A Non-Canonical Interface of SNX9 PX Domain Selectively Sequesters PI(3,4)P2 Lipids, Protecting Them from Hydrolysis

Plasma membrane remodeling processes are tightly regulated by the spatiotemporal distribution and dynamic conversion of phosphoinositidyl lipids (PIPs). This regulation is controlled by the recruitment of proteins such as sorting nexin 9 (SNX9), a key mediator of late-stage endocytosis and macropinocytosis. Using live cell imaging, in vitro reconstitution assays, and molecular dynamics simulations, we investigated how SNX9 distinguishes between PI(3,4)P ₂ and PI(4,5)P ₂ , and the physiological relevance of this selectivity. Our results revealed that during macropinocytic membrane ruffling, SNX9 is recruited in a spatiotemporally coordinated manner with PI(3,4)P ₂ , but not with PI(4,5)P ₂ . While SNX9 induces comparably weak mechanical remodeling on model membranes containing either PIP ₂ species, it exhibits a clear selective binding to PI(3,4)P ₂ , mediated by a non-canonical interface. Through mutational analysis of key residues involved in this sequestration, we further demonstrated that SNX9 protects PI(3,4)P ₂ from hydrolysis. Together, these results reveal a previously unrecognized mechanism of SNX9-PIP ₂ lipid interaction that underscores the pivotal role of SNX9 in coordinating membrane remodeling processes.