FIGURE 2.

PI4P biosensor is enriched on the plasma membrane following inositol starvation. A, wild type (BY4742) cells, expressing a PI(4,5)P₂-binding probe (GFP-2XPH^PLCδ1) from the constitutive PRC1 promoter carried on a 2μ plasmid (pRS426), were pre-grown at 30 °C in synthetic media containing 75 μm inositol. At mid-logarithmic growth phase, cells were harvested by filtration, washed, and resuspended in medium with (I⁺) or without (I⁻) inositol. Cells were subsequently incubated for 120 min at 30 °C and examined by fluorescence microscopy. Images in the left and middle panels represent >90% of cells observed. Closed arrows indicate plasma membrane. The image in the right panel represents ∼5% of cells growing in the absence of inositol. Arrowheads point to endosomes. Overlay with differential interference contrast (DIC) image is shown. Scale bar, 5 μm. B, wild type cells (BY4742), constitutively expressing a PI4P-binding probe (GFP-2XPH^Osh2) from pRS426-PRC1, were pre-grown, harvested, washed, and resuspended in I⁺ or I⁻ media as described in A. Following the medium shift, cells were incubated at 30 °C and examined by fluorescence microscopy at the indicated times. Images are representative of >95% of cells observed. Arrowheads indicate Golgi. Closed arrows show polarized plasma membrane distribution in bud tips (I⁺ and I⁻ 60 min) and uniform plasma membrane distribution in mother and daughter cells (I⁻ 120 min). Open arrow shows early appearance in plasma membrane. Scale bar, 5 μm. C, wild type (BY4742) cells were labeled to steady state with myo-[³H]inositol in the presence of 75 μm inositol. One-third of the culture (I⁺) was collected, and lipids were extracted, deacylated, and separated by HPLC. The remaining two-thirds of the culture was harvested by filtration, washed, resuspended in medium lacking inositol (I⁻) without label, and incubated at 30 °C. At 60 and 120 min, cells were harvested and processed as for the I⁺ sample.