Endocytic Internalization in Budding Yeast Requires Coordinated Actin Nucleation and Myosin Motor Activity

Abstract

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

“…Myo3 and Myo5 were tagged with monomeric RFP at their C-termini by integration at their respective chromosomal loci. The Myo5-RFP had a lifetime similar to that in previous reports (Jonsdottir and Li, 2004;Sun et al, 2006), and unlike Pan1-GFP, the Myo5-RFP remained at the cell cortex for its duration. We observed Myo3-RFP to behave similarly to Myo5-RFP, as expected, also showing no internal movement.…”

“…Rabbit skeletal muscle actin, hemagglutinin (HA)-tagged Arp2/3 complex, Myo5, and Vrp1 were purified as described previously (Martin et al, 2005;Toshima et al, 2005;Sun et al, 2006). GST-Pan1 PRD was expressed in and purified from the protease-deficient DDY1810 yeast strain.…”

“…Actin assembly was performed using 2 M rabbit skeletal muscle actin (5% pyrene labeled) as described previously (Sun et al, 2006), and assembly was monitored using a FluoroMax 3 fluorometer (Jobin-Yvon Horiba, Edison, NJ) with an excitation and emission of 365 and 407 nm, respectively. Barbed end concentrations were calculated using the rate of actin polymerization at 50% polymerization and a rate constant of 8.7 M Ϫ1 s Ϫ1 , as described previously (Higgs and Pollard, 1999).…”

“…Myo3 and Myo5 both localize to cortical actin patches (Goodson et al, 1996), and a recent study reports that Myo5 appears transiently at cortical endocytic patches, immediately preceding the fast movement of vesicles away from the membrane, suggesting a function in vesicle scission (Jonsdottir and Li, 2004). Both the motor and Arp2/3 activities of Myo5 have been implicated in completing the late stages of endocytosis (Sun et al, 2006). In support of their endocytic roles, the C-terminal tails of Myo3 and Myo5 interact with multiple endocytic proteins: Las17, a yeast homologue of Wiskcott Aldrich Syndrome protein (WASP) (Evangelista et al, 2000); Vrp1, a yeast homologue of human WASP-interacting protein (Anderson et al, 1998); and Arc19 and Arc40, components of the Arp2/3 actin nucleating complex (Evangelista et al, 2000;Lechler et al, 2000).…”

“…In support of their endocytic roles, the C-terminal tails of Myo3 and Myo5 interact with multiple endocytic proteins: Las17, a yeast homologue of Wiskcott Aldrich Syndrome protein (WASP) (Evangelista et al, 2000); Vrp1, a yeast homologue of human WASP-interacting protein (Anderson et al, 1998); and Arc19 and Arc40, components of the Arp2/3 actin nucleating complex (Evangelista et al, 2000;Lechler et al, 2000). Vrp1 is an activator of the Arp2/3 actin polymerization activity of type I myosins in both S. cerevisiae and Schizosaccharomyces pombe (Sirotkin et al, 2005;Sun et al, 2006).…”