Structuring of the yeast endolysosomal pathway by the Rab5 guanine nucleotide exchange factors Muk1 and Vps9

The endolysosomal pathway, with its interconnected endosomes and lysosomes, has key functions in cellular nutrient and ion uptake, metabolic adaptation, as well as protein and organelle turnover via autophagy. Rab5 GTPases are organelle identity markers on endosomes, though it remains unclear why cells have several Rab5 isoforms and guanine nucleotide exchange factors (GEFs) as their activators. Using yeast, we demonstrate that the key Rab5 GEFs Vps9 and Muk1 overlap in their Rab5 specificity in vitro but cover distinct cellular territories in vivo. Vps9 functions between the Golgi and endosomes, while Muk1 is primarily found in the early endocytic pathway. Using targeting approaches, we show that Rab5 GEFs can only partially replace each other, demonstrating that each GEF is specific for its cellular niche. Intriguingly, Muk1 functions in vivo as an oligomer through its C-terminal domain, which is sufficient to also oligomerize a chimeric Vps9. Overall, our data suggest the spatial distribution of Rab5 GEFs and their substrate Rab5s fine-tune the endolysosomal system for cellular needs and metabolic cues.