The CLS defects in the <i>rim15Δyak1Δ</i> or <i>rim15Δmck1Δ</i> mutants are rescued by <i>GSY2</i>/<i>TSL1</i> overexpression and/or trehalose supplementation.

<p><b>6A</b> and <b>6B</b>: Stress resistance (6A) and storage carbohydrates (6B) accumulated in the <i>rim15Δyak1Δ</i> or <i>rim15Δmck1Δ</i> cells bearing the empty vectors, <i>GSY2</i>/<i>TPS1</i> or <i>GSY2/TSL1</i> overexpression plasmids. <b>6C</b>, <b>6D</b> and <b>6E:</b> Relative CFU of WT (6C), the <i>rim15Δyak1Δ</i> (6D) and <i>rim15Δmck1Δ</i> (6E) mutants overexpressing <i>GSY2</i>/<i>TPS1</i> or <i>GSY2/TSL1</i>. <b>6F</b>, <b>6G</b> and <b>6H:</b> Supplementation of trehalose enhances the ability of WT cells to exit from quiescence (6F), ameliorates or rescues the CFU defects of the <i>rim15Δyak1Δ</i> (6G) and <i>rim15Δmck1Δ</i> (6H) cells. Cells were grown in YPD supplemented with 1% Trehalose from inoculation.</p

Identifying signaling mutants displaying defects in starvation-induced gene expression.

<p><b>1A:</b> 272 signalling mutants and WT cells transformed with the pSSA3-RFP construct were arrayed in quadruplets on SMM medium containing 0.6% glucose and supplements, grown for 2–3 days. Images with a dark background obtained by scanning were analysed with Cell Profiler (<a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a>). Those showing significant increased or decreased expression of pSSA3-RFP were subjected to quantitative assay of both reporters and cell growth (OD_595nm) using a plate reader. <b>1B:</b> Relative expressions of pSSA3-RFP and pHSP12-HSP12-VFP in mutants to those in WT cells after normalising to OD_595nm. Contained in the red box are WT, <i>bck1Δ</i> and <i>slt2Δ</i> deletion cells bearing the pHSP12-HSP12-VFP or pSSA3-RFP constructs, patched on SMM (agar) medium and grown for 2 days before imaging.</p

The working model showing that quiescence exit and chronological lifespan in yeast is dependent on the accumulation of storage carbohydrates and the intracellular ROS levels controlled by the kinases negatively regulated by TORC1/PKA.

<p>Yak1, Mck1 and Rim15 may also act together to negatively control the G₁/S transition to limit the population entering the stationary phase. Arrow indicates activation; bar denotes inhibition and the dashed lines are regulations to be confirmed. Trehalose plays a dominant role over glycogen in CLS extension and therefore indicated by a broad arrow.</p

Phenotypic analysis of the single, double and triple mutants of <i>RIM15</i>, <i>YAK1</i> and <i>MCK1</i>.

<p><b>3A:</b> Expression levels of pHSP12-HSP12-VFP at 20h and of pSSA3-RFP at 48h in the above mutants. <b>3B:</b> Heat and oxidative stress resistance displayed by WT and mutant cells grown for 3 days in YPD. <b>3C:</b> Glycogen and trehalose accumulated in WT and mutant cells grown for 3 days in YPD. <b>3D:</b> The number of colonies formed by WT and mutant cell cultures grown in YPD for 3 days. <b>3E:</b> Median, 1^st and 3^rd quartiles of cell size of the G_d and G₁ populations in the day 3 culture. <b>3F:</b> Median, 1^st and 3^rd quartiles of cell size of the S-G₂-M and >2C populations in the day 3 culture. At least 100,000 G_d/G₁ and 15,000 ≥2C cells from each culture were scored. The pairwise comparisons between WT and each mutant in 3e and 3f reveal that all p values calculated from the t-test scores are less than 0.0001.</p

The levels of intracellular ROS in early stationary-phase cells are controlled by Rim15, Yak1 and Mck1.

<p><b>7A</b>: ROS levels determined in WT, the single, double and triple mutants of <i>RIM15</i>, <i>YAK1</i> and <i>MCK1</i>. <b>7B</b>: ROS levels detected in WT, the single and double mutants of <i>GSY2</i> and <i>TPS1</i>. WT and mutant cells were grown in YPD for 3 days before staining by DHR. The mean value represents the average of two biological and two technical replicates. In each replicate, at least 10, 000 cells were scored. The p values, listed below each mutant, were calculated by pairwise t-test between the WT and mutant.</p

The accumulation of both trehalose and glycogen is necessary for quiescence exit.

<p><b>5A:</b> Glycogen and trehalose levels determined in the <i>gsy2Δ</i>, <i>tps1Δ</i> and <i>tsl1Δ</i> single and the <i>gsy2Δtps1Δ</i> and <i>gsy2tsl1Δ</i> double mutants. <b>5B:</b> Heat and oxidative stress resistance displayed by the above mutants. <b>5C:</b> Relative CFU of the above mutants over 28 days. <b>5D:</b> Comparison between relative CFU and cell viability of the <i>tps1Δ</i> and <i>gsy2Δtps1Δ</i> mutants over 28 days.</p

A model showing that transition-phase cell cycle, cell size and the acquisition of quiescence-related characteristics are coordinated by Mck1 and Rim15.

<p>Arrows indicate activation and bars denote inhibition. Dashed line means suggested interaction. Blue squares indicate chromosomal DNA.</p

CLS of the signaling mutants is well correlated with the amount of storage carbohydrates.

<p><b>2A:</b> Relative CFU of WT and ‘signaling’ mutants at 7, 14 and 21 days normalised to that at day 0. Day 0 data is collected after cells were grown in YPD for three days to early stationary phase. <b>2B, 2C, 2D and 2E:</b> Correlation coefficient (R) between relative CFU (at day 14) and trehalose (2B), the sum of trehalose and glycogen (2C), glycogen (2D) or the ratio of G_d/G₁ (1C) cells at 24h (2E).</p

Chronological lifespan of the single, double and triple mutants of <i>RIM15</i>, <i>YAK1</i> and <i>MCK1</i> is well correlated with the accumulation of storage carbohydrates.

<p><b>4A:</b> Relative CFU measured over 21 days. <b>4B:</b> Comparison between relative CFU and cell survival displayed by WT and mutant cultures at day 12. <b>4C</b>, <b>4D</b> and <b>4E:</b> Correlation between relative CFU (at day 15) and total amount of storage carbohydrates (4C), trehalose (4D) or glycogen (4E). <b>4F:</b> Correlation between relative CFU and the ratio of G_d/G₁ (1C) cells at 24h in YPD.</p

<i>TOR1</i>-regulated quiescence establishment and exit is mediated by <i>RIM15</i>, <i>YAK1</i> and <i>MCK1</i>.

<p><b>8A</b>, <b>8B</b>, <b>8C</b> and <b>8D</b>: Stress resistance (8A), storage carbohydrates (8B), relative CFU in H₂O (8C) or relative CFU in spent medium (8D) displayed by the <i>tor1Δ</i> mutants carrying also the single, double or triple deletions of <i>RIM15</i>, <i>YAK1</i> and <i>MCK1</i>. <b>8E</b> and <b>8F</b>: Comparison of cell survival of WT, <i>rim15Δ</i>, <i>mck1Δ</i> and <i>rim15Δmck1Δ</i> cells in spent medium carrying <i>tor1Δ</i> deletion (8E) or an intact <i>TOR1</i> (8F).</p