The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy*

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.

Plasmid Construction-We cloned every ATG gene from S. cerevisiae for the expression of recombinant proteins. For expression of the Atg12-Atg5 conjugate in Escherichia coli, Atg7 and Atg10 were cloned into the pET-11a vector, and Atg12 and Atg5 were cloned into the pACYC184 vector. Briefly, an Atg7 fragment including the Shine-Dalgarno sequence was amplified from pHT1-Atg7 by PCR using primers having an NdeI site (5Ј-GGAATTCCATATGTCGTCAGAA-AGGGTCT-3Ј) and a BamHI site (5Ј-GGGGATCCGCTAGC-TTAAGCAATCTCATCAGATTC-3Ј). PCR product digested with NdeI and BamHI was ligated into pET-11a. An Atg10 fragment was prepared from pET-11a-Atg10 by XbaI and BamHI digestion and was ligated into the above pET-11a-Atg7 construct. In a similar fashion, an Atg5 fragment including the Shine-Dalgarno sequence was amplified from pHT1-Atg5 by PCR using primers having an NdeI site (5Ј-GGGGGCGCATA-TGAATGACATTAAACAATT-3Ј) and a PstI site (5Ј-GGCT-GCAGACTAGTTTAGAGCTCAGAGGAAGCTT-3Ј). PCR product digested with NdeI and PstI was ligated into pACYC184. An Atg12 fragment was prepared from pHT1-Atg12 by XbaI and PstI digest and ligated into the above pACYC184-Atg5 construct. Expression plasmids encoding Atg7Myc (C-terminal Myc 3 -tagged Atg7), Atg3 (thrombincleavage from GST), and Atg8 Gly-116 (C-terminal glycine-exposed form of Atg8) were used as described previously (18). Atg5 was cloned into the pHT1 vector, and Atg16 and Atg10 were cloned into the pGEX6p vectors. Atg12 was cloned into the pYNGHisB vector for expression in the superworm system (Katakura industries). DNA sequences were confirmed using an automated DNA sequencer.
In Vitro Atg8-PE Conjugation Reaction-Atg8 lipidation reactions were carried out as described previously (18) with the modification that the ATP regeneration system (phosphocreatine and creatine kinase) is omitted (see the exception in Fig. 1A). Reactions were stopped by the addition of SDS sample buffer.

RESULTS AND DISCUSSION
The Atg12-Atg5 Conjugate Directly Accelerates the Lipidation of Atg8-We have reported that Atg8-PE can be formed in an in vitro reaction containing yeast Atg8 (a processed form), Atg7, Atg3, ATP, and liposomes containing PE (18). This result suggests that Atg3 is competent for Atg8-PE formation without an E3-like enzyme unlike ubiquitin and other ubiquitin-like protein conjugation reaction. However, efficient formation of Atg8-PE in vitro required high PE content within liposomes (optimal PE content is 70%), which is much higher than PE content in yeast organelle membranes (15-30%) (19). Very little Atg8-PE is formed in reactions with low PE-containing liposomes (10 -40%). This observation, in addition to the significance of the Atg12 system for the formation of Atg8-PE in vivo (15,20), prompted us to directly examine the molecular function of the Atg12-Atg5 conjugate in Atg8-PE formation using an in vitro system. Recombinant Atg12-Atg5 conjugate was formed in E. coli co-expressing four components of the yeast Atg12 system (Atg12, Atg7, Atg10, and Atg5) and was purified by successive chromatography to near homogeneity (see "Experimental Procedures"). In the absence of the Atg12-Atg5 conjugate, as reported, little Atg8-PE was formed in the reaction containing 20% DOPE/80% POPC liposomes (Fig. 1A, lane 2), whereas most Atg8 was conjugated to PE in the reaction containing 70% DOPE/30% POPC liposomes (Fig. 1A, lane 4). However, in the presence of the Atg12-Atg5 conjugate, the level of Atg8-PE formed in the reaction containing 20% DOPE/80% POPC liposomes was strikingly enhanced (Fig. 1A, lane 1), and Atg8 was completely converted to Atg8-PE in the reaction containing 70% DOPE/30% POPC liposomes (Fig. 1A, lane 3). In contrast, individually prepared components of the Atg12 system, Atg12, Atg5, or Atg10 alone, or a mixture of Atg12 and Atg5 were unable to enhance Atg8-PE formation (Fig. 1B, lanes 5-8), indicating that the conjugation between Atg12 and Atg5 is essential for the function. Additionally, we confirmed that the Atg12-Atg5 conjugate had neither E1 nor E2 activities for Atg8 lipidation (Fig. 1B, lanes 1 and 2).

JOURNAL OF BIOLOGICAL CHEMISTRY 37299
We also examined the rate of Atg8-PE formation in reactions containing 20% DOPE/80% POPC liposomes in the presence or absence of the Atg12-Atg5 conjugate. The rate of Atg8-PE formation increased in an Atg12-Atg5 dose-dependent manner and reached maximum at 0.5 M (Fig. 1C). The clear enhancement of Atg8-PE formation was still observed in the presence of 1 ⁄ 10 of the amount of the Atg12-Atg5 conjugate when compared with the E1 and E2 enzymes (0.1 M). These results show that the Atg12-Atg5 conjugate repeatedly functions in the acceleration of Atg8-PE formation.
The Atg12-Atg5 Conjugate Functions as an E3-like Enzyme-To further define the function of the Atg12-Atg5 conjugate in Atg8 lipidation, first we examined the rate of the Atg8-Atg3 complex formation with or without the Atg12-Atg5 conjugate. For this experiment, we used the Atg3 C234S mutant, in which the active-site cysteine at position 234 is replaced by serine, which forms a stable Atg8-Atg3 C234S complex linked through an ester bond with the C-terminal glycine of Atg8 (supplemental Fig. S1) because the wild-type Atg8-Atg3 intermediate is unstable in SDS-PAGE even under nonreducing conditions. As shown in Fig. 2A, the rates of Atg8-Atg3 complex formation with or without the Atg12-Atg5 conjugate were indistinguishable. Thus, the Atg12-Atg5 conjugate acts on the step after the Atg8-Atg3 intermediate formation.
We previously reported that Atg8-PE is not formed from the Atg8-Atg3 C234S complex by itself (18). This would be reasonable considering that Atg8 is stably linked with Atg3 C234S mutant through an ester bond in the Atg8-Atg3 C234S complex. Unexpectedly, however, a significant amount of Atg8 was transferred from Atg3 C234S to PE by the addition of the Atg12-Atg5 conjugate (supplemental Fig. S2). We performed time course experiments of Atg8 lipidation after ATP depletion to observe single round reaction from the Atg8-Atg3 C234S complex. It was shown that Atg8-PE was gradually formed in the presence of the Atg12-Atg5 conjugate, accompanied by a decrease of Atg8-Atg3 C234S as well as an increase of free Atg3 C234S (Fig. 2B). These results clearly show that the Atg12-Atg5 conjugate accelerates the step of Atg8 transfer from the E2 enzyme Atg3 to the substrate molecule PE, which corresponds  to that catalyzed by E3 enzyme in ubiquitin and other ubiquitinlike protein conjugation reactions (21).
It is suggested that the Atg12-Atg5 conjugate stimulates the conjugating activity of Atg3. This result provides an explanation for the observation that the C terminus of Atg8 was covalently bound to the N terminus of Atg3 upon the addition of the Atg12-Atg5 conjugate (Fig. 1, A and B, asterisk); 4 Atg3 stimulated by the Atg12-Atg5 conjugate mis-transfers Atg8 to its own N terminus when insufficient PE is present.
It has not been reported that ubiquitin linked with the E2 enzyme through the ester bond is transferred to the substrate. The core structure of Atg3 resembles the canonical E2 enzymes but lacks the conserved residues around the active cysteine (22). Atg3 may take the characteristic mechanism in conjugation differed from other E2 enzymes.
The Atg12-Atg5 Conjugate Associates with Both Atg3 and PEcontaining Liposomes-Further, we assessed the interaction among proteins and liposomes. First, we examined the interaction between the Atg12-Atg5 conjugate and Atg3 or Atg8 using a pull-down assay. The Atg12-Atg5 conjugate firmly associated with GST-Atg3 but not with GST-Atg8 (Fig. 3A), suggesting that the Atg12-Atg5 conjugate works on Atg8 lipidation via an interaction with Atg3.
Next, we tested whether the Atg12-Atg5 conjugate binds to PE-containing liposomes. We were not able to demonstrate an interaction using sedimentation analysis. 5 Therefore, we utilized a cross-linker that reacts with amino and sulfhydryl groups (the head group in PE and cysteine residues in proteins, respectively). The Atg12-Atg5 conjugate was incubated with liposomes containing PE modified at the fatty acid moiety with the fluorescent dye NBD. We used NBD-sphingosine having a free amine group at the lipid head as a negative control. The Atg12-Atg5 conjugate showed NBD fluorescence depending on the PE content of the liposomes and no fluorescence signal with NBD-sphingosine (Fig. 3B). Together, these results suggest that the Atg12-Atg5 conjugate promotes Atg8-PE conjugation by interacting with both Atg3 and PE-containing liposomes.
Effect of Atg16 on the Stimulation of Atg8 Lipidation by the Atg12-Atg5 Conjugate-In yeast, the Atg12-Atg5 conjugate forms a multimeric complex with Atg16, which is essential for autophagosome formation (12) and important for efficient production of Atg8-PE in vivo (15,23). Recombinant Atg12-Atg5 conjugate incubated with recombinant Atg16 formed an ϳ350-kDa complex, similar in size to the complex observed in yeast lysates (Fig. 4A) (12). This suggests that the ϳ350-kDa complex formed in vivo consists of only the Atg12-Atg5 conjugate and Atg16. We tested whether Atg16 is involved in the facilitation of Atg8-PE formation. As shown in Fig. 4B, the coexistence of Atg16 with the Atg12-Atg5 conjugate only slightly increased the rate of Atg8-PE formation. Atg16 alone had no effect on Atg8 lipidation (data not shown). Therefore, Atg16 is not essentially required for the effective formation of Atg8-PE in vitro.
However, Atg16 is required for Atg8-PE formation in vivo. Microscopic studies have shown that both Atg5 and Atg16 locate to the pre-autophagosomal structure (PAS) or isolation membranes, where Atg8-PE is suggested to exist (14,15,23,24). These localizations of Atg5 and Atg16 require the interaction between Atg5 and Atg16 (15,23). Thus, we propose that interaction with Atg16 regulates the localization of the Atg12-Atg5 conjugate, which is required for the formation of Atg8-PE in vivo. Since the Atg12-Atg5 conjugate seems to be irreversible, interaction with Atg16 would be important for the control of its E3-like activity for Atg8 lipidation.
As reported for a mammalian homolog of Atg8, LC3 (25), we have independently found that yeast Atg8 can also form a conjugate with phosphatidylserine (PS) in vitro, although only PEconjugated Atg8 is detected in yeast lysates (6). It is possible that the Atg12-Atg5 conjugate or the Atg12-Atg5⅐Atg16 complex might provide specificity toward PE against PS; however, this hypothesis was not supported by in vitro experiments (sup- FIGURE 3. The Atg12-Atg5 conjugate associates with both Atg3 and PE-containing liposomes. A, the Atg12-Atg5 conjugate (36 pmol) were incubated with 190 pmol of GST-Atg3, GST-Atg8, and GST at 30°C for 1 h in buffer containing 100 mM PIPES/NaOH (pH 6.4) and 150 mM NaCl and further incubated at 4°C for 1 h after the addition of glutathione-Sepharose 4B (20 l slurry, GE Healthcare). Sepharose beads were washed four times by high salt buffer (50 mM PIPES/NaOH (pH 6.4), 1 M NaCl, 2% Triton X-100). Since Atg12-Atg5 and GST-Atg3 migrate in close proximity at 75 kDa, a sample of Atg12-Atg5 with GST-Atg3 was treated by PreScission protease, which cleaves GST-Atg3 into Atg3 and GST. Sepharose beadprecipitated fractions were visualized by CBB staining (upper result) or Western blotting with anti-His antibodies (lower result). B, liposomes consisting of 50% NBD-PE/50% POPC, 20% NBD-PE/80% POPC or 50% NBDsphingosine/50% POPC (50 M lipids) and cross-linker AMAS (2 mM) were incubated at 30°C for 30 min and further incubated for 15 min after the addition of 1 M glycine. The Atg12-Atg5 conjugates (36 pmol) were added to the reaction mixtures containing liposomes modified with cross-linker and incubated at 30°C for 30 min. Cross-linking was terminated by the addition of 1 M cysteine. Samples were resolved by SDS-PAGE and analyzed by LAS image analyzer to detect NBD fluorescence (upper) followed by CBB staining (lower).
plemental Fig. S3). Instead, local lipid composition of membranes to which the Atg12-Atg5⅐Atg16 complex is targeted might provide substrate specificity for Atg8 lipidation in vivo.
We have shown that the Atg12-Atg5 conjugate has an E3-like activity for Atg8 lipidation. Intriguingly, one conjugate facilitates the other conjugation. It has been suggested that Atg8, probably in the form of Atg8-PE, functions in autophagosome formation (17), and more recently, we have shown that Atg8-PE acts on membrane tethering and hemifusion, which are required for the expansion of autophagosomal membrane (26). The Atg12-Atg5 conjugate, forming the Atg12-Atg5⅐Atg16 complex, would contribute to the expansion of autophagosomal membrane by promoting Atg8 lipidation. In mammalian cells, it has been observed that the Atg12-Atg5⅐Atg16 complex preferentially localizes on the outer membrane of the forming autophagosome (14,24). Possibly, the Atg12-Atg5⅐Atg16 complex coordinates asymmetric production of Atg8-PE on the autophagosomal membrane.
There are two major families of E3 ligases for ubiquitin, HECT-, and RING-type E3 ligases. Both Atg12 and Atg5 lack authentic features of ubiquitin E3 ligases, conserved cysteine, or RING finger motif. As revealed by structural analysis, Atg12 takes a ubiquitin-like fold (27), whereas Atg5 consists of two ubiquitin-like folds that flank a helix-rich region (23). Accordingly, the Atg12-Atg5 conjugate comprises three ubiquitin-like folds, which structurally differs from known E3 ligases for other ubiquitin-like proteins (28). The Atg12-Atg5 conjugate promotes "protein-to-lipid" conjugation, whereas E3 ligases promote "protein-to-protein" conjugation. The Atg12-Atg5 conjugate would exert a novel action on protein-lipid conjugation reaction. Further characterization of the E3-like catalysis will lead to new mechanistic and structural insights.