59 ID to promote GFP–LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP–LC3A or GFP–LC3B (as indicated), and subjected to anti-CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP–LC3. Quantification and data expression were done as in Figure 2H. (E) Amino acids 282–323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA–LC3A or HA–LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels. (F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C–E) are not caused by differential surface expression. (G) Scheme of TMEM59 showing amino-acid positions and the minimal active subdomain (amino acids 263–281). ED, extracellular domain; TM, transmembrane domain.Source data for this figure is available on the online supplementary information page. Source data fig 3 [embj20138-sup-0003-SourceData-S3.jpg] Download figure Download PowerPoint The active subdomain induces LC3 labelling of its own vesicular compartment To explore the nature of the autophagic response induced by this minimal peptide, we first tested its function in cells depleted of the essential mediators ATG5 or ATG7. Reduced levels of these effectors decreased the autophagic potential of CD16:7 constructs containing the minimal active fragment (CD16:7–263–281; Supplementary Figure S4A–C), indicating that the activity proceeds through an ATG5/ATG7-dependent route. We next determined the subcellular distribution of the autophagic process. Unexpectedly, both overexpressed TMEM59 and aggregated, endocytosed CD16:7–263–281 chimeras tightly colocalized with activated GFP–LC3 (Supplementary Figure S5A and B). Again, 293 cells displayed a clustered signal (see Supplementary Figure S5B), but when the chimera experiment was repeated in JAR cells we detected individual vacuoles stained for both markers (Figure 4A). Most chimera-positive vesicles were labelled with GFP–LC3 in these cells (Supplementary Figure S5C), and GFP–LC3 appeared to stain the vesicle periphery (see inset Figure 4A). These vacuoles colocalized with EEA1 and/or CD63 (Figure 4B), suggesting that, once aggregated, the construct follows the regular endocytic route to the lysosome. In addition, electron microscopy studies showed that the LC3-labelled vesicles containing endocytosed chimera presented single membranes (Figure 4C), and so did the vacuoles induced by straight TMEM59 overexpression (Supplementary Figure S5D). These data point to the notion that single-membrane endosomes become decorated with LC3 in what appears to be an atypical autophagic event not involving canonical double-membrane autophagosomes. Consistent with this idea, TMEM59 does not seem to influence conventional autophagy. For instance, neither aggregation of the active chimera nor TMEM59 overexpression altered the expression levels of three recognized autophagic substrates: p62, NBR1 or GFP–huntingtin-Q74 (Mizushima et al, 2010) (Supplementary Figure S6). Similarly, TMEM59 depletion did not impact on the way the levels of these substrates (or LC3II) were changed by standard modulators of autophagy (bafilomycin or starvation, Supplementary Figures S7 and S8). Therefore, the autophagic activity of TMEM59 seems unrelated to canonical autophagy, and involves LC3 labelling of the same single-membrane endosomes where the molecule becomes activated. Figure 4.The minimal active subdomain of TMEM59 promotes LC3 labelling and lysosomal degradation of its own vesicular compartment. (A) Endocytic vesicles containing aggregated CD16:7–263–281 become labelled with GFP–LC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFP–LC3A, and subjected to anti-CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFP–LC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HA–LC3A conversion in JAR cells. (B) Colocalization of endocytosed CD16:7–263–281 with EEA1 and CD63. JAR cells were transfected with CD16:7–263–281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC-coupled primary antibodies), as indicated. Representative confocal pictures are shown. (C) Immunoelectron microscopy assay showing that endocytosed CD16:7–263–281 localizes to LC3-labelled, single-membrane vesicles. JAR cells were transfected with the CD16:7–263–281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti-CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1–LC3. Arrows indicate single membrane (black) or IgG1–LC3A (white). Scale bar: 400 nm. (D) Lysosomal inhibition increases HA–LC3II levels promoted by CD16:7–263–281. 293 cells were transfected with the indicated chimera and HA–LC3A, and subjected to anti-CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules. (E) Aggregation of CD16:7–263–281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti-CD16 WBs. (F) Degradation of CD16:7–263–281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti-CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page. Source data fig 4 [embj20138-sup-0004-SourceData-S4.jpg] Download figure Download PowerPoint An important question in the field is whether an observed accumulation of active LC3 reflects a real increase in LC3 lipidation and autophagic flux, or it simply represents arrest of autophagosomal degradation (Mizushima and Yoshimori, 2007; Klionsky et al, 2008; Mizushima et al, 2010). To discern between both possibilities, we conducted aggregation studies in the absence or presence of a lysosomal inhibitor (bafilomycin). The presence of this drug increased the levels of LC3II generated by the active CD16:7 construct (CD16:7–263–281; Figure 4D), suggesting that the minimal peptide adds LC3II beyond the degradation blockade imposed by the inhibitor. In addition, the fact that TMEM59 does not promote accumulation of autophagic substrates (see Supplementary Figure S6), argues against the idea that the subdomain blocks autophagic degradation. Taken together, these data suggest that the active subdomain of TMEM59 mainly functions by promoting LC3II synthesis. Since virtually all activated GFP–LC3 colocalized with the endocytosed chimera (see Figure 4A and Supplementary Figure S5B), we interiorized the notion that this synthesis is induced in situ by the aggregation event. We next examined whether LC3 labelling targets the marked vesicles for degradation. Anti-CD16 western blots (WBs) showed that the stimulated CD16:7–263–281 chimera produced a lower molecular weight smear indicative of protein destruction (Figure 4E). This decay was reduced by E64d and pepstatin (Supplementary Figure S9), implying lysosomal involvement and suggesting again that the active subdomain does not obstruct vesicle maturation. Importantly, the degradation was inhibited by ATG5 depletion (Figure 4F), arguing that it is promoted by the autophagic activity triggered in response to aggregation. Consequently, the active subdomain of TMEM59, if stimulated, suffices to direct its own endocytic compartment for autophagic degradation. The active subdomain defines a novel ATG16L1-binding motif We hypothesized that the active peptide might conceal an underlying protein motif that retains function. To test this idea, we performed an alanine scanning study where all residue