FIGURE 2: Atg1 influences the cell viability independently on the Pmk1 MAPK pathway. (A) Influences of overexpression of Atg1 and the kinase-defective variant Atg1^D193A on cell growth. Cells transformed with the thiamine repressible-expression vectors, pREP1-GFP, pREP1-atg1⁺-GFP, or pREP1-atg1^D193A-GFP were streaked onto an EMM plate with or without 4 µM thiamine and incubated for 5 days at 27°C. (B) Measurement of the viability of the cells subjected to Atg1 overexpression. Cells transformed with pREP1-atg1⁺-GFP or its control vector were incubated in liquid EMM in the absence of thiamine for 48 hours at 27°C. The cells were then 10-fold serially diluted as indicated (10⁰ to 10⁻⁴) starting from OD₆₆₀ = 0.5, and 5 µL were spotted onto EMM plates with or without thiamine. Plates were incubated for 4.5 days at 27°C. Two independent transformant clones (#1 and #2) for pREP1-atg1⁺-GFP were tested. (C) Overexpression of Atg1 induces cytotoxicity independently on Pmk1. Δpmk1 cells harboring pREP1-GFP or pREP1-atg1⁺-GFP and wild-type cells harboring pREP1-GFP were streaked onto an EMM plate with or without 4 µM thiamine and incubated for 4 days at 27°C.