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Evolution from covalent conjugation to non-covalent interaction in the ubiquitin-like ATG12 system


Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12–ATG5 complex retains the ability to facilitate ATG8–phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.

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Fig. 1: ATG10 and the C-terminal glycine in ATG12 are lost in some eukaryotes.
Fig. 2: Apicomplexan ATG12 and ATG5 form a non-covalent complex.
Fig. 3: The non-covalent TgATG12–TgATG5 complex promotes TgATG8 lipidation.
Fig. 4: K. phaffii has a non-covalent Atg12 system.

Data availability

Uncropped blot images are shown in Supplementary Dataset 1. All other original data are available from the corresponding authors upon reasonable request.


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We are grateful to M. Shirakawa for technical assistance. We thank K. Hikosaka for assistance in the culture of P. falciparum, M. Komatsu (Juntendo University) for Atg7+/+ and Atg7-/- MEFs, S. Sato (Universiti Malaysia Sabah) for the pSSPF2 vector, M. Meissner (University of Glasgow) for the P5RT70loxPKillerRedloxPYFP-HX vector, S. Yamaoka (Tokyo Medical and Dental University) for the retroviral pMRXIP vector, T. Kitamura (The University of Tokyo) for the retroviral pMXs-IP vector, and T. Yasui (Osaka University) for the pCG-gag-pol and pCG-VSV-G plasmids. This work was supported by funding from the National Key Research and Development Program of China (No. 2017YFD0500400 to H.J.); The Tokyo Biochemical Research Foundation (to M.H.S.); Grants-in-Aid for Scientific Research on Innovative Areas (No. 25111005, to N.M., and No. 16H0101200, to Y.S.) from the Japan Society for the Promotion of Science; and Exploratory Research for Advanced Technology (ERATO) (No. JPMJER1702, to N.M.) and Core Research for Evolutional Science and Technology (CREST) (No. JPMJCR13M7, to N.N.N.) from the Japan Science and Technology Agency (JST).

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Authors and Affiliations



H.J. and N.M. conceived the project. Y.P. and H.J. performed Toxoplasma experiments. J.K.M. and M.H.S performed the Plasmodium experiments under the supervision of K.K. H.S. and H.J. performed the phylogenetic analysis. M.O., Y.S., and H.Y. performed the Komagataella experiments. Y.K. performed mass spectrometry. N.N.N. performed structural analysis. Y.P., H.Y., H.S., J.K.M., and H.J. performed other experiments. Y.P., H.Y., H.S., M.O., J.K.M., Y.S., H.J., and N.M. wrote the manuscript.

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Correspondence to Honglin Jia or Noboru Mizushima.

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Supplementary Figure 1 Conservation of ATG10 and ATG12 in Alveolata and Pichiaceae species.

a,b, ATG10 and ATG12 genes and the C-terminal amino acid sequences of ATG12 of representative Alveolata species including apicomplexan parasites (a) and Pichiaceae (b) species are shown. Losses of the ATG10 gene (magenta) and the C-terminal glycine in ATG12 (green) are shown. Black boxes indicate the presence of these genes. n.d., not detected. The residues of ATG12 on a putative binding interface with ATG5 and the C-terminal end are indicated (magenta).

Supplementary Figure 2 Detection of a covalent conjugate and non-covalent complex of Atg12 and Atg5 by mass spectrometry.

a, Schematic representation of mass spectrometry of Atg12 and Atg5 in K. phaffii and S. cerevisiae. Lysates from K. phaffii and S. cerevisiae cells expressing FLAG-KpAtg12 and FLAG-ScAtg12, respectively, were subjected to immunoprecipitation using anti-FLAG antibody. The gels were stained with CBB and three portions at the positions corresponding to the Atg12 monomer, Atg5 monomer, and Atg12–Atg5 conjugate were excised for in-gel trypsin digestion. Trypsin digests from each sample were labeled by stable isotope dimethyl labeling method. Mixtures of labeled peptides were subjected to LC-MS/MS and peptides were identified and quantified. b, List of sequences and quantities of peptides identified. The number indicates the area of the chromatogram of the peptides identified.

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Supplementary Figures 1–2 and Supplementary Dataset 1

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Pang, Y., Yamamoto, H., Sakamoto, H. et al. Evolution from covalent conjugation to non-covalent interaction in the ubiquitin-like ATG12 system. Nat Struct Mol Biol 26, 289–296 (2019).

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