Research Perspective Volume 2, Issue 4 pp 231—237

Figure 4. Quiescence establishment upon nitrogen, sulphate or phosphate starvation is glucose- and Sty1-dependent. (A) Sty1 and carbon source are necessary for lifespan extension upon nutrients starvation. 972 (WT) and AV18 (Δsty1) strains grown in minimal media until OD₆₀₀ 0.5 were harvested by centrifugation. Then, cells were washed twice with minimal media without nutrients and resuspended to a final OD600 of 0.1 in minimal media lacking glucose (MM-G), nitrogen (MM-N), nitrogen and glucose (MM-N/G), sulphate (MM-S), sulphate and glucose (MM-S/G), phosphate (MM-P), phosphate and glucose (MM-P/G). Five minutes (5 min) and 25 days (Day 25) after the shift, serial dilutions of the cultures were plated onto YE plates. (B) Sty1 is activated upon nutrients depletion. Wild type cells treated like in A and resuspended in MM, MM-N, MM-S and MM-P with a final OD600 of 0.5 were recollected 10 or 60 minutes after the shift and Sty1 phosphorylation was determined from TCA extracts using anti-p38-P antibody. Anti-Sty1 antibody was used as a loading control. (C) Lack of Cgs1 impairs quiescence maintenance upon nitrogen depletion. Strains 972 (WT), AZ74 (h- pka1::kanMX6), AZ73 (h- sck2::kanMX6), DC3 (h- cgs1::kanMX6) and AZ107, harbouring the as mutation sty1.T97A [1] and treated or not with 5µM ATP analogue 1NM-PP1 (sty1as + 1NM-PP1 and sty1as respectively) were grown in minimal media until OD₆₀₀ 0.5. Addition of the ATP analogue specifically inhibits the Sty1.T97A kinase activity. Cells were harvested by centrifugation, washed twice with milli Q water and resuspended to a final OD₆₀₀ of 0.1 in minimal media lacking nitrogen (MM-N). Five minutes (5 min), 10 and 20 days (Day 10 and Day 20) after the shift/resuspention serial dilutions of the cultures were plated onto YE plates.