Figure 5. Overexpression of ubiquitin prevents Kog1 degradation in the tor2–2041 mutant cells.

The tor1 tor2–2041 (Y864) cells expressing KOG1-HA were transformed with empty vector or vector expressing ubiquitin. The transformed cells were grown to exponential phase at 23°C and shifted to 37°C or remained at 23°C. A. The levels of Kog1-HA in the cell extracts from the cells collected at different time points after the shift were determined by western blotting. Wild type cells expressing KOG1-HA (Y985) were used as control. The levels of Tpd3 were used as the loading control. B. Cell extracts were partitioned into membrane (P) and soluble (S) by centrifugation at 100, 000 × g. The distribution of Kog1-HA in each fraction was examined by western blotting. C. The tor1 tor2–2041 (Y864) cells were transformed with the GFP-ATG8 gene together with a control vector or vector expressing ubiquitin. The transformed cells were grown to exponential phase at 23°C and shifted to 37°C for indicated times. The cell extracts were analyzed by western blotting with anti-GFP antibody for the autophagic cleavage of GFP-Atg8 fusion protein.