特异性结构域突变的Rab11转基因与野生型Rab11的功能差异分析

本项目将尝试对Rab11亚家族的特异性的结构域展开功能研究。Rab GTPase家族在膜转运中起重要的调节作用。目前的模型提示每个Rab结合独特的一组四个效应蛋白来完成其膜转运功能，但某个Rab如何通过与其效应蛋白的结合来实现其功能特异性尚不清楚。而对Rab家族的研究多使用显性失活或组成活性的Rab作为突变，无法单独研究Rab某个特定的效应蛋白结合域。在果蝇中，可以利用已有的rab11无效突变，引入某个结构域突变的Rab11转基因来对这个结构域进行功能研究，进而探索研究Rab家族结构和功能关系的策略。前期试验发现Rab11的38至42号氨基酸残基在与其效应蛋白dRip11的结合中起作用。我们将构建该结构域突变的Rab11-38/42转基因，来对rab11无效突变进行拯救，并以野生型Rab11作为对照，分析两者在干细胞维持、细胞极化和类肿瘤生长抑制等已知的Rab11重要生物功能中的作用差异。

We propose a functional study of a specific effector-binding domain of Rab11. Rab family of GTPases play key roles in regulating membrane trafficking. The emerging view is that each Rab recruits a unique set of four different effectors (EI-EIV) in mediating membrane trafficking. Due to the limitations of using dominant negative or constitutively active Rabs as mutant alleles, an effector-binding domain of a certain Rab can not be studied independently. While in Drosophila, we can use an existing rab11 null allele and a rab11 transgene with a mutated effector-binding domain to study the functions of this domain, and further develope a strategy to investigate the relationship between the structure and the function of a Rab. Initial study showed Rab11's amino acid residues 38-42 are involved in binding to one of its effectors, dRip11. We plan to construct a Rab11-38/42 transgene which has Rab11's 38-42 domain mutated and test whether it can rescue the rab11 null mutation phenotypes. By using the Rab11 wild-type transgene as a control, we will study the differences between these two transgenes in known Rab11 important functions such as stem cell maintenance, cell polarization, and tumor-like growth suppression.