Inhibition of InsP7 formation in WT yeast cells mimics the
defectin vacuole morphology reported for ipk2ΔIPK2 and
kcs1Δ strains. WT, kcs1Δ,
vip1Δ, and kcs1vip1ΔΔ cells were grown in
yeast minimal medium supplemented with 50 μCi/ml [3H]inositol
overnight at 30 °C. Inositol phosphates were extracted using a protocol
similar to that with mammalian cells. HPLC traces are shown in Fig. S3. For
vacuole labeling experiments, WT, ipk2Δ, or
kcs1Δ strains of haploid yeast cells were grown in YPD. WT
cells were grown in the presence of vehicle (DMSO) or TNP. They were then
stained with cell tracker CMAC (blue), which stains the vacuolar
lumen and membrane marker MDY-64 (green) according to the
manufacturer's instructions. Shown is a comparison of InsP7 levels
in WT, WT plus TNP, and kcs1Δ cells (A);
vip1Δ, vip1Δ+ TNP, and
kcs1vip1ΔΔ cells (B); and ddp1Δ,
ddp1Δ+TNP and ddp1kcs1ΔΔ cells
(C). D (i), WT cells grown in the presence of
vehicle (DMSO). D (ii), WT cells grown in the presence of
TNP (10 μm). D (iii), ipk2Δ
grown in the presence of vehicle (DMSO). D (iv), kcs1Δ
grown in presence of vehicle (DMSO).