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. 2021 Dec 9;118(50):e2113789118. doi: 10.1073/pnas.2113789118

Fig. 3.

Fig. 3.

Puncta formed by PLD-containing proteins are necessary for actin-independent endocytosis. (A) Quantification of the membrane uptake of a lipophilic membrane-bound fluorescent dye (FM4-64) in wild-type BY4741 (Left) and GPD1Δ cells (eliminates turgor pressure; Right) treated with either DMSO, Lat A (prevents F-actin polymerization), or HD (nonspecifically alters solvent quality). Each boxplot shows the relative fluorescence units of n = 50 cells. GPD1Δ cells can undergo endocytosis in the absence of F-actin polymerization (blue) because there is no turgor pressure in these cells. (B) At fixed temperature, HD dissolves endocytic puncta, and this in turn inhibits endocytosis. The bar plot quantifies the percentage of cells that contain Sla1-GFP foci (dark gray), or not (light gray), as a function of HD concentration monitored as counts of endocytic puncta labeled with Sla1-GFP (n = 150 cells). Plot overlay: fluid uptake of water-soluble fluorescent dye LY into vacuoles (mean ± SD; n = 25 foci; logistic fit). (C) PLDs of endocytic coat proteins are essential for their localization to endocytic puncta. Fluorescence images of cortical localization of Ent1, Ent2, Sla1, Yap1801, and Yap1802 fused to Venus YFP. Full-length (Upper) versus C-terminal PLD truncation mutants of the proteins (Lower). Sequence ranges of deleted PLDs indicated (Lower) and grayscale dynamic ranges for image pairs. (Scale bar, 2 μm.) (D) LY dye uptake for strains that express either full-length or PLD deletion mutants of Ent1, Ent2, Yap1801, Yap1802, and Sla1 (as detailed in C). Reductions in Ent1 and Sla1 mutants are significant (n = 100 cells; two-sided t test; see Materials and Methods). (E) Illustration of the membrane topology (dark gray) and remodeling into the cell during endocytosis in the absence of actin. Electron microscopy data suggest that clathrin-coated plasma membrane patches are surrounded by a cortical body of ∼200 nm diameter (light gray) before appearance of actin structures (7). Clathrin heavy and light chains (Chc1 and Clc1) interact with initiator proteins (Ede1 and Syp1) to form a lattice on the membrane (in green). Subsequently, PLD-containing coat proteins (light gray), such as Sla1/2, Ent1/2, and Yap1801/2, directly bind to the initiator–clathrin lattice and form the endocytic puncta (in gray).