PDF(Pubmed)
Abstract：
Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0\'s localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0\'s essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.
摘要：
弓形虫通过内分泌分裂，其中使用内膜复合物（IMC）作为支架在母体细胞的细胞质内形成两个子芽。在内分泌期间，合成IMC的成分，并以严格调节的方式依次添加到新生的子芽中。我们先前表明，早期募集蛋白IMC32和IMC43形成了必需的子芽组装复合物，为弓形虫的子细胞支架奠定了基础。在这项研究中,我们确定了必要的，通过在邻近标记和酵母双杂交筛选中使用IMC32作为诱饵，早期招募IMC蛋白BCC0作为该复合物的第三个成员。我们证明BCC0对子芽的定位取决于IMC32和IMC43的存在。缺失分析和功能互补研究表明,BCC0的残基701-877对于其定位和功能都是必需的,并且尽管有轻微的错误定位,残基1-899对于功能是足够的。成对酵母双杂交测定法还证明BCC0的必需结构域与IMC32的卷曲螺旋区结合，并且BCC0和IMC43不直接相互作用。该数据支持复杂组装的模型，其中IMC32-BCC0亚复合物最初通过IMC32的棕榈酰化作用募集到新生芽，并且一旦通过IMC32与IMC43的结合开始芽伸长就锁定在支架中。一起,这项研究解剖了三种早期募集子蛋白的复合物的组织和功能，这对于内分泌学期间IMC的正确组装至关重要。
