Abstract
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.
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The data that support the findings of the current study are included in Supplementary Figs. 1–10 and Supplementary Movies 1–5. The source imaging data presented in this paper are available from the corresponding author upon reasonable request. The full gel source data for western blot and PCR have been provided in Source data are provided with this paper.
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The download links for published codes were provided in the Methods. Other custom MATLAB routines are available upon request from the corresponding author.
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Acknowledgements
We thank Dr. Pietro De Camilli for the de-identified Lowe syndrome patient fibroblasts and control fibroblasts. We thank Dr. Martin Loose and Ivana Matijevic for their expertise and input. We thank Drs. Tom Kirchhausen, Joan Brugge, and Luke Lavis for generous gifts of reagents. This research was funded by the National Key R&D Program of China (2021YFA0804802 and 2022YFA1304500 to K.H., and 2021YFA1300301 to H.C.) and the National Natural Science Foundation of China (32525023, 32321004, 92354305, and 91957106 to K.H., and 32425018 to H.C.).
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Y.D. and Y.G. performed imaging experiments; Y.D., Y.L., Z.A., and K.H. performed imaging analysis; Y.D., S.D., and Y.G. generated genome-edited cell lines; X.M., H.C., X.B., Y.D., and Z.Z. purified proteins and performed in vitro assays; Y.D. and Z.W. performed co-IP experiments; Y.D., X.Z. and Y.Y. performed cargo uptake experiments; K.H. and H.C. supervised the work.
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Du, Y., Miao, X., Gao, Y. et al. Lipid turnover and GEF recruitment collectively determine Rab5 recruitment and activation during the first step of early endosome formation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70543-8
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DOI: https://doi.org/10.1038/s41467-026-70543-8
