Abstract
BACKGROUND: Genetic analyses of budding and fission yeast identified >50 proteins that assemble at sites of clathrin-mediated endocytosis in structures called actin patches. These proteins include clathrin, clathrin-interacting proteins, actin binding proteins, and peripheral membrane proteins such as F-BAR proteins. Many questions remain regarding the interactions of these proteins, particularly the participation of F-BAR proteins in the assembly of actin filaments. RESULTS: Our microscopic and genetic interaction experiments on fission yeast show that F-BAR proteins Cdc15p and Bzz1p accumulate in two distinct zones on invaginating membrane tubules and interact with Myo1p and Wsp1p, nucleation-promoting factors for Arp2/3 complex. The two F-BAR proteins peak prior to movement of the actin patch and their accumulation in actin patches depends on the nucleation-promoting factors. At their peak local concentrations, we estimated the stoichiometries of the proteins in actin patches to be one Bzz1p per two Wsp1p and one Cdc15p per Myo1p. Purified Bzz1p has two SH3 domains that interact with Wsp1p and stimulate actin polymerization by Arp2/3 complex. Cells lacking either Cdc15p or Bzz1p assemble 3- to 5-fold less actin in patches (in spite of normal levels of Wsp1p, Myo1p, and Arp2/3 complex), and patches move shorter distances from the plasma membrane. CONCLUSION: We propose that during clathrin-mediated endocytosis, F-BAR proteins interact with nucleation-promoting factors to stimulate Arp2/3 complex in two different zones along the invaginating tubule. We further propose that polymerization of actin filaments in these two zones contributes to membrane scission.