In Vivo Dynamics of Clathrin and Its Adaptor-Dependent Recruitment to the Actin-Based Endocytic Machinery in Yeast

Abstract

Clathrin-mediated transport is a major pathway for endocytosis. However, in yeast, where cortical actin patches are essential for endocytosis, plasma membrane-associated clathrin has never been observed. Using live cell imaging, we demonstrate cortical clathrin in association with the actin-based endocytic machinery in yeast. Fluorescently tagged clathrin is found in highly mobile internal trans-Golgi/endosomal structures and in smaller cortical patches. Total internal reflection fluorescence microscopy showed that cortical patches are likely endocytic sites, as clathrin is recruited prior to a burst of intensity of the actin patch/endocytic marker, Abp1. Clathrin also accumulates at the cortex with internalizing alpha factor receptor, Ste2p. Cortical clathrin localizes with epsins Ent1/2p and AP180s, and its recruitment to the surface is dependent upon these adaptors. In contrast, Sla2p, End3p, Pan1p, and a dynamic actin cytoskeleton are not required for clathrin assembly or exchange but are required for the mobility, maturation, and/or turnover of clathrin-containing endocytic structures.

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

“…However, Newpher et al (24) reported that in sla2Δ cells Ent1-GFP exhibited Merged images of Sla1-GFP and Abp1-RFP in epsin-depleted (tetENT1, ent2Δ) and sla2Δ strains are shown. The images of the epsin-depleted strain were taken before (− doxycycline) and after (+ doxycycline) the depletion of Ent1.…”

“…To perform FRAP experiments in wild-type cells, we arrested endocytosis with latA, which allows the assembly of the endocytic coat but blocks vesicle budding (11,19,24). Ent1-GFP recovery in latA-treated wild-type cells was very slow, indicating that Ent1 binds to the endocytic site in a stable manner (Fig.…”

Clathrin Functions in the Absence of the Terminal Domain Binding Site for Adaptor-associated Clathrin-Box Motifs

“…To generate cells expressing GFP-Clc1, a 3.5-kb EcoRI/NotI fragment from pTMN17 (Newpher et al, 2005) containing the coding sequence for GFP-Clc1 was ligated with EcoRI/NotI digested pRS306 to generate pJRC7. pJRC7 was digested with PacI to linearize the plasmid in the CLC1 region for targeted integration.…”

“…GFP-LC is a suitable marker for localizing HC within yeast cells as GFP-Clc1 has been shown to completely colocalize with the fluorescent signal of Chc1-RFP (Newpher et al, 2005). GFP-LC localizes to the TGN, endosomes, and small cortical patches.…”

“…GFP-LC localizes to the TGN, endosomes, and small cortical patches. Cortical actin patches are the sites of endocytosis in yeast, and clathrin recruitment to these sites is dependent upon the endocytic adaptors Ent1/2 and Yap1801/2 (Newpher et al, 2005), which have C-terminal clathrin-box motifs. However, the clathrin in these cortical patches is often masked by the intense fluorescent clathrin signal on TGN/ endosomal structures.…”