Regulation of the Rab7 guanine nucleotide exchange factor in endolysosomal biogenesis

细胞器的内膜系统结构的分泌和内吞途径。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体和溶酶体。蛋白质的运输需要通过膜分裂和它们在细胞器中的融合形成囊泡。对于出芽，外壳蛋白使膜变形，而融合依赖于局部Rab GTP酶，它们相互作用的拴系因子如HOPS和SNARE作为融合机制。在内吞途径中，内吞囊泡与早期内体融合，早期内体在与溶酶体（酵母中的液泡）融合之前成熟为晚期内体。早期内体携带Rab 5，其在晚期内体上被Rab 7取代。鸟嘌呤核苷酸交换因子Mon 1-Ccz 1是Rab 7定位于晚期内体和溶酶体所必需的。它的活性需要在时间和空间上协调，因为早期内体成熟到晚期内体。在这一点上，我们已经建立了条件，以确定Rab 5依赖MC 1活性的膜，并遵循蛋白质的功能，在酵母和果蝇细胞。然而，我们缺乏对MC 1活性及其在膜上的取向和功能的精确调节的机制见解。拟议的实验旨在解决这些方面的三个不同的目标。在目标1中，我们将解决MC 1如何识别膜相关Rab 7的问题。我们假设Rab 7使用特定的门来结合MC 1，这可能是受调控的。在目标2中，我们将询问Mon 1的N-末端结构域和MC 1中的其他位点如何通过磷酸化调节。揭示这些位点的相关性可能会揭示MC 1是如何保持沉默的，直到内体或自噬体准备招募Rab 7。最后，我们将在目标3中讨论MC 1复合物如何与Rab 7及其招募者结合并在膜上定向。为此，MC 1复合物将在携带相应相互作用物的纳米盘或小脂质体上重构，并且结构将通过冷冻电子显微镜解析。新界面的相关性将通过突变蛋白分析来解决。这些分析将提供MC 1在内体成熟和溶酶体功能之间的界面处作为受调节的GEF的功能的详细描述。为此，这三个小组将联合收割机结合他们的生物化学，细胞生物学和结构的专业知识，对MC 1复合物和Rab GTP酶。

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.