Figure 2:

Mutation of 6 Msn2 PKA–targeted serines to alanine is detrimental for growth. (A) Scheme of Msn2A6. Induced expression of Msn2A6 by a copper-regulated promoter (CUP1) represses growth on (B) solid medium and in (C) liquid culture (50 µM CuSO₄). (D) FACS analysis of the terminal phenotype of arrested Msn2A6 cells. Expression of CUP1-MSN2 and CUP1-MSN2A6 was induced in W303 msn2∆msn4∆ cells by addition of CuSO₄ (6.25 µM) to liquid cultures at an OD_600nm of 0.7. Cells were grown further to an OD_600nm of 1 (first time point T = 0) and kept in logarithmic growth by continuously diluting the culture with prewarmed media containing copper sulfate. At four additional time points (T = 1–4 h), samples were taken. An increasing number of cells expressing Msn2A6 are in the G1/S phase of the cell cycle. Induced expression of Msn2A6 in W303 msn2∆msn4∆ cells by a 17-ß-estradiol–induced system represses growth on (E) solid medium and in (F) liquid culture, showing a transition from exponential to linear growth.