Fig 5. Calcineurin activity and intracellular localization of ion transporters in ESCRT mutants.

(A) Real time monitoring of calcineurin activity in ESCRT mutants. Wild-type and ESCRT mutant cells harboring the multicopy plasmid 3×CDRE::luc(2.2) reporter vector were incubated with D-luciferin sodium salt and treated with 100mM NaCl or 100mM CaCl₂, as indicated. Using a luminometer, light emission levels expressed as relative light units (RLU) were measured per minutes for 3 hours. Graph shows the Area Under Curve (AUC) of 3×CDRE::luc(R2.2) reporter activity untreated or treated with NaCl and CaCl₂, respectively. The data were averaged from three independent experiments. Error bars, means±SD. ***P<0.001 compared with values from wild-type cells. (B) Intracellular localization of Trp663 and Trp1322 in ESCRT mutants. Wild-type, Δvps25 and Δdid4 cells harboring Trp663-GFP or GFP-Trp1322 were grown to early log phase in EMM plus adenine and uracil media containing 4 μM thiamine, and then were analyzed by fluorescence microscopy. Bar, 10 μm.