Figure 4.

Derepression of the INO1 gene and localization of Opi1p-GFP in wild type, OPI1^ffat, and scs2Δ strains following a shift to medium lacking inositol and choline. A, overnight cultures of YCY3 (wild type), YCY5 (OPI1^ffat), and YCY7 (scs2Δ) expressing genomic Opi1p-GFP were diluted to A_{600 nm} = 0.2 in I⁺C⁻ medium and allowed to grow to mid-logarithmic phase at 30 °C. Cells were harvested by centrifugation and washed and resuspended in I⁻ medium, followed by incubation for 3 h at the same temperature. Inositol was added back after 3 h of inositol starvation. Samples were taken at 0, 1, 2, and 3 h of inositol starvation and 30 min after adding back inositol. Total RNA was isolated and analyzed by RT-PCR as described under “Experimental procedures.” Solid triangles, wild type; solid squares, OPI1^ffat; solid crosses, scs2Δ. B, Opi1p-GFP localization over the same time course. Cells were imaged by fluorescence microscopy. A representative z-section is chosen for each image. Scale bar, 5 μm. White arrows, Opi1p-GFP associated with distinctive puncta.