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

内溶酶体系统通过内体摄取将质膜连接到细胞的溶酶体蛋白降解和再循环机制。因此，内溶酶体途径控制其限制膜的完整性和嵌入的膜蛋白的功能性，而且还控制细胞信号级联。内体从早期内体成熟到晚期内体，这需要早期内体Rab 5与晚期内体Rab 7 GT3的协调交换，作为晚期内体与溶酶体融合的先决条件。我们的数据显示，三聚体果蝇Mon 1-Ccz 1-布利复合物不需要布利的Rab 7 GEF活性或Rab 5依赖性激活。然而，布利的缺失导致Rab 5在晚期内体的大量积累，这导致布利突变动物的生理缺陷。我们的数据表明，布利在内体成熟中的作用，通过抑制Rab 5 GEF或通过招募Rab 5 GAP，导致在两种情况下的膜结合的过度活跃Rab 5的积累。在这个建议中，我们将采取结合在体内和体外的方法来详细剖析果蝇Rab 7 GEF复合物的功能。在目标1中，我们将确定Rab 7 GEF复合物的分子环境，通过使用改进的布利蛋白纯化和相邻交联，然后通过质谱法在果蝇和昆虫细胞中搜索特异性相互作用。目标2和目标3将对可能的候选人和地点进行评估。在目标2中，我们将通过分析野生型和布利突变体动物中Rab 5 GAP或GEF缺失或过表达对Rab 5和Rab 7定位的后果来关注布利的体内功能，并将阐明新型相互作用物的作用。在目标3中，我们将利用我们的体外系统（i）概括我们是否发现Rab 7 GEF复合物中布利在Rab 5依赖性Rab 7活化中的任何特定作用，（ii）确定Rab 7 GEF复合物对Rab 5 GAP或GEF的催化活性的作用，以及（iii）确定新的相互作用物和翻译后修饰在GEF和GAP测定中的作用。最后，我们的目标是重建Rab 5失活和Rab 7激活之间的可能电路，使用支持的脂质膜，以确定如何详细调节这种Rab级联。我们希望，这项分析将澄清协调EE LE和溶酶体成熟的后生动物细胞作为一个先决条件，可能的干预措施，在生物医学。

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.