Regulation of actin capping protein during clathrin-mediated endocytosis

Abstract

Clathrin-mediated endocytosis (CME) is a major endocytic pathway that is essential in all eukaryotic cells. In the budding yeast S. cerevisiae, polymerization of actin into a branched network is critical to provide the force necessary for membrane invagination during CME. Polymerization of this branched actin network is a highly regulated process, reliant on a multitude of endocytic factors for proper formation. A key regulator is actin capping protein (CP), which binds to the barbed end of actin filaments with high affinity to prevent the loss or addition of actin subunits. While regulation of CP by proteins containing a capping protein-interacting (CPI) motif has been demonstrated in higher eukaryotes, it has not been described in yeast or during endocytosis. Here, we identify and dissect the roles of three CPI motif-containing endocytic factors, Aim21, Bsp1 and Twf1, in CP regulation. Aim21 was the first CPI motif we identified, and the first CPI motif described in yeast. Together with its binding partner Tda2, Aim21 binds to CP through its CPI motif with nanomolar affinity. We demonstrate that Tda2 functions as a dimerization engine for Aim21, bringing two molecules of Aim21 together to form a hetero-tetrameric complex that we term the Tda2-Aim21 complex. Formation of the Tda2-Aim21 complex is essential for a strong interaction with CP, as Aim21 alone binds to CP with more than a 10 fold weaker affinity. Mutating the CPI motif of Aim21 in the yeast genome leads to a recruitment defect in CP and an over-accumulation of F-actin at CME sites, suggesting Aim21 aids in the recruitment of CP to endocytic sites. The little-studied endocytic factor, Bsp1, displays the same phenotype when its CPI motif is mutated in yeast. In addition, the Bsp1 and Aim21 CPI motifs allosterically inhibit the capping function of CP during in vitro actin polymerization assays. When mutations to both the Aim21 and Bsp1 CPI motifs are combined in yeast, CP localization to CME sites is severely reduced, demonstrating that Aim21 and Bsp1 have redundant functions during yeast CME in recruiting a transiently active CP to cortical actin patches. In contrast, the well-conserved actin disassembly factor, twinfilin (Twf1), is not important for recruitment of CP, but is itself reliant on its interaction with CP to localize to CME sites. While the CPI motifs of Aim21 and Bsp1 inhibit the capping function of CP, the Twf1 CPI motif has no effect, despite binding to CP with nanomolar affinity. Mutation of the Twf1 CPI motif results in an accumulation of CP and F-actin at endocytic sites, suggesting that it functions downstream of CP recruitment to recycle CP and actin network components. Together, these findings shed light on how CPI motifs regulate CP in in a step-wise manner during yeast endocytosis.