Lipid turnover and GEF recruitment collectively determine Rab5 recruitment and activation during the first step of early endosome formation

Early endosomes are the pivotal sorting station in eukaryotic cells. A longstanding critical question is how the small GTPase Rab5 is precisely targeted to the correct membrane to initiate early endosome formation. Here, we identify Rabex5 and hRME6 as the two guanine-nucleotide exchange factors (GEFs) that together regulate Rab5 recruitment during early endosome formation. Single-molecule imaging of genome-edited cells reveals that Rabex5 and hRME6 are recruited continuously or transiently to nascent uncoated endocytic carriers, respectively. However, in contrast to uncoated endocytic carriers and other intracellular organelles, directing Rabex5 or its GEF domain to clathrin-coated pits or the plasma membrane fails to trigger Rab5 recruitment. Both in vivo and in vitro experiments show that the plasma membrane-enriched phospholipid PI(4,5)P₂ prevents Rab5 association with the plasma membrane. Importantly, we found that impaired hydrolysis of PI(4,5)P₂ led to reduced early endosome formation in Lowe syndrome cells. Therefore, the spatiotemporal recruitment and activation of Rab5 during early endosome formation are collectively determined by Rabex5/hRME6 recruitment and PI(4,5)P₂ depletion during uncoated endocytic carrier formation.