ABSTRACT
WASP family proteins control actin polymerization by activating the Arp2/3 complex. Several subfamilies exist, but their regulation and physiological roles are not well understood, nor is it even known if all subfamilies have been identified. Our extensive search reveals few novel WASP family proteins. The WASP, WASH, and SCAR/WAVE subfamilies are evolutionarily ancient, with WASH the most universally present, whereas WHAMM/JMY first appears in invertebrates. An unusual Dictyostelium WASP homologue that has lost the WH1 domain has retained its function in clathrin-mediated endocytosis, demonstrating that WASPs can function with a remarkably diverse domain topology. The WASH and SCAR/WAVE regulatory complexes are much more rigidly maintained; their domain topology is highly conserved, and all subunits are present or lost together, showing that the complexes are ancient and functionally interdependent. Finally, each subfamily has a distinctive C motif, indicating that this motif plays a specific role in each subfamily's function, unlike the generic V and A motifs. Our analysis identifies which features are universally conserved, and thus essential, and which are branch-specific modifications. It also shows the WASP family is more widespread and diverse than currently appreciated and unexpectedly biases the physiological role of the Arp2/3 complex toward vesicle traffic.
Subject(s)
Dictyostelium/genetics , Protozoan Proteins/genetics , Wiskott-Aldrich Syndrome Protein Family/genetics , Wiskott-Aldrich Syndrome Protein/genetics , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites/genetics , Conserved Sequence , Dictyostelium/cytology , Dictyostelium/metabolism , Evolution, Molecular , Green Fluorescent Proteins/classification , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/physiology , Humans , Microscopy, Fluorescence/methods , Molecular Sequence Data , Phylogeny , Protozoan Proteins/metabolism , Protozoan Proteins/physiology , Sequence Homology, Amino Acid , Wiskott-Aldrich Syndrome Protein/classification , Wiskott-Aldrich Syndrome Protein/metabolism , Wiskott-Aldrich Syndrome Protein Family/classification , Wiskott-Aldrich Syndrome Protein Family/metabolismABSTRACT
Actin plays an essential role in many eukaryotic cellular processes, including motility, generation of polarity, and membrane trafficking. Actin function in these roles is regulated by association with proteins that affect its polymerization state, dynamics, and organization. Numerous proteins have been shown to localize with cortical patches of yeast actin during endocytosis, but the role of many of these proteins remains poorly understood. Here, we reveal that the yeast protein Ysc84 represents a new class of actin-binding proteins, conserved from yeast to humans. It contains a novel N-terminal actin-binding domain termed Ysc84 actin binding (YAB), which can bind and bundle actin filaments. Intriguingly, full-length Ysc84 alone does not bind to actin, but binding can be activated by a specific motif within the polyproline region of the yeast WASP homologue Las17. We also identify a new monomeric actin-binding site on Las17. Together, the polyproline region of Las17 and Ysc84 can promote actin polymerization. Using live cell imaging, kinetics of assembly and disassembly of proteins at the endocytic site were analyzed and reveal that loss of Ysc84 and its homologue Lsb3 decrease inward movement of vesicles consistent with a role in actin polymerization during endocytosis.
