Apicoplast biogenesis mediated by ATG8 requires the ATG12-ATG5-ATG16L and SNAP29 complexes in Toxoplasma gondii

In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in Toxoplasma and Plasmodium, ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12-ATG5 complex in vivo in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12-ATG5-ATG16L complex exists in Toxoplasma gondii (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in T. gondii. TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12-TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in T. gondii.

在顶复门寄生虫中，巨自噬/自噬机制被重新利用以维持类质体细胞器——顶质体。此前，我们发现在弓形虫和疟原虫中，ATG12以非共价方式与ATG5相互作用，这与大多数生物中的共价相互作用不同。然而，顶复门寄生虫是否具有ATG16L1的功能性直系同源物仍不清楚，ATG16L1是一种在其他生物体内对共价的ATG12 - ATG5复合物的功能至关重要的蛋白质。此外，自噬机制用于维持顶质体的机制尚不清楚。我们报道在刚地弓形虫（Tg）中存在ATG12 - ATG5 - ATG16L复合物。该复合物依赖于TgATG16L定位在顶质体周边的孤立结构上。TgATG12、TgATG5或TgATG16L的诱导性缺失导致顶质体丢失并影响寄生虫生长。我们发现一种假定的可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白，即突触体相关蛋白29（TgSNAP29，Qbc SNARE），是刚地弓形虫维持顶质体所必需的。TgSNAP29的缺失破坏了TgATG8在顶质体上的定位。此外，我们鉴定了TgATG8的一个假定的泛素相互作用基序对接位点（UDS）。UDS位点的突变消除了TgATG8在顶质体上的定位，但不影响其脂化。这些发现表明，TgATG12 - TgATG5 - TgATG16L复合物是顶质体生物发生所必需的，在刚地弓形虫中，TgATG8通过囊泡以依赖SNARE的方式转运至顶质体。