Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.

Actin polymerization often occurs at the plasma membrane to drive the protrusion of lamellipodia and filopodia at the leading edge of migrating cells. A role for actin polymerization in another cellular process that involves the reshaping of the plasma membrane--namely endocytosis--has recently been established. Live-cell imaging studies are shedding light on the order and timing of the molecular events and mechanisms of actin function during endocytosis.

A genome-scale specificity and interaction map for yeast SH3 domain-containing proteins reveal how family members show selective binding to target proteins and predicts the dynamic localization of new candidate endocytosis proteins.

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase–interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein ζ1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.

Recent studies have revealed the detailed timing of protein recruitment to endocytic sites in budding yeast. However, little is understood about the early stages of their formation. Here we identify the septin-associated protein Syp1p as a component of the machinery that drives clathrin-mediated endocytosis in budding yeast. Syp1p arrives at endocytic sites early in their formation and shares unique dynamics with the EH-domain protein Ede1p. We find that Syp1p is related in amino acid sequence to several mammalian proteins one of which, SGIP1-␣, is an endocytic component that binds the Ede1p homolog Eps15. Like Syp1p, SGIP1-␣ arrives early at sites of clathrin-mediated endocytosis, suggesting that Syp1p/Ede1p and SGIP1-␣/Eps15 may have a conserved function. In yeast, both Syp1p and Ede1p play important roles in the rate of endocytic site turnover. Additionally, Ede1p is important for endocytic site formation, whereas Syp1p acts as a polarized factor that recruits both Ede1p and endocytic sites to the necks of emerging buds. Thus Ede1p and Syp1p are conserved, early-arriving endocytic proteins with roles in the formation and placement of endocytic sites, respectively. INTRODUCTIONThe dynamics of protein recruitment to sites of clathrinmediated endocytosis have been revealed by live-cell microscopy in budding yeast and mammalian cells (Merrifield et al., 2002;Kaksonen et al., 2003;Kaksonen et al., 2005). These studies have identified numerous proteins that sequentially assemble at endocytic sites and have shown that actin polymerization can power internalization. It is now evident that the dynamic recruitment and disappearance of endocytic proteins are precisely coordinated for productive internalization and that each protein has defined dynamics at endocytic sites. In Saccharomyces cerevisiae four endocytic modules have been defined that each contain proteins with similar dynamics: the coat, WASP/myo, amphiphysin, and actin modules (Kaksonen et al., 2005).Despite detailed knowledge of events at endocytic sites, little is understood about the early stages of their formation. The best candidates for proteins that initiate endocytic site formation are those that arrive earliest. In mammalian cells the classical coat protein clathrin marks the earliest known stage of endocytic site formation (Merrifield et al., 2002). Additionally, the adapter AP-2, is critical for site formation and has similar dynamic behavior to clathrin and so is thought to arrive early (Hinrichsen et al., 2003;Motley et al., 2003;Ehrlich et al., 2004;Keyel et al., 2004). In yeast, the role of AP-2 is unclear, but clathrin, a coat module component, marks the first stages of endocytosis, and its deletion causes severe defects in the number of sites formed (Huang et al., 1999;Kaksonen et al., 2005;Newpher et al., 2005;Newpher and Lemmon, 2006). An additional yeast protein, Ede1p, arrives early and plays a role in endocytic site formation, although its dynamics and function have yet to be fully investigated (Kaksonen et al., 2005;Toshima et al., 200...