Fig. 5. Loss of Vps20p or Vta1p causes defects in -factor degradation. Wild-type (RH1800) and vps20 (AMY174) (A), and wild-type (AMY165) and vta1 (AMY162) (B) cells were grown to early exponential phase and assayed for [³⁵S]-factor transport to the vacuole and degradation at 30°C. [³⁵S]-factor was prebound to the cells on ice and then the cells were harvested at 4°C and resuspended in fresh YPUAD and incubated at 30°C. At the time points shown, samples were taken in duplicate and washed in either phosphate buffer pH6 (removes unbound [³⁵S]-factor) or citrate buffer pH 1 (removes bound non-internalised [³⁵S]-factor). Lysates were prepared from each sample of cells and subjected to thin layer chromatography to separate intact (i) and degraded (d) [³⁵S]-factor, which were visualised by fluorography at -80°C. 6, washed in pH 6 buffer; 1, washed in pH 1 buffer; o, origin where samples were loaded.