Function of the trimeric Rab7 GEF complex in endolysosomal biogenesis in Drosophila

三聚体 Rab7 GEF 复合物在果蝇内溶酶体生物合成中的功能

• 批准号: 248987912
• 负责人: Professor Dr. Achim Paululat
• 金额: --
• 依托单位: Arbeitsgruppe Zoologie-Entwicklungsbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/248987912?language=en
• 关键词: Function; trimeric; Rab7; GEF; complex

The endolysosomal system connects the plasma membrane via endosomal uptake to the lysosomal protein degradation and recycling machinery of the cell. Thereby, the endolysosomal pathway controls both the integrity of its limiting membrane and functionality of the embedded membrane proteins, but also cellular signaling cascades. Endosomes mature from early to late endosomes, which requires a coordinated exchange of the early endosomal Rab5 by the late endosomal Rab7 GTPase as a prerequisite of late endosome fusion with the lysosome. Our data revealed that the trimeric Drosophila Mon1-Ccz1-Bulli complex does not requires Bulli for its Rab7 GEF activity or Rab5-dependent activation. However, loss of Bulli causes a massive accumulation of Rab5 at late endosomes that causes physiological deficits in bulli mutant animals. Our data suggest a role of Bulli in endosomal maturation by either inhibiting the Rab5 GEF or by recruiting the Rab5 GAP resulting in both cases in an accumulation of membrane-bound hyperactive Rab5. Within this proposal we will take a combined in vivo and in vitro approach to dissect the function of the Drosophila Rab7 GEF complex in detail. Within Aim 1, we will determine the molecular environment of the Rab7 GEF complex by searching for specific interactions in Drosophila and insect cells using improved Bulli protein purification and next-neighbor crosslinking followed by mass spectrometry. Possible candidates and sites will be evaluated in Aim 2 and 3. In Aim 2, we will focus on the in vivo function of Bulli by analyzing the consequences of Rab5 GAP or GEF deletions or overexpression on Rab5 and Rab7 localization, both in wild-type and bulli mutant animals, and will clarify the role of novel interactors. In Aim 3, we will take advantage of our in vitro system (i) to recapitulate if we find any specific role of Bulli within the Rab7 GEF complex in Rab5-dependent Rab7 activation, (ii) determine the role of the Rab7 GEF complex on the catalytic activity of the Rab5 GAP or GEF, and (iii) determine the role of new interactors and posttranslational modifications in GEF and GAP assays. We finally aim to reconstitute a possible circuit between Rab5 inactivation and Rab7 activation using supported lipid membranes to determine how this Rab cascade is regulated in detail. We expect that this analysis will clarify the coordination of EE to LE and lysosome maturation in metazoan cells as a prerequisite for possible interventions in biomedicine.

内酶体系统通过内酶体摄取将质膜与细胞的溶酶体蛋白降解和再循环机制连接起来。因此，内溶酶体途径不仅控制其限制膜的完整性和嵌入膜蛋白的功能，而且还控制细胞信号转导。内体从早期到晚期成熟，这需要晚期内体Rab7 GTP酶与早期内体Rab5的协调交换，这是晚期内体与溶酶体融合的先决条件。我们的数据表明，三聚体果蝇Mon1-Ccz1-Bulli复合体不需要Bulli就能实现其Rab7全球环境基金活性或Rab5依赖的激活。然而，Bulli的丢失会导致Rab5在内体晚期大量积累，从而导致Bulli突变动物的生理缺陷。我们的数据表明Bulli通过抑制Rab5的环境基金或招募Rab5的间隙在内体成熟中发挥作用，在这两种情况下都导致了膜结合的高活性Rab5的积累。在这项建议中，我们将采取体内和体外相结合的方法来详细剖析果蝇Rab7全球环境基金复合体的功能。在目标1中，我们将通过改进的Bulli蛋白纯化和邻接交联法寻找果蝇和昆虫细胞中的特定相互作用，并随后进行质谱分析，从而确定Rab7-gef复合体的分子环境。在目标2和目标3中，我们将评估可能的候选和位点。在目标2中，我们将通过分析Rab5 GAP或Global缺失或过表达对Rab5和Rab7定位的影响，重点研究Bulli在体内的功能，并阐明新的相互作用因子的作用。在目标3中，我们将利用我们的体外系统：(I)总结我们是否在Rab7全球环境基金复合体中发现Rab5依赖的Rab7激活中的任何特定作用；(Ii)确定Rab7全球环境基金复合体对Rab5 GAP或全球环境基金的催化活性的作用；以及(Iii)确定新的相互作用子和翻译后修饰在全球环境基金和GAP分析中的作用。最后，我们的目标是使用支持的脂膜重建Rab5失活和Rab7激活之间的可能电路，以确定该Rab级联反应是如何被详细调控的。我们期望这项分析将阐明EE对LE的协调和后生动物细胞中溶酶体的成熟，作为可能的生物医学干预的先决条件。