Figure 4.

Ultrastrucure of ubiquitin proteasome (UP) components and their presence within ATG-like vacuoles. UP components were identified by immunocytochemistry against either P20S or 19S/PA700 as shown in representative micrograph (A) where arrowheads show P20S-bound immunogold particles and representative micrograph (B) where arrowheads show 19S/PA700-bound immunogold particles. In both (A,B) these proteasome components are shown in the context of multiple membranes ATG-like vacuoles shown by arrows. However, the presence of UP component also occurred in the cytosol. In fact, as measured in graph (C), we carried out an extensive count of total cytosolic UP component. For these counts we used P20S immunogold particles. Again, even in this case we measured a dose-dependent increase in the number of P20S particles for increasing doses of rapamycin (1 nM; 10 nM; 100 nM and 1 μM) number of ATG-like compared with baseline conditions (B,C). When we compared such a rapamycin-dependent increase in cytosolic UP components with the dose-dependent increase in ATG-like vacuoles primarily expressed by graph Figure 1F we obtain graph (D) showing that the ratio between P20S immunogold particles out of total ATG-like vacuoles was steady for increasing doses of rapamycin (1 nM; 10 nM; 100 nM and 1 μM) number of ATG-like compared with baseline conditions (B,D). Values are given as the mean ± S.E.M. Comparisons between groups were made by using one-way ANOVA. *P ≤ 0.05 compared with baseline condition. Scale bars (A,B) = 112 nm.