The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii

Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network inT. gondii respectively.

高尔基体后囊泡运输对于蛋白质精确运输到顶复门原虫的表膜、表膜下网络和顶端分泌细胞器是必不可少的。然而，在刚地弓形虫中仅鉴定出少数参与囊泡运输、拴系和融合的分子复合物。因此，尚不清楚这种寄生虫是如何完成复杂的囊泡运输的。Sec1/Munc18样（SM）蛋白是参与囊泡融合的蛋白质复合物的重要组成部分。在此，我们利用基于生长素诱导型降解决定子的条件性敲除策略发现，SM蛋白TgSec1的缺失会导致质膜蛋白错误定位。相比之下，SM蛋白TgVps45的条件性缺失会导致形态变化、内膜复合物不对称缺失以及在反复内二芽殖过程中表膜下微管成核、子代寄生虫极化和对称组装方面的缺陷。TgVps45与SNARE蛋白TgStx16和TgVAMP4 - 1相互作用。TgStx16的条件性缺失会导致与TgVps45缺失类似的生长缺陷，这表明它们共同作用于高尔基体反面网状结构（TGN）处的囊泡融合。这些发现表明这两种SM蛋白分别对刚地弓形虫的表膜和表膜下网络的组装至关重要。