The Rab GEF VINE couples phosphatase recruitment to GAP-mediated Rab5 inactivation

Rab5-family GTPases cycle between active and inactive forms to regulate endosomal membrane identity and protein trafficking. VPS9-family guanine nucleotide exchange factors (GEFs) promote Rab activation at endosomes, whereas GTPase-activating proteins (GAPs) oppose Rab signaling. Here, we identify an unexpected role for the yeast VPS9-family GEF complex VINE in promoting the inactivation of the Rab5 homolog Vps21. Through genome-wide proximity screening, predictive modeling, targeted mutagenesis, and in vivo assays, we show that VINE recruits the protein phosphatase Glc7 through the ankyrin repeat–containing domain of its GEF subunit Vrl1. Our results suggest this directs the dephosphorylation of Kxd1, a subunit of the GAP adaptor BLOC-1, which in turn enhances its interaction with the Vps21-specific GAP Msb3 and accelerates GAP-mediated Vps21 inactivation. Thus, VINE is a VPS9-family GEF complex that selectively limits endosomal Rab signaling. These findings reveal a novel mechanism integrating positive and negative Rab regulation, providing insight into how Rab5 signaling is fine-tuned during endosomal trafficking and maturation.