A living cell must remove unhealthy or excess material from its interior in order to remain healthy and operational. Cells pack this waste into membrane-bound compartments named autophagosomes in a process called autophagy. So-called autophagy proteins make sure that only the unwanted material is eliminated. However, it was not completely clear how these proteins achieve this.

What was known was that proteins called cargo receptors recognize and bind to specific waste materials. At the same time, so-called autophagy enzymes tag the membranes of the autophagosome with a protein known as Atg8, so that cargo receptor molecules can bind this membrane. Now, Fracchiolla, Sawa-Makarska et al. report that, in yeast, an autophagy enzyme links these two events by binding to the cargo receptor and promoting the tagging of the autophagosome’s membrane at the same place. The enzyme in question is a complex made from three autophagy proteins (called Atg12, Atg5 and Atg16), and its activity ensures that the membrane is tagged only next to those materials that need to be disposed of.

Although it is now clearer how a cell’s waste ends up in the autophagosome, it is still puzzling how this process is regulated and how the other autophagy-related components contribute to this highly coordinated process. In particular, an important next step will be to find out what is the source of membrane that gives rise to the autophagosome.

Macro-autophagy (hereafter autophagy) is a conserved pathway for the delivery of cytoplasmic material into the lysosomal system for degradation (Kraft and Martens, 2012). Upon induction of autophagy, double membrane organelles termed autophagosomes are formed in a de novo manner. Initially, autophagosome precursors appear as small membrane structures referred to as isolation membranes (or phagophores). As isolation membranes expand, they gradually enclose cytoplasmic cargo material. Upon closure of the isolation membranes, autophagosomes are formed within which the cargo is isolated from the rest of the cytoplasm. Autophagosomes subsequently fuse with the lysosomal compartment where the inner membrane and the cargo are eventually degraded.

When induced by starvation autophagy can be relatively non-selective with regard to the cargo that is sequestered within autophagosomes. However, it has become clear that autophagy can be highly selective and even exclusive when induced by the presence of intracellular cargo material (Zaffagnini and Martens, 2016). Substances including aggregated proteins, cytosolic pathogens and damaged or surplus organelles have all been shown to be selectively degraded by autophagy (Khaminets et al., 2016). Autophagy thereby protects the organism from pathological conditions such as neurodegeneration, cancer and infection (Levine and Kroemer, 2008; Mizushima and Komatsu, 2011). In S. cerevisiae the cytoplasm-to-vacuole-targeting (Cvt) pathway mediates the delivery of the oligomeric prApe1 enzyme as well as Ams1 and Ape4 into the vacuole via small autophagosomes that are referred to as Cvt vesicles (Nakatogawa et al., 2009).

Selectivity of autophagic processes is mediated by cargo receptors that link the cargo to isolation membranes due to their ability to simultaneously bind the cargo and Atg8-family proteins on the isolation membrane (Johansen and Lamark, 2011; Rogov et al., 2014; Stolz et al., 2014). The interaction of the cargo receptors with Atg8-family proteins is mediated by LC3-interacting regions (LIRs) (Pankiv et al., 2007;Ichimura et al., 2008) also known as Atg8 interacting motifs (AIMs) in the cargo receptors (Noda et al., 2010). Atg8-family proteins are ubiquitin-like proteins that are conjugated to the headgroup of the membrane lipid phosphatidylethanolamine (PE) rendering the otherwise soluble proteins membrane-bound (Ichimura et al., 2000). This conjugation reaction is also referred to as lipidation. The Atg8 conjugation cascade is analogous to the chain of reactions that mediate the conjugation of ubiquitin to its substrates. Thus, Atg8 is activated by the E1-like enzyme Atg7 under consumption of ATP and subsequently transferred to the E2-like enzyme Atg3 from which Atg8 is ultimately transferred to the headgroup of PE (Ichimura et al., 2000; Klionsky and Schulman, 2014). This last step is strongly facilitated by a complex composed of the Atg12~Atg5 protein conjugate and Atg16. The Atg12~Atg5-Atg16 complex acts in an E3-like manner and determines the site of Atg8 conjugation (Fujita et al., 2008b; Hanada et al., 2007). The Atg8 conjugation machinery acts in concert with other proteins of the autophagic machinery including the Atg1/ULK1 complex, the class III PI3K complex 1, Atg9 and the WIPIs to mediate the efficient generation of autophagosomes or Cvt vesicles (Dooley et al., 2014; Fujita et al., 2008a; Juris et al., 2015; Kishi-Itakura et al., 2014; Komatsu et al., 2005; Kraft et al., 2012; Mizushima et al., 1998, 2001; Sou et al., 2008). The precise mechanisms by which the Atg12~Atg5-Atg16 complex and Atg8 aid the formation, elongation or closure of the autophagosomal membranes are unclear.

Recent work has provided important information about how the presence of an autophagic cargo induces the formation of an isolation membrane. In particular, it was shown that the Atg19 cargo receptor recruits the Atg11 scaffold protein to the prApe1 cargo for Atg1 kinase activation (Kamber et al., 2015; Torggler et al., 2016). In addition, it was demonstrated that the cargo receptors Optineurin and NDP52 recruit the ULK1 complex to damaged mitochondria (Lazarou et al., 2015). Furthermore, TRIM proteins were shown to localize the ULK1, PI3K complexes and ATG16L1 to their cargo in a process referred to as precision autophagy (Chauhan et al., 2016; Kimura et al., 2015).

A major question is how the presence of an autophagic cargo is coupled to Atg8 conjugation and thus isolation membrane formation in space and time. Here we show that the S. cerevisiae Atg19 and Atg34 as well as the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like Atg12~Atg5-Atg16 complex. Employing Atg19 as a model in a fully reconstituted system we show that it is capable of recruiting Atg12~Atg5-Atg16 to the prApe1 cargo. This recruitment is mediated by a direct interaction of the AIM motifs in Atg19 with the Atg5 subunit. In our in vitro system the recruitment of the Atg12~Atg5-Atg16 complex is sufficient to drive accumulation of lipidated Atg8 at the cargo. Since the interaction of the Atg19 cargo receptor with the E3-like Atg12~Atg5-Atg16 complex is outcompeted by Atg8, the system may have an inherent directionality whereby the final product in form of Atg8~PE could displace the upstream conjugation machinery at the concave side of the isolation membrane.

During classical ubiquitination reactions the localization of the E3 ligase determines where ubiquitin is conjugated to its substrates (Deshaies and Joazeiro, 2009; Komander and Rape, 2012). We therefore asked if autophagic cargo receptors could interact with the Atg12~Atg5-Atg16 E3-like complex and thereby recruit it to the cargo. Indeed, in pull down experiments GST-Atg19 used as a bait successfully pulled down Atg12~Atg5-Atg16, demonstrating a direct interaction between these two components (Figure 1A). In a complementary approach we imaged the recruitment of Atg12~Atg5-Atg16-mCherry to beads coated with GST-Atg19 under equilibrium condition (Figure 1B). Atg12~Atg5-Atg16-mCherry was robustly and specifically recruited to these beads (Figure 1B). The α-mannosidase (Ams1) receptor Atg34 was also able to bind the Atg12~Atg5-Atg16 complex (Figure 1C), suggesting that this interaction is a more general property of cargo receptors. In order to test if this interaction occurs in cells we performed immunoprecipitation experiments using Atg5-TAP to pull down 6xmyc-Atg19 (Figure 1D). Atg19 was specifically pulled down by Atg5-TAP. Employing the M-Track assay, which is based on the methylation of the human histone 3 N-terminus by the human SUV39H1 methyltransferase when the two components come into close contact (Brezovich et al., 2015; Zuzuarregui et al., 2012), we confirmed that Atg19 and the Atg12~Atg5-Atg16 complex are in close proximity in living cells (Figure 1E). It was previously shown that overexpression of a methyltransferase does not result in unspecific methylation of the histone 3 N-terminus (Brezovich et al., 2015). Next, we tested if the interaction of cargo receptors with the E3-like complex is conserved. To this end, we co-expressed human GFP-ATG5 and mCherry-p62 in HeLa cells in which the endogenous p62 had been knocked-down by RNAi. mCherry-p62 was efficiently co-precipitated by GFP-ATG5 (Figure 1F). We confirmed this result by using a microscopy-based assay in which we imaged the recruitment of mCherry-p62 to GFP-ATG5 coated beads in HeLa cell lysates (Figure 1G). We extended this analysis by investigating other human cargo receptors and found that NDP52 was also pulled down by GFP-ATG5 (Figure 1H). In addition, we detected a weak but consistent co-precipitation of Optineurin (OPTN) (Figure 1H). In summary, the S. cerevisiae Atg19 and Atg34 cargo receptors directly interact with the Atg12~Atg5-Atg16 E3-like enzyme and an interaction with this complex is also detectable for the human cargo receptors p62, OPTN and NDP52.

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Further focusing on Atg19, we tested which of the Atg12~Atg5-Atg16 complex subunits interacts with the Atg19 cargo receptor by using GST-Atg19 as bait to pull down Atg5 stabilized with the N-terminal helix of Atg16 (Atg5-Atg16 (1–46)), the Atg12~Atg5 conjugate, the Atg5-Atg16 complex and the full Atg12~Atg5-Atg16 complex (Figure 2A and Figure 2—figure supplement 2A). Atg12 could not be tested in isolation since we were unable to purify the protein. All proteins tested showed interaction with Atg19 suggesting that Atg5 is sufficient for the binding to Atg19 (Figure 2A,B). We confirmed this result in size exclusion chromatography experiments using Atg5-Atg16 (1–46) and Atg19. Indeed, a fraction of Atg5-Atg16 (1–46) shifted to higher molecular weight fractions in the presence of Atg19 (Figure 2—figure supplement 1).

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The presence of Atg16 had a stimulatory effect on the interaction of Atg5 with Atg19 suggesting that Atg16 could directly interact with Atg19 (Figure 2A,B and Figure 2—figure supplement 2B). However, when tested in isolation Atg16 did not show any detectable binding to Atg19 (Figure 2C). Full length Atg16 may therefore enhance the interaction by an allosteric effect on Atg5 or by increasing the avidity of the interaction due to its ability to self-associate (Fujioka et al., 2010; Kaufmann et al., 2014).

In order to identify the regions in Atg19 that are required for the interaction with Atg12~Atg5-Atg16 we tested a series of Atg19 truncation mutants (Figure 2D) for their interaction with the entire complex and components thereof (Figure 2E). Atg5 and Atg5-Atg16 showed robust interaction with all Atg19 truncations including the C-terminal domain encompassing amino acids 365–415 (Figure 2E and Figure 2—figure supplement 2C). The presence of Atg12, either in context of the Atg12~Atg5 conjugate or the Atg12~Atg5-Atg16 complex, changed the properties of the interaction and required the presence of amino acids 124–254, which include the cargo binding domain of Atg19 (Yamasaki et al., 2016). We corroborated the results of the pull down experiments for the full Atg12~Atg5-Atg16 complex in a microscopy-based assay under equilibrium conditions (Figure 2F). This assay confirmed that the coiled-coil domain of Atg19 is required for the interaction when Atg5 is conjugated to Atg12 (Figure 2F). To interrogate the role of the C-terminal region of Atg19 we performed further microscopy-based interaction experiments (Figure 2G). Consistent with the pull down experiments the Atg12~Atg5-Atg16 complex bound strongly to full length Atg19 but not to the isolated C-terminus (amino acids 365–415) (compare Figure 2E and G) . Intriguingly, deletion of the last eight amino acids containing the canonical AIM motif (Noda et al., 2008; Sawa-Makarska et al., 2014) from the C-terminus of Atg19 strongly reduced the interaction (Figure 3A) suggesting that this AIM motif contributes to the interaction of Atg19 with the Atg12~Atg5-Atg16 complex. Next, we further dissected the contribution of the AIM motifs in the Atg19 C-terminus to the interaction with Atg5. While deletion of the canonical AIM motif in the extreme C-terminus resulted in strong but incomplete reduction in Atg5 binding, further mutation of two additional AIM-like sequences (Abert et al., 2016; Sawa-Makarska et al., 2014) completely abolished the interaction (Figure 3B and Figure 3C Figure 3—figure supplement 1). The AIM-dependent association of Atg19 with Atg5 is relevant for the interaction of the two proteins in vivo as the W412A mutation in the canonical AIM motif of Atg19 results in markedly reduced interaction of the two proteins in co-immunoprecipitation experiments (Figure 3D).

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The dependence of the Atg19 - Atg12~Atg5-Atg16 interaction on the AIM motifs suggested that it is mutually exclusive with the interaction of Atg19 with Atg8. To test this possibility, we immobilized the C-terminus of Atg19 on beads and added Atg5-mCherry-Atg16 (1–46). Subsequently, we added GFP-Atg8 or Atg8 to the beads and determined the signal of Atg5-mCherry-Atg16 (1–46) on the beads (Figure 3E). Indeed, Atg8 outcompeted the Atg19-bound Atg5-mCherry-Atg16 (1–46) in a dose dependent manner. The loss of the Atg5-mCherry signal correlated with an increased GFP-Atg8 signal at the bead (Figure 3E,F). Thus, the interaction of Atg5 with the C-terminus of Atg19 is AIM-dependent and mutually exclusive with Atg8 binding. Next, we asked if the interaction of Atg19 with Atg5 is also mutually exclusive with the Atg19 - Atg8 interaction in the context of the entire Atg12~Atg5-Atg16 complex and when Atg19 is bound to the prApe1 cargo. First, we generated cargo mimetic beads by attaching the prApe1 propeptide to them via a GST-tag. Consistent with previous results Atg19 was robustly recruited to these beads (Figure 3—figure supplement 2A) (Pfaffenwimmer et al., 2014; Sawa-Makarska et al., 2014). Next we tested if Atg19 recruited the Atg12~Atg5-Atg16 complex to the artificial cargo. Indeed, the Atg12~Atg5-Atg16 complex showed a strong Atg19 dependent signal at the beads (Figure 3—figure supplement 2B). Employing this experimental setup, we then went on by adding GFP-Atg8 to the cargo mimetic beads (Figure 3G). The complex was displaced in a concentration dependent manner confirming that Atg12~Atg5-Atg16 and Atg8 compete for the same binding sites on Atg19.

The Atg5 subunit of the Atg12~Atg5-Atg16 complex and the AIM-like motifs in Atg19 are both essential for the binding of these two components. Moreover, the interaction of Atg19 with Atg5 and Atg8 is mutually exclusive, strongly suggesting that the AIM motifs in Atg19 directly interact with Atg5. To identify potential binding sites in Atg5 for the AIM motif we employed computational modeling and molecular dynamics simulations. To this end, we used the structure of Atg5-Atg16 (1–46) from S. cerevisiae (PDB:2DYO) (Matsushita et al., 2007) and performed molecular dynamics simulations which allowed us to capture the flexibility of the Atg5-Atg16 (1–46) complex. Subsequently, in silico docking was performed for 10 randomly selected snapshots from the molecular dynamics trajectory using a peptide encompassing the canonical AIM motif (⁴¹¹TWEEL⁴¹⁵) of Atg19. Modelling analysis suggested three possible binding sites for the peptide, two of which mapped to Atg5 (Figure 4A,B) and one to a site formed by Atg16. Since our pull down experiments (Figure 2C) did not detect any direct interaction of Atg16 with Atg19 the latter site was excluded from further analysis. The other two sites were analyzed. Both contained residues that were persistently involved in forming salt bridges and/or hydrogen bonds with the TWEEL peptide. These were K57 and K137 in the first binding site (Figure 4A, pose 1) and N84 and R208 in the second binding site (Figure 4A, pose 2). These two sites show a similar architecture composed of a hydrophobic pocket surrounded by positively charged residues. Specifically, molecular dynamics simulations showed that the hydrophobic pocket serves to dock W412 and L415 of the TWEEL peptide, while lysine, arginine and asparagine residues on the surface of Atg5 contact the peptide via salt bridges and hydrogen bonds with the glutamic acid residues E413 and E414. Structural superposition showed that the two sites are unrelated to the AIM-binding sites of Atg8 (Figure 4—figure supplement 1).

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In order to validate the predictions from the molecular dynamics simulations we set out to interfere with the interaction. To this end, we mutated the predicted binding sites in Atg5 at residues K57, K137, N84 or R208 to E. We refrained from mutating the hydrophobic pockets of Atg5 in order to avoid non-specific loss of function effects due to disruption of the hydrophobic core of the protein. The mutant proteins were tested for their ability to bind to full length GST-Atg19 in pull down experiments (Figure 4C). Atg5 K137E as well as Atg5 R208E still efficiently bound to Atg19, while the Atg5 K57E and Atg5 N84E mutants showed no detectable binding in this assay.

We went on to test the effect of the K57E and N84E mutations on the recruitment of the Atg12~Atg5-Atg16 complex to cargo mimetic beads (Figure 5A–C). The wild type Atg12~Atg5-Atg16-mCherry complex robustly localized to the beads. Introduction of the K57E into Atg5 resulted in a strongly reduced recruitment of the Atg12~Atg5-Atg16 complex, while the N84E mutation rendered the recruitment undetectable (Figure 5B,C). When combined, the two mutations also resulted in a loss of Atg12~Atg5-Atg16-mCherry complex recruitment to the beads. Thus, introducing negative charge at positions 57 and 84 in the two predicted AIM binding sites interfered with the interaction of the two proteins. These residues are therefore likely to be directly involved in the formation of binding sites for the AIM motif. Interestingly, in the context of the Atg12~Atg5 conjugate N84 in pose two would be largely covered by Atg12 (Figure 4B). This may explain why the C-terminal domain of Atg19 containing the AIM motif is not sufficient for the interaction with Atg12~Atg5 and requires the coiled-coil domain (Figure 2D–G), which may reorient Atg12 away from pose 2.

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The K57 and N84 mutations did not abolish the conjugation of Atg5 to Atg12 (Figure 5A). We also did not observe significant defects of the mutant Atg12~Atg5-Atg16 complexes with respect to liposome binding (Figure 5D) or of the single and double mutant Atg12~Atg5 conjugates with respect to promoting Atg8 lipidation (Figure 5E and Figure 5—figure supplement 1).

We went on to test if the K57E,N84E double mutation would interfere with the Atg5 - Atg19 interaction in vivo by performing co-immunoprecipitations (Figure 5F). Consistent with the results shown above (Figure 3D), wild type Atg5 robustly co-precipitated Atg19. The K57E,N84E double mutant Atg5 showed a strongly reduced ability to interact with Atg19, but the interaction was still detectable (Figure 5F). It was previously shown that Atg16 interacts with Atg21 and that this interaction recruits the Atg12~Atg5-Atg16 complex to the pre-autophagosomal structure (PAS) (Juris et al., 2015). We therefore asked if the residual interaction of the K57E,N84E mutant Atg5 with Atg19 could be dependent on the recruitment of the Atg12~Atg5-Atg16 complex to the PAS by Atg21. To this end, we introduced the E102A mutation into Atg16, which was reported to abolish the Atg16 - Atg21 interaction (Juris et al., 2015). Indeed, the interaction of the Atg5 K57E,N84E with Atg19 became undetectable in context of the Atg16 E102A mutation (Figure 5F).

Next, we tested the impact of the single mutations or their combination on the functionality of the Cvt pathway by monitoring prApe1 processing. The N84E mutation, which had the stronger effect on the Atg19 - Atg5 interaction also affected prApe1 processing more pronouncedly compared to the K57E mutation while the K57E,N84E double mutation had the strongest effect on prApe1 processing (Figure 5G,H and Figure 5—figure supplement 2). For the cells expressing Atg5-TAP (Figure 5H and Figure 5—figure supplement 2) we consistently noticed a somewhat lower levels of the Atg12~Atg5 for the K57E,N84E double mutant under rich conditions. This effect was not seen for the myc-tagged version. prApe1 processing was also affected by the K57E,N84E double mutation under starvation condition in the context of the Atg16 D101A, E102A mutation (Figure 5—figure supplement 3 Figure 5H). In contrast, starvation-induced bulk autophagy as measured by GFP-Atg8 cleavage and the Pho8∆60 assay was not significantly affected by the Atg5 K57E,N84E double mutation (Figure 5I,J), even when tested in combination with the Atg16 D101A, E102A mutant (Figure 5—figure supplement 4).

The data presented so far have shown that the Atg19 cargo receptor can recruit the E3-like Atg12~Atg5-Atg16 complex to the prApe1 cargo and that mutations that abolish this interaction reduce the efficiency of the Cvt pathway. The cargo receptor - E3 interaction might therefore be a minimal axis to couple Atg8 conjugation to the cargo. To test this hypothesis, we developed a fully reconstituted system to recapitulate these reactions. In analogy to the experiment shown in Figure 3—figure supplement 2, we added the Atg12~Atg5-Atg16 complex to cargo mimetic beads bound by Atg19. The Atg12~Atg5-Atg16 complex directly binds to membranes (Figure 5D) (Romanov et al., 2012) and it may thus be able to link membranes and the cargo. To test this, we added small unilamellar vesicles (SUVs) labeled by the incorporation of ATTO390-PE or Rhodamine to Atg19-bound cargo mimetic beads in the presence or absence of the Atg12~Atg5-Atg16 complex (Figure 6A, Figure 6—figure supplement 1). The SUVs were recruited to the beads in an Atg12~Atg5-Atg16 complex dependent manner (Figure 6A, Figure 6—figure supplement 1). The complexes containing the Atg19 binding defective mutants of Atg5 were less efficiently recruited to cargo mimetic beads and were also less efficient in membrane recruitment (Figure 6—figure supplement 2). Thus, the complex brings the membrane substrate for Atg8 conjugation in proximity of the cargo. Next we analyzed if this minimal system would allow local accumulation of conjugated Atg8 by adding the ubiquitin-like molecule GFP-Atg8, the E1-like enzyme Atg7, the E2-like enzyme Atg3, and the cofactors MgCl₂ and ATP to the system. Intriguingly, GFP-Atg8 showed increased signal at the cargo in the presence of ATP (Figure 6A). We also detected an increased signal for the membrane and the Atg12~Atg5-Atg16 complex (Figure 6A), likely due to its association with Atg8 and the membranes (Kaufmann et al., 2014; Romanov et al., 2012). Since the presence of ATP increased the GFP-Atg8 signal at the beads, this effect may be due to lipidation of Atg8 and thus its stable anchoring and concentration on the membranes. If so, then the lipidated Atg8 should be more strongly bound by the Atg19 receptor (Abert et al., 2016; Sawa-Makarska et al., 2014). To test this, we conducted FRAP experiments on the cargo mimetic beads (Figure 6B). Indeed, Atg8 recovered more slowly in the presence of ATP and recovery was slowest for the Atg8 positive puncta, which we interpret as larger Atg8-positive vesicular structures (Figure 6B).

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In the experiments shown in Figure 6A,B the vesicles were also present in solution and we could therefore not exclude the possibility that conjugation occurred remotely from the cargo, followed by attachment of the vesicles harboring lipidated Atg8. For this reason, we recruited vesicles to cargo mimetic beads via the Atg12~Atg5-Atg16 complex and washed away unbound vesicles (Figure 6C). Subsequently, we added the Atg8 conjugation machinery. In the presence of ATP the bead associated signal was increased, consistent with local lipidation of Atg8 at the cargo (Figure 6C). To corroborate this result, we performed FRAP experiments on these beads (Figure 6D). Indeed, the recovery of Atg8 was much reduced in the presence of ATP, consistent with its stable attachment to the membrane via lipidation. Furthermore, upon addition of the Atg4 protein, which removes Atg8 from PE the GFP-Atg8 signal decreased (Figure 6—figure supplement 3).

Here we have shown that the cargo receptor Atg19 directly interacts with the Atg5 subunit of the E3-like Atg12~Atg5-Atg16 complex and that this interaction is sufficient to direct Atg8 conjugation to the cargo in a reconstituted system. The recruitment of the Atg12~Atg5-Atg16 complex requires the AIM motifs of Atg19 and it is likely that these motifs directly bind Atg5 since our modeling analysis and molecular dynamics simulations predicted two binding sites for the C-terminal AIM motif of Atg19 and mutation of these sites abolished Atg19 binding. In addition, the interaction of Atg19 with Atg5 is competitive with the interaction with Atg8, which also directly interacts with the C-terminal AIM motif of Atg19 (Noda et al., 2008). Additional regulation of the two mutually exclusive binding events may occur due to phosphorylation as it was shown that Atg19 is phosphorylated in its C-terminal domain (Pfaffenwimmer et al., 2014; Tanaka et al., 2014).

Since Atg19 contains multiple AIM-like sequences, it is possible that both sites in Atg5 contribute to Atg19 binding. Mutation of N84 in site two had a more pronounced effect on the interaction with Atg19. In the context of the Atg12~Atg5 conjugate this site would be buried by the Atg12 subunit (Noda et al., 2013). Since the coiled-coil domain of Atg19 was required for the interaction of Atg19 with Atg5 when conjugated to Atg12, we hypothesize that the coiled-coil domain is required to expose binding site two by changing the position of Atg12.

The interaction of cargo receptors with the Atg12~Atg5-Atg16 complex is conserved since we detected an interaction of the S. cerevisiae Atg34 and human p62, NDP52 and Optineurin cargo receptors with Atg12~Atg5-Atg16 and ATG5, respectively, suggesting that the recruitment of the E3-like ligase for ATG8-family members conjugation to the cargo is a more general property of cargo receptors. Future work will have to elucidate the biochemical details of the interaction of the human cargo receptors with ATG5 and the ATG12~ATG5-ATG16L complex. It is possible that this interaction is at least for p62 in part mediated indirectly by ALFY since it interacts with p62 and ATG5 (Filimonenko et al., 2010). A similar mechanism has been described for C. elegans where EPG-7 binds both, p62/SQST-1 and ATG12/LGG-3 (Lin et al., 2013).

The results presented in this study suggest the following sequence of events, at least for the Cvt pathway (Figure 7). When bound to their respective cargo, cargo receptors cluster and provide a high-avidity binding platform that recruits the autophagy machinery, including the E3-like Atg12~Atg5-Atg16 complex. This machinery is able to bring and keep membranes in close proximity to the cargo and to act catalytically by promoting several rounds of Atg8 conjugation. Consistent with the idea of local Atg8 lipidation the E2-like Atg3 enzyme localizes to the site of autophagosome formation (Ngu et al., 2015). The Atg12~Atg5-Atg16 complex is able to directly bind lipids (Romanov et al., 2012) and thus it may link the cargo to the membrane. However, in vivo additional interactions with PROPPINs/WIPIs will render the system more robust (Dooley et al., 2014; Juris et al., 2015).

Figure 7

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Due to the action of the Atg8 conjugation machinery Atg8 accumulates at the isolation membrane and may subsequently outcompete the Atg12~Atg5-Atg16 complex on the concave side of the isolation membrane. In conflict with this hypothesis is the finding that the signal of the Atg12~Atg5-Atg16 complex at the bead was not decreased upon addition of ATP (Figure 6C). We interpret this result with the previously described interactions of the Atg12~Atg5-Atg16 complex with the membrane and lipidated Atg8 on the convex side (Kaufmann et al., 2014; Romanov et al., 2012). Consistent with exclusion of Atg12~Atg5-Atg16 from the concave side of the isolation membrane, the Atg12~Atg5-Atg16 complex is excluded from the autophagosomal lumen in vivo (Mizushima et al., 2003). On the concave side Atg8 may be subsequently bound with high avidity by the cargo receptors, which could result in close apposition of the membrane and the cargo and thus exclusion of non-cargo material from the autophagosome (Figure 7) (Abert et al., 2016; Sawa-Makarska et al., 2014; Wurzer et al., 2015). As a consequence, this mechanism would localize the Atg8 conjugation machinery to the highly curved edge of the membrane where the isolation membrane has not yet formed, which may additionally stimulate Atg8 conjugation (Nath et al., 2014). Thus, the intricate AIM/LIR-based interplay between the cargo, the cargo receptors, the Atg8 conjugation machinery and Atg8 may serve to confer directionality to the Atg8 lipidation resulting in robust membrane growth exclusively around the cargo. In vivo, this may occur at a permissive site such as the vacuole or the endoplasmic reticulum (Lamb et al., 2013; Nakatogawa et al., 2009).

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Author details

1. Dorotea Fracchiolla

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   DF, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Justyna Sawa-Makarska

   Competing interests

   The authors declare that no competing interests exist.

2. Justyna Sawa-Makarska

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   JS-M, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Dorotea Fracchiolla

   Competing interests

   The authors declare that no competing interests exist.

3. Bettina Zens

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   BZe, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Anita de Ruiter

   Department of Structural and Computational Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Present address

   Institute for Molecular Modelling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria

   Contribution

   AdR, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Gabriele Zaffagnini

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   GZ, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Andrea Brezovich

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   AB, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Julia Romanov

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   JR, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Kathrin Runggatscher

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   KR, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Claudine Kraft

   Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

   Contribution

   CK, Conception and design, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

10. Bojan Zagrovic

    Department of Structural and Computational Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

    Contribution

    BZa, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

11. Sascha Martens

    Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

    Contribution

    SM, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

    For correspondence

    sascha.martens@univie.ac.at

    Competing interests

    The authors declare that no competing interests exist.

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