Abstract
SummaryClathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells’ limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2’s cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.
Highlights
The levels of a vast array of transmembrane proteins of widely differing functions must be tightly but dynamically regulated
During mammalian Clathrin-mediated endocytosis (CME), a reversible phosphorylation event occurs on Thr156 of the m2 subunit of the main endocytic clathrin adaptor, AP2
We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. m2Thr156 phosphorylation favors a new, cargobound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2’s cargo affinity in vitro
Summary
Wrobel et al show that phosphorylation of the mammalian endocytic AP2 adaptor causes a conformational change that allows it to efficiently bind the protein NECAP. This, in turn, recruits membraneremodeling proteins into clathrin-coated pits, which drive their formation toward final scission from the parent membrane. Highlights d AP2 m2T156 phosphorylation starts early at CCP inception and increases as CCP grows d Phosphorylation favors an AP2 conformational change and triggers NECAP binding d NECAP PHear simultaneously binds P-AP2 and membraneremodeling proteins on opposite faces d Disrupting the P-AP2:NECAP:membrane-remodeling protein network reduces CME rates. 2019, Developmental Cell 50, 494–508 August 19, 2019 a 2019 The Authors.
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