Abstract
Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization.
Highlights
In specific secretory cell types, endocytosis compensates for the expansion of cell surface area caused by vesicular transport to the plasma membrane (PM) [1]
In vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis
There are several criteria to be met if Sec4p is proposed to play a direct role coordinating actin patch assembly with its established role during the late stages of exocytosis
Summary
In specific secretory cell types, endocytosis compensates for the expansion of cell surface area caused by vesicular transport to the plasma membrane (PM) [1]. In polarized cell types, polarized exocytosis is linked to a reciprocal endocytic event where membrane traffic to and from adherens junctions maintain epithelial apical-basal polarity by recycling junctional proteins [4]. These examples raise the more general question of whether cycles of exocytosis and endocytosis are directly coupled in all cell types. The exocyst complex, which tethers post-Golgi vesicles to the PM during exocytosis, was described as a “network hub” for coordinating the two trafficking pathways at the cell surface [8] These results suggest the possibility of a direct regulatory mechanism for integrating the late stages of post-Golgi exocytosis with endocytic internalization
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