Characterization of differentiated quiescent and nonquiescent cells in yeast stationary-phase cultures

Abstract

Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental changes. Our results indicate that Q and NQ cells differentiate significantly, with Q cells providing genomic stability and NQ cells providing nutrients to Q cells and a regular source of genetic diversity through mutation and transposition. These studies are relevant to chronological aging, cell cycle, and genome evolution, and they provide insight into complex responses that even simple organisms have to starvation.

Figure 1.

Distribution of gene sets in VxInsight Q/NQ gene-expression and cell cycle topographies (Spellman et al., 1998). Clustering is described in Materials and Methods. Hill height is a function of the number of genes in that cluster. (A) Clustering of gene expression for 178 arrays from Q and NQ fractions. Green dots, genes whose mRNAs are significantly increased in Q fractions; white dots, genes that are significantly increased in NQ fractions. (B) Localization of Q (green dots) and NQ (white dots) from A on the gene expression topography from Spellman's cell cycle data set. Yellow spots in upper right are G1-regulated genes. (C) Distribution of G1-regulated genes from B as a function of Q/NQ gene expression topography. (D) Green dots, ribosomal hill; white dots, distribution of the top 200 aging genes (Powers et al., 2006) as a function of the Q/NQ gene-expression topography.

Figure 2.

mRNA abundance in Q and NQ samples treated with or without proteinase K. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) was used to cluster ∼2400 transcripts. Samples include, unseparated SP culture (SP) and Q and NQ cell fractions. Cell lysates from samples were incubated with buffer alone (−) or proteinase K (+). Before clustering, treated samples were normalized to untreated samples (black lanes). The color scale at the bottom indicates the log₂ values for changes in mRNA abundance.

Figure 3.

Venn diagram of transcripts that increased after proteinase K treatment of cell lysates. Transcripts were evaluated that had a ≥1.5-fold increase in abundance after proteinase K treatment of SP culture and Q and NQ cell fraction lysates. The transcripts that were evaluated were required to be present in 80% of the data points.

Figure 4.

Model for cell differentiation in yeast grown in rich, glucose-based medium (YPD) after glucose and carbon starvation. Glucose exhaustion leads to the formation of daughter cells, which will become quiescent, and mother cells, 50% of which will become apoptotic by day 14 after inoculation. The NQ cells rapidly lose the ability to reproduce, and many of the NQ cells that can reproduce lose mitochondrial function, i.e., are petite. We hypothesize that this is a result of genomic instability in these cells. Q cells contain large P-bodies (Ray, unpublished) and the majority of protease-released mRNAs. In addition, based on abundant mRNAs, these cells are also poised to respond to a variety of environmental changes. Because a major group of protease-released mRNAs includes those encoding proteins involved in recombination and transposition, it seems likely that these cells are also poised to become apoptotic. We hypothesize that the differentiation of these cells, including the group of NQ cells that undergoes genomic rearrangements, is an evolutionarily conserved process that serves to provide both stability in the quiescent cells and innovation in the nonquiescent cells during times of nutrient limitation.