FM4-64 staining

Yeast cells were grown overnight in YPD to late log phase. 1–1.5 ml of culture was collected, washed once with 1 ml YNB complete media, and resuspended with 100 ul of YNB complete media. Yeast cells were then labeled with FM4-64 (T3166, Invitrogen, 10 ug/ml final concentration) for 10 min at room temperature in the dark (Vida and Emr, 1995). For the endosome staining, cells were immediately washed with 1 ml ice cold YNB complete media to remove the FM4-64 and kept on ice to stop the membrane trafficking. For vacuole membrane staining, 1 ml room temperature YNB complete media was added to the 10 min FM4-64 incubating cells and the incubation was continued for another 50 min in the dark to ensure the vacuole membrane staining. The cells were then collected, and washed with 1 ml ice cold YNB complete media, and kept on ice. Cells were resuspended in milliQ water and imaged by fluorescence microscopy.