Regulation of the Rab7 guanine nucleotide exchange factor in endolysosomal biogenesis

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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.