Identification And Functional Characterization Of ACAP4BAR Domain In Membrane Dynamics

Cellular membranes function as walls, fences and barriers which are highly dynamic structures to segregate and transport intracellular components. Their dynamics are coordinated with the cytoskeleton and with metabolic changes in the bilayer. Specific proteins are employed to tightly modulate these processes.One remarkable cellular behavior needs various membrane dynamic processes is cell migration, a critical process throughout development and wound repair. During cell migration, coordination of membrane traffic, actin skeleton remodeling and formation of new adhesion complexes is required for protrusive activity at the leading edge of the migrating cells. Small GTPase, such as ARF and Rho, play a central role in coupling actin dynamics to membrane trafficking. ARF6and its GTPase-activating protein ACAP4play a key role in membrane traffick and the actin-based cytoskeletal remodeling. Most ARF GAP proteins contain an N-terminal BAR domain which contributes to membrane traffic by either inducing or sensing membrane curvature. Many BAR domain proteins function to coordinate membrane sculpture and various membrane-skeletal dynamics.We have investigated the role of the putative BAR domain in the function of ACAP4. Here, we show that the ACAP4BAR domain associates with and tubulates large unilamellar vesicles and drives membrane tubulation in vivo. The BAR domain dimer is critical for the recruitment of ACAP4to the endosomal vesicles. Mutations that impair the membrane binding abilities of ACAP4BAR domain cannot suppress AlFx or EGF induced membrane remodeling, suggesting that the recruitment is critical for the coordination with ARF6at the membrane ruffles of the leading edge. We have also identified a novel phosphorylation site in the BAR domain participating in the regulation of EGF induced migration. Thus, our studies shed new lights on the coordination of membrane traffic and actin-cytoskeleton remodeling in the ARF6-mediated cell migration.The Niemann-Pick type C is a cholesterol trafficking disorder caused by the mutations in the NPC1and NPC2gene. NPC2is believed to be responsible in the trafficking of low-density lipoprotein-derived cholesterol in the late-endosomal/lysosomal pathway. However, the precise role of NPC2in this process remains unknown. Here we report the BAR domain of ACAP4interacts with NPC2in vitro and in vivo. We find that ACAP4regulates NPC2trafficking at the perinuclear endosomal structures. Our study suggest a new role of ACAP4in NPC2sorting and LDL-derived cholesterol trafficking.To sum up, the membrane-sculpting properties of the BAR domain contributes to the regulatory role of ACAP4in the membrane-cytoskeleton dynamics and endocytic trafficking.