Clathrin-mediated endocytosis selects proteins at the plasma membrane for internalization in membrane-bound vesicles. In mammalian cells, cargo is initially concentrated at endocytic sites by adaptor proteins that promote clathrin recruitment and assembly. A variety of adaptors, such AP2, AP180/clathrin assembly lymphoid myeloid leukemia (CALM), epsins, arrestins, and Dab2, recognize different endocytic motifs and select distinct classes of cargo (Maldonado-Báez and Wendland, 2006
). The subsequent recruitment of accessory factors including Wiscott-Aldrich syndrome protein (WASP), dynamin, and amphiphysin stimulates actin polymerization, which results in membrane deformation and vesicle scission (Smythe and Ayscough, 2006
Studies in yeast have demonstrated that conserved endocytic modules are recruited to sites of internalization in a similar temporal sequence (Kaksonen et al., 2005
; Newpher et al., 2005
; Perrais and Merrifield, 2005
). Clathrin and adaptors are part of the early coat module that establishes the initial site of uptake, whereas the late coat module (Sla1/Sla2/End3/Pan1) couples coat formation to actin polymerization through recruitment and stimulation of yeast WASP (Las17) and Myo5. This activates the Arp2/3 complex to form an actin network that, stabilized by the actin regulatory module (Cap1/2, Sac6, Abp1), promotes rapid inward movement of the internalizing vesicle. Finally, the amphiphysins Rvs161/167 are thought to drive scission of endocytic vesicles from the plasma membrane. Recycling of the endocytic machinery by vesicle uncoating requires additional proteins, including the synaptojanin homologue Inp52.
This sequence of events, defined by live cell imaging studies, suggests that endocytic processes are largely conserved. However, striking differences in the functional requirements for certain regulatory proteins argue that internalization may be differentially regulated in yeast and mammalian cells. For example, the dynamic regulation of actin polymerization is essential for uptake in yeast but not in most mammalian cell types (Engqvist-Goldstein and Drubin, 2003
; Smythe and Ayscough, 2006
). Conversely, clathrin has an important role in higher cells, yet loss of yeast clathrin causes only a partial reduction in receptor internalization, and yeast homologues of major clathrin adaptor proteins such as AP2 and AP180 are not required for the uptake of known cargo (Wendland and Emr, 1998
; Huang et al., 1999
). Several other yeast genes are homologous to components of the mammalian endocytic machinery, yet do not lead to observable endocytosis defects when mutated (Engqvist-Goldstein and Drubin, 2003
; Kaksonen et al., 2005
). Some of these factors may have functionally redundant homologues, or have cargo-specific roles. Additional, unrecognized factors may also regulate cargo uptake in yeast. To better understand the endocytic process in both yeast and mammalian cells, it will be important to identify the complete set of structural and regulatory proteins, and systematically define their specific roles.
The yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1, which regulates the fusion of exocytic vesicles at the cell surface, is a widely studied endocytic cargo protein. After delivery to the plasma membrane of the growing bud, it is rapidly internalized and transported to endosomal and Golgi compartments, where it is incorporated into new secretory vesicles (Lewis et al., 2000
). Here, we perform a genome-wide analysis of Snc1 localization in yeast to uncover genes with functional roles in endocytosis, and quantify their relative contribution to this process. We identify functions for known and novel proteins not previously shown to be required for uptake, and demonstrate that the yeast AP180 homologues and clathrin have a cargo-specific role in Snc1 internalization. In addition, we describe an endocytic function for the previously uncharacterized protein low dye binding 17 (Ldb17).