ISOLATION OF YEAST MUTANTS DEFECTIVE FOR LOCALIZATION OF VACUOLAR VITAL DYES

An application of flow cytometric sorting is used for isolation of Sac charomyces cerevisiae mutants that mislocalize vacuolar vital dyes. Th is screen is based on the ability of a lipophilic styryl compound, mpr opyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide ( FM4-64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15 degrees C. Cells stained at 15 degrees C fo r both FM4-64 and carboxydichlorofluorescein diacetate (a vacuolar lum inal vital stain), had a pronounced shift in red/green fluorescence fr om cells stained at 30 degrees or 38 degrees C. Flow cytometric select ion based on this characteristic shift allowed the isolation of 16 mut ants, These comprised 12 complementation groups, which we have designa ted SVL for styryl dye vacuolar localization. These groups were put in to three classes. Class I mutants contain very large vacuoles; class I I mutants have very fragmented vacuoles; and class III mutants show th e strongest svl phenotype with punctate/diffuse FM4-64 staining. Limit ed genetic overlap was observed with previously isolated mutants, name ly svl2/vps41, svl6/vps16, and svl7/fab1, The remaining svl mutants ap pear to represent novel genes, two of which showed temperature-sensiti ve vacuole staining morphology, Another mutant, svl8, displayed defect s in uptake and sorting of phosphatidylcholine and phosphatidylethanol amine. Our flow cytometric strategy may be useful for isolation of oth er mutants where mislocalization of fluorescent compounds can be detec ted.