Isolation of yeast mutants defective for localization of vacuolar vital dyes.
An application of flow cytometric sorting is used for isolation of Saccharomyces cerevisiae mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrie nyl )pyridinium dibromide (FM4-64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15 degreesC. Cells stained at 15 degreesC for both FM4-64 and carboxydichlorofluorescein diacetate (a vacuolar luminal vital stain), had a pronounced shift in red/green fluorescence from cells stained at 30 degrees or 38 degreesC. Flow cytometric selection based on this characteristic shift allowed the isolation of 16 mutants. These comprised 12 complementation groups, which we have designated SVL for styryl dye vacuolar localization. These groups were put into three classes. Class I mutants contain very large vacuoles; class II mutants have very fragmented vacuoles; and class III mutants show the strongest svl phenotype with punctate/diffuse FM4-64 staining. Limited genetic overlap was observed with previously isolated mutants, namely svl2/vps41, svl6/vps16, and svl7/fab1. The remaining svl mutants appear to represent novel genes, two of which showed temperature-sensitive vacuole staining morphology. Another mutant, svl8, displayed defects in uptake and sorting of phosphatidylcholine and phosphatidylethanolamine. Our flow cytometric strategy may be useful for isolation of other mutants where mislocalization of fluorescent compounds can be detected.
Zheng
B
B
Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Medical School, Houston, TX 77030, USA.
Wu
J N
JN
Schober
W
W
Lewis
D E
DE
Vida
T
T
D001693:Biological Transport, Active / Q000235:genetics; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004734:Energy Metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007425:Intracellular Membranes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010641:Phenotype; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014617:Vacuoles / Q000378:metabolism*
C092350:FM 4-64; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds