Spatio-temporal regulation of endocytic protein assembly by SH3 domains in yeast

Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60 different proteins. SH3 domains are the most abundant protein–protein interaction domain in this process, but the function of most SH3 domains in protein dynamics remains elusive. Using mutagenesis and live-cell fluorescence microscopy in the budding yeast Saccharomyces cerevisiae, we dissected SH3-mediated regulation of the endocytic pathway. Our data suggest that multiple SH3 domains regulate the actin nucleation–promoting Las17–Vrp1 complex, and that the network of SH3 interactions coordinates both Las17–Vrp1 assembly and dissociation. Furthermore, most endocytic SH3 domain proteins use the SH3 domain for their own recruitment, while a minority use the SH3 domain to recruit other proteins and not themselves. Our results provide a dynamic map of SH3 functions in yeast endocytosis and a framework for SH3 interaction network studies across biology.