Figure 3.Role of the Sty1, Pka1 and TOR-Sck2 pathways in stationary phase. (A)
Lack of Pka1 and Sck2 kinases promotes stationary phase cell survival under
glucose rich conditions in a Sty1-, Atf1-dependent manner. Strains 972
(WT), AV18 (Δsty1), MC22 (Δpka1), MC24 (Δpka1
Δsty1), MC25 (Δsck2), MC27 (Δsck2Δsty1),
AV15 (Δatf1) and AZ118 (h- sck2::kanMX6
atf1::natMX6; Δsck2Δatf1) were grown in YE-4% glucose
media. Serial dilutions of the logarithmic (Log) and stationary phase (Day
4) cultures were spotted onto YE plates. (B) Loss of function of
the glucose dependent Pka1 kinase triggers enhanced intracellular H2O2
levels and Sty1 activation. Strains 972 (WT) and MC22 (Δpka1) were
grown in YE-4% glucose media. Cells were harvested at an OD600
of 0.5 and relative intracellular H2O2 levels were
analysed as described in Figure 2A. The same cultures were used to
characterize Sty1 phosphorylation from TCA extracts, using anti-p38-P
antibody. Wild type cells treated with 1 mM H2O2 for 10 min (H2O2) were used as a control of
activated Sty1. Anti-Sty1 antibody was used as a loading control. (C)
Activation of the transcriptional stress response at stationary phase is
Sty1-dependent and Cgs1-independent. Strains 972 (WT), AV18 (Δsty1),
AV15 (Δatf1), MC22 (Δpka1) and AZ106 (h-
cgs1::kanMX6 ura4-D18; Δcgs1) were grown in YE-1% glucose media.
The time points of the five growth curve were recorded approximately at the
same percentages of the maximum OD600 of each culture. Standard
deviation for every point is indicated. At the time points indicated (A to
D), cells were collected and RNA samples were obtained and hybridized
against fbp1, atf1, gpx1, cta1 and gpd1.
(D) Pka1 pathway is required for stationary phase survival upon
calorie restriction. Strains 972 (WT) and AZ106 (Δcgs1) were
grown in YE-1% glucose media. At the logarithmic phase (Log) or 72 hours
after reaching the stationary phase (Day 3) serial dilutions of the
cultures were plated onto YE plates. (E) Strains 972 (WT), AZ106 (Δcgs1),
AZ103 (Δpyp1) and AZ115 (h- pyp1::kanMX6
cgs1::natMX6; Δpyp1 Δcgs1) were grown in YE-4% glucose
media. At the logarithmic phase (Log) or several days after reaching
stationary phase (Day 2 and Day 5) serial dilutions of the cultures were
plated onto YE plates.
