The unfolded protein response is not necessary for the G1/S transition, but it is required for chromosome maintenance in Saccharomyces cerevisiae

Abstract

Background: The unfolded protein response (UPR) is a eukaryotic signaling pathway, from the endoplasmic reticulum (ER) to the nucleus. Protein misfolding in the ER triggers the UPR. Accumulating evidence links the UPR in diverse aspects of cellular homeostasis. The UPR responds to the overall protein synthesis capacity and metabolic fluxes of the cell. Because the coupling of metabolism with cell division governs when cells start dividing, here we examined the role of UPR signaling in the timing of initiation of cell division and cell cycle progression, in the yeast Saccharomyces cerevisiae.

Methodology/principal findings: We report that cells lacking the ER-resident stress sensor Ire1p, which cannot trigger the UPR, nonetheless completed the G1/S transition on time. Furthermore, loss of UPR signaling neither affected the nutrient and growth rate dependence of the G1/S transition, nor the metabolic oscillations that yeast cells display in defined steady-state conditions. Remarkably, however, loss of UPR signaling led to hypersensitivity to genotoxic stress and a ten-fold increase in chromosome loss.

Conclusions/significance: Taken together, our results strongly suggest that UPR signaling is not necessary for the normal coupling of metabolism with cell division, but it has a role in genome maintenance. These results add to previous work that linked the UPR with cytokinesis in yeast. UPR signaling is conserved in all eukaryotes, and it malfunctions in a variety of diseases, including cancer. Therefore, our findings may be relevant to other systems, including humans.

Figure 1. ER stress in the absence of UPR signaling blocks cell cycle progression and leads to increased ploidy.

A, the density (in cells/ml) of IRE1⁺ (strain X2180-5B) and ire1Δ (strain SCMSP176) cultures was monitored at regular intervals in the presence (or absence, as indicated) of 1 mM DTT. B, the DNA content of the same cultures and at the time-points shown in (A), was determined by flow cytometry. The x-axis of these histograms indicates fluorescence per cell, while the y-axis indicates the number of cells analyzed. The portion of the histograms in DTT-treated ire1Δ cells with increased ploidy (>2N) is indicated.

Figure 2. Loss of UPR signaling does not affect the timing of the G1/S transition.

A, the rate of cell size increase for each elutriation experiment of the indicated strains is shown (they were the same strains as in Fig. 1). From these graphs, we determined the rate of size increase (shown as fl/min). The average (± SD) is shown in each case. These experiments were done in standard “rich” media with slightly lower glucose content (1% yeast extract, 2% peptone, 0.5% dextrose). B, from the same elutriation experiments shown in (A), we also measured the percentage of budded cells as a function of cell size (shown in fl). The data points shown were from the linear portion of each experiment, when the percentage of budded cells began to increase, and used to determine the critical size for division. C, and D, are the same type of analyses described in (A), and (B), respectively, except that the medium used was white grape juice.

Figure 3. ER stress in the absence of UPR signaling decreases the rate of cell size increase, but it does not affect the critical budding size.

Synchronous early-G1 cultures of IRE1⁺ (strain X2180-5B) and ire1Δ (strain SCMSP176) were obtained by elutriation. Half of the elutriated culture for each strain was exposed to 1 mM DTT. The rate of cell size increase (A), and (C), and the critical budding size (B), and (D), were then monitored for IRE1⁺, and ire1Δ cells, respectively.

Figure 4. Loss of UPR signaling does not affect the nutrient or growth rate dependence of the G1/S transition.

A, from steady-state glucose-limited (0.08% glucose) chemostat cultures of IRE1⁺ (strain X2180-5B) or ire1Δ (strain SCMSP176) cells, we monitored the fraction of unbudded cells (G1 fraction), as a function of the dilution rate. B, a similar experiment as in (A), was done using cultures limited for nitrogen, containing 0.002% nitrogen ammonium sulfate.

Figure 5. The yeast metabolic cycle of ire1Δ cells.

A, oscillations of dissolved oxygen concentrations (shown as % saturation, DO₂) in continuous cultures of ire1Δ cells (strain SCMSP207). The average (± SD) period, T, of these oscillations is indicated. The arrow indicates the point of addition of glucose-limited media (0.08% glucose) and initiation of steady-state chemostat conditions. The rectangle placed around the 120 h time point indicates a cycle that was analyzed in further detail in (B). B, at regular intervals as indicated, samples were taken and analyzed for DNA content by flow cytometry (shown at the bottom). The portion of the histogram with increased ploidy (>2 N) is indicated in the last time point (#15), but similar sub-populations of cells with increased ploidy were evident at other time points as well.

Figure 6. Loss of UPR signaling leads to sensitivity to hydroxyurea.

IRE1⁺ and ire1Δ cells (in the CEN.PK strain background) were spotted at 10-fold dilutions on YPD plates (1% yeast extract, 2% peptone, 2% dextrose), under various genotoxic conditions, as indicated. The plates were incubated at 30°C for 3 days, and photographed.

Figure 7. Loss of UPR signaling leads to chromosome loss.

Sectoring assay for chromosome loss, with IRE1⁺ or ire1Δ cells, in the YPH363 strain background. Formation of red sectors indicates chromosome loss. Representative plates for each strain are shown, at 0 mm and 1 mM DTT. There were very few viable ire1Δ cells in the presence of 5 mM DTT, and this is illustrated by a streak of the two strains on the same plate containing 5 mM DTT. The total number of colonies counted and the percentage of sectored colonies in each case are shown.