Figure 4. Simultaneous inactivation of CORVET and HOPS prevents GFP-Atg8 proteolysis but not the formation of autophagosomes. (A) Growth tests of a wild-type strain compared with hbrA2/ vps33^ts. (B) GFP-Atg8 processing assays in hbrA2/vps33^ts. When indicated, strains were shifted to 42°C for 45 min before inducing autophagy. (C) Two examples of autophagosome cycles in hbrA2 (vps33^ts) cells in which autophagy had been induced after shifting cells to 42°C in an incubator. To prevent the (minor) decrease in the incubation temperature that might occur during image acquisition from permitting recovery of functional HbrA2/Vps33, the culture was photographed within 10 min after the incubation chamber was transferred from the 42°C incubator to the microscope set-up adjusted to 37°C. (D) Examples of GFP-Atg8 autophagosomes (marked with arrows) in a hbrA2/vps33^ts cell shifted to nitrogen-starvation conditions at 42°C. (Note that, upon shifting cells to the restrictive temperature, the mutation arrests apical extension, leading to tip swelling.) One cell was counterstained with CMAC (magenta pseudocolor) to show that none of the numerous mini-vacuoles seen under these conditions overlaps with the autophagosome. (E) Our experiments show that autophagosomes fuse with late endosomes/vacuoles in a RabS^RAB7-dependent manner, although they can also fuse with early endosomes inefficiently (discontinuous line).