(A) Structure of a Pan1p phosphorylation mutant. The two amino-terminal Eps15 homology (EH) domains, long repeat (LR) regions, predicted coiled-coil (CC), acidic region (A), and carboxy-terminal prolin-rich domain (PR) domain, are indicated. The fifteen consensus phosphorylation sites previously mutated in Pan1-15TA are indicated below the protein in black. The three additional sites mutated in Pan1-18TA are in red. (B) Plate showing the growth phenotype of pan1-15TA and pan1-18TA mutants. A dilution series of cells was plated on YPD plates and incubated for 2–3 days at 25 or 37°C, respectively. (C) Analysis of the phosphorylation state of the Pan1-18TA mutant. Protein expression was analyzed by immunoblotting 20 μg of total cell lysate (TCL) with an anti-GFP antibody (left panel). Phosphorylated proteins were purified from TCL with Phos-tag agarose, run on SDS-PAGE and immunoblotted with the anti-GFP antibody (right panel) as described in Materials and methods. Lane 1, JJTY369; lane 2, JJTY509; lane 3, JJTY5486. (D) Alexa Fluor 488-phalloidin staining of fixed wild-type and pan1-18TA cells to visualize actin. (E) The effect of Pan1p phosphorylation-site mutations on endocytic internalization. Radiolabeled α-factor internalization assays performed on wild-type (blue), pan1-15TA (yellow), pan1-18TA (magenta), pan1-18TA sla1-10TA (green), or ark1△ prk1△ (black) cells at 25°C. Each curve represents the average of three independent experiments, and error bars indicate the SD at each time point. (F) The localization of Pan1-GFP in wild-type and pan1-18TA cells. Cells expressing Pan1-GFP and Abp1-mCherry were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy. Merged images of GFP and mCherry channels are shown in the right panels. (G) Endocytic cargo is transported to the vacuole through the actin clumps in pan1-18TA. pan1-18TA cells were labeled with A594-α-factor as described in the Methods. The images were acquired simultaneously at 1, 5, 15, and 30 min after washing out unbound A594-α-factor and warming the cells to 25°C. Scale bars, 2.5 μm.