Regulation of the Rab7 guanine nucleotide exchange factor in endolysosomal biogenesis

内膜系统的细胞器构成了分泌和内吞途径。这些细胞器包括分泌途径中的内质网或高尔基体，以及内吞途径中的内小体和溶酶体。蛋白质的运输需要通过膜的分裂和细胞器的融合形成囊泡。对于萌发，外壳蛋白使膜变形，而融合依赖于局部的Rab GTP酶，它们相互作用的拴系因子如啤酒花和陷阱作为融合机制。在内吞途径中，内吞小泡与早期内小体融合，后者在与溶酶体(酵母中的液泡)融合之前成熟到晚期内小体。早期内体携带Rab5，在晚期内体上被Rab7取代。鸟嘌呤核苷酸交换因子Mon1-Ccz1是Rab7定位到晚期内小体和溶酶体所必需的。它的活性需要在时间和空间上进行协调，因为早期的内体成熟到晚期的内体。在这一点上，我们已经建立了条件来确定膜上依赖Rab5的MC1活性，并跟踪该蛋白在酵母和果蝇细胞中的功能。然而，我们缺乏对MC1活性的精确调控及其在膜上的定位和功能的机械性见解。拟议的实验旨在通过三个不同的目标来解决这些方面。在目标1中，我们将解决MC1如何识别膜相关的Rab7的问题。我们假设Rab7使用一个特定的门与MC1结合，这可能是受调控的。在目标2中，我们将询问Mon1的N-末端结构域和MC1中的其他位点是如何被磷酸化调节的。揭示这些位点的相关性可能会揭示MC1是如何保持沉默的，直到内吞体或自噬体内准备好招募Rab7。最后，我们将在目标3中解决MC1复合体如何与Rab7及其招募者结合并在膜上定位。为此，MC1复合体将被重组到携带各自相互作用的纳米盘或小脂质体上，结构将通过冷冻电子显微镜进行解析。新界面的相关性将通过突变蛋白质分析来解决。这些分析将提供MC1作为内体成熟和溶酶体功能之间的调节环境基金的功能的详细图景。为此，这三个小组将结合他们在MC1复合体和Rab GTP酶方面的生化、细胞生物学和结构专业知识。

Organelles of the endomembrane system structure the secretory and endocytic pathway. These organelles include the endoplasmic reticulum or the Golgi in the secretory pathway, and endosomes and lysosomes in the endocytic pathway. Transport of proteins requires vesicle formation by membrane fission and their fusion at organelles. For budding, coat proteins deform membranes, whereas fusion depends on localized Rab GTPases, their interacting tethering factors such as HOPS and SNAREs as the fusion machinery. In the endocytic pathway, endocytic vesicles fuse with early endosomes, which mature to late endosomes before their fusion with the lysosome (the vacuole in yeast). Early endosomes carry Rab5, which is replaced by Rab7 on late endosomes. The guanine nucleotide exchange factor Mon1-Ccz1 is required for Rab7 localization to late endosomes and lysosomes. Its activity needs to be coordinated in time and space as early endosomes mature to late endosomes. At this point, we have established conditions to determine Rab5-dependent MC1 activity on membranes and follow the protein’s function in yeast and Drosophila cells. However, we lack mechanistic insights into the precise regulation of MC1 activity and its orientation and function on membranes. The proposed experiments aim to address these aspects in three distinct aims. In Aim 1, we will address the question how MC1 recognizes the membrane-associated Rab7. We postulate that Rab7 uses a specific gate to bind to MC1, which is likely regulated. In Aim 2, we will ask how the N-terminal domain of Mon1 and other sites in MC1 are regulated by phosphorylation. Uncovering the relevance of these sites may reveal how MC1 is kept silent, until endosomes or autophagosomes are ready to recruit Rab7. Finally, we will address in Aim 3, how MC1 complexes bind to Rab7 and its recruiters and are oriented on membranes. For this, MC1 complexes will be reconstituted on nanodiscs or small liposomes carrying the respective interactors, and the structure will be resolved by cryo-electron microscopy. The relevance of novel interfaces will be addressed by mutant protein analysis. These analyses will provide a detailed picture of MC1 function as a regulated GEF at the interface between endosomal maturation and lysosomal function. For this, the three groups will combine their biochemical, cell biological, and structural expertise on MC1 complexes and Rab GTPases.