Identification of TgAtg8–TgAtg3 interaction in Toxoplasma gondii

• TgAtg8–TgAtg3 interaction occurs in Toxoplasma gondii.
• The TgAtg8 can bind directly TgAtg3 in vitro.
• There is higher affinity between TgAtg8 and TgAtg3.
• TgAtg8 possesses the conserved structure to interact with other TgAtg containing AIM.
• TgAtg3 contains core E2 enzymatic activity structure and a truncated handle region.

Autophagy is a catabolic process in eukaryotic cells involved in the targeted degradation of cellular organelles and the cytoplasm. Recent works in Toxoplasma gondii suggest that the autophagy processes may serve as an important pathway in modulating parasite survival or death. As an important modulator of Atg8 lipidation and autophagy, Atg8–Atg3 interaction has been attracting increasing attention. However, there is no direct evidence that TgAtg8–TgAtg3 interaction occurs in the parasite. In this study, we firstly found TgAtg8 partially colocalized with TgAtg3 in GFP-TgAtg8 transgenic strains using IFA. Then, lysates from GFP-TgAtg8 tachyzoites were directly subject to large-scale tandem affinity purification with anti-GFP antibody. Western blot and tandem mass spectrometry (MS/MS) analysis determined the interaction between TgAtg8 and TgAtg3. Additionally, we performed real-time interaction analysis with a surface plasmon resonance biosensor using BIAcore system. As expected, the result demonstrated a concentration-dependent increases in resonance signals and indicated the TgAtg8 could bind directly TgAtg3 in vitro. Noteworthily, A KD of 34.9 nM obtained from TgAtg8–TgAtg3 interaction indicate a high-affinity between Atg8-Atg3 in Toxoplasma. Furthermore, homology modeling and sequence alignment showed that TgAtg8 has greatest sequence and structural conservation. Within TgAtg3, this protein possesses the core E2 enzymatic activity structure and a truncated handle region which may contain AIM sequence. Taken together, our findings would help elucidate the formation mechanism of autophagosome in Toxoplasma and provide a possibility for looking into parasitic drug targets.

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