Selection and stabilization of endocytic sites by Ede1, a yeast functional homologue of human Eps15

Abstract

During clathrin-mediated endocytosis (CME), endocytic-site maturation can be divided into two stages corresponding to the arrival of the early and late proteins at the plasma membrane. The early proteins are required to capture cargo and position the late machinery, which includes proteins involved in actin assembly and membrane scission. However, the mechanism by which early-arriving proteins select and stabilize endocytic sites is not known. Ede1, one of the earliest proteins recruited to endocytic sites, facilitates site initiation and stabilization. Deletion of EDE1 results in fewer CME initiations and defects in the timing of vesicle maturation. Here we made truncation mutants of Ede1 to better understand how different domains contribute to its recruitment to CME sites, site selection, and site maturation. We found that the minimal domains required for efficient Ede1 localization at CME sites are the third EH domain, the proline-rich region, and the coiled-coil region. We also found that many strains expressing ede1 truncations could support a normal rate of site initiation but still had defects in site-maturation timing, indicating separation of Ede1 functions. When expressed in yeast, human Eps15 localized to the plasma membrane, where it recruited late-phase CME proteins and supported productive endocytosis, identifying it as an Ede1 functional homologue.

FIGURE 1:

Localization of Ede1-GFP truncation mutants. (A) Schematic diagram of full-length Ede1 and the truncation constructs that were integrated at the EDE1 locus. A C-terminal GFP tag, present on all constructs, is not shown. (B) Representative images from the first frame of movies of cells expressing the indicated constructs imaged in the same field as Ede1-GFP reference cells labeled with FM4-64 (magenta). Movies of the GFP channel were taken at 2 s/frame for 4 min, immediately followed by capture of a still image of FM4-64. Scale bar, 5 μm. (C) Magnification of the cell outlined in a white box from B followed by circular kymographs of the mother and bud cortex. Scale bars, 2 μm and 20 s.

FIGURE 2:

A construct containing the coiled-coil domain, third EH domain, and PP region is sufficient to recruit coat proteins. (A) Lifetimes of individual Ede1 patches from cells of the indicated genotype calculated from three independent experiments (EDE1-GFP, 328 patches; ede1^EH3PPCConly-GFP, 480 patches). Mean ± SD. The p values were calculated using the Student’s t test. (B) Ede1-GFP variants were imaged with Sla1-mCherry for 4 min at 2 s/frame using simultaneous dual-color imaging. The first frame of a movie of a representative cell, along with circular kymographs of the mother and bud cortex. Scale bars, 2 μm and 40 s. (C) The patch traces identified in kymographs from three independent experiments were segregated into one of four categories depending on the relative arrival of GFP and mCherry: GFP or mCherry alone, GFP followed by mCherry, or mCherry followed by GFP. Mean percentage and SD of each category per cell. (D) Ede1-GFP variants were imaged with Sla2-mCherry for 4 min at 2 s/frame using simultaneous dual-color imaging. The first frame of a movie of a representative cell is shown along with circular kymographs of the mother and bud cortex. Scale bars, 2 μm and 40 s.

FIGURE 3:

ede1^EH3PPCConly and ede1^ΔCCinternal facilitate endocytic initiation but not normal maturation timing. (A) The 90-s movies of Sla1-GFP in cells expressing 13xmyc-tagged Ede1 truncations were taken at 1 s/frame. First frame from a representative cell. Scale bar, 5 μm. This is followed by circular kymographs of the bud and mother cortex of the cells shown in A; scale bars, 2 μm and 20 s. (B) Quantification of kymograph analysis from three independent experiments of the number of patches that start during a 90-s movie in cells of the indicated genotype. The p values were calculated using Student’s t test. (C) Quantification of kymograph analysis from three independent experiments of the proportion of Sla1-GFP patches that internalize per cell (25 cells/strain). (D) Quantification of the lifetime of Sla1-GFP from three independent experiments in cells of the indicated genotype. (E) ede1^ΔCCinternal-GFP and Sla1-mCherry were imaged for 4 min at 2 s/frame with ∼300-ms delay between channels. The first frame of a movie of a representative cell, in addition to a circular kymograph of the bud and mother cortex. Scale bars, 2 μm and 40 s.

FIGURE 4:

Coiled coils contribute to endocytic-site localization by aggregating Ede1 molecules. (A) Representative images of Ede1-GFP or ede1^ΔCCinternal-GFP tagged with a prenylation motif (CCIIS) or a control sequence (SSIIS). Scale bar, 5 μm. (B) Constructs from A were expressed with Sla1-mCherry and imaged for 4 min at 2 s/frame with ∼300-ms delay between channels. The first frame of a movie of a representative cell, followed by a circular kymograph of the bud and mother cortex. Scale bars, 2 μm and 40 s. (C) Whole-cell lysates of diploid cells with the indicated genotype were subjected to immunoadsorption using mouse anti-GFP antibody, followed by SDS–PAGE and Western blotting, using rabbit anti-GFP antibody to detect Ede1-GFP and mouse anti-myc antibody to detect the Ede1-13xmyc-tagged truncations.

FIGURE 5:

Eps15 can function as an endocytic-site initiator in the absence of Ede1. (A) Representative still images of cells expressing the Eps15-GFP using the indicated promoter. (B) GFP constructs from A were expressed with Sla1-mCherry and imaged for 4 min at 2 s/frame with ∼300-ms delay between channels. The first frame of a movie of a representative cell, followed by a circular kymograph of the bud and mother cortex. Scale bars, 2 μm and 40 s. (C) The patch traces identified in kymographs from B in two independent experiments were segregated into one of four categories depending on the relative arrival of GFP and mCherry: GFP or mCherry alone, GFP followed by mCherry, or mCherry followed by GFP. The mean percentage and SD of each category per cell is indicated. (D) Quantification of kymograph analysis from two independent experiments of the number of patches that start during a 90-s movie in cells of the indicated genotype. The p values were calculated using Student’s t test. (E) Quantification of the Sla1-mCherry lifetime from two independent experiments in cells of the indicated genotype. (F) Quantification of kymograph analysis of the proportion of Sla1-mCherry patches that internalize from two independent experiments per cell (25 cells/strain).