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，内溶酶体途径既控制其限制膜的完整性和嵌入膜蛋白的功能，也控制细胞信号级联。内体从早期到晚期成熟，这需要后期内体Rab7 GTPase协调交换早期内体Rab5，作为后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Mon1-Ccz1-Bulli复合物的Rab7 GEF活性或rab5依赖性激活不需要Bulli。然而，Bulli基因缺失导致Rab5在后期核内体大量积累，从而导致Bulli突变动物的生理缺陷。我们的数据表明Bulli在内体成熟中的作用是通过抑制Rab5 GEF或募集Rab5 GAP，从而导致膜结合过度活跃的Rab5的积累。在这个提议中，我们将采取体内和体外相结合的方法来详细剖析果蝇Rab7 GEF复合物的功能。在Aim 1中，我们将通过使用改进的Bulli蛋白纯化和邻交联以及质谱法在果蝇和昆虫细胞中寻找特异性相互作用来确定Rab7 GEF复合物的分子环境。可能的候选人和地点将在第二部分和第三部分进行评估。在Aim 2中，我们将通过分析Rab5 GAP或GEF缺失或过表达对野生型和Bulli突变动物中Rab5和Rab7定位的影响来关注Bulli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Rab7 GEF复合物中Bulli在Rab5依赖的Rab7激活中的任何特定作用，（ii）确定Rab7 GEF复合物对Rab5 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分析中的作用。我们最终的目标是利用支持的脂质膜重建Rab5失活和Rab7激活之间的可能回路，以确定Rab5级联是如何被详细调节的。我们希望这一分析将阐明后生动物细胞中EE与LE和溶酶体成熟的协调，作为可能干预生物医学的先决条件。

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.