Nutritional modulation of cell size at s phase initiation in the buddine yeast saccharomyces cerevisiae: a role for glucose sensing and the cyclin dependent kinase inhibitor

The budding yeast Saccharomyces cerevisiae is a model organism for studies on cell cycle. For the survival of this cells a tight coordination of cell growth and division occurs at Start, a regulatory area of the cell cycle positioned immediately before beginning of S phase, at the G1-S boundary. Start is the event, or set of events, that commits a cell to a round of division. This mechanism is based on achieving of a critical cell size (protein content per cell at the onset of DNA replication, Ps) to enter into S phase. Ps increases in proportion with ploidy and is modulated by nutrients. In fact, in bach cultures, the average cell size remains at low levels during growth on non-fermentable substrates, while the average size of cells increases in a linear way with the specific growth rate only during growth on fermentable substrates. A genome-wide genetic analysis has suggested that cell size control could be due to ribosome biogenesis rate, one of the most energetic demanding processes in a cell and it is modulated according to nutrient availability. Indeed, a large cluster of genes involved in ribosome biogenesis, have been identified in a screen for small size (whi) mutants. This includes SFP1 and SCH9 genes. The first encode a zinc finger protein, promoting the transcription of a large cluster of genes involved in ribosome biogenesis, where the latter is serine threonine protein kinase involved in stress response and nutrient-sensing signaling pathway. Recent work from our laboratory allowed to identify that Far1, a cyclin kinase dependent inhibitor, and Cln3, a G1 phase cyclin, may form a nutritional modulated threshold controlling the entrance into S phase. Two parallel pathways downstream from the TORC1 complex regulate expression of genes encoding ribosomal proteins (RP) and the so-called RiBi regulon, composed by genes involved in ribosome biogenesis. The two pathways involve Sfp1, and Sch9. Therefore it was of interest to see whether the increase in size (RNA and protein) brought about by FAR1 overexpression was mediated by Sfp1 and Sch9. The effect of FAR1 overexpression on cell size parameters in the wild type BY4741 strain (isogenic to the sch9Δ and sfp1Δ mutants), grown in synthetic complete media supplemented with either ethanol or glucose as a carbon source, was similar to that reported in the W303 background. sfp1Δ and sch9Δ mutants were much smaller than wild type both in glucose - confirming previous data (Jorgensen et al., 2002; Jorgensen et al., 2004) - and ethanol-supplemented media. As observed in wild type cells, in both mutant strains FAR1 overexpression had only minor effects on cell cycle and cell size related parameters on glucose-grown cells. FAR1 overexpression did not affect duplication time in ethanol-grown sch9Δ cells, while sfp1Δ mutants overexpressing the FAR1 gene product were quite unhealthy with a large increase in duplication time. Overall increase in cell size was dramatic in ethanol-grown cells: however, while in wild type cells and sch9Δ mutants the increase in cell size derived from a balanced increase in RNA and protein content, in the sfp1Δ mutant the increase in protein content was not accompanied by an increase in RNA content, as shown by both FACS and chemical analysis, indicating that the Sfp1 is required to maintain proper coupling of RNA and protein syntheses when the Far1 protein is overexpressed in ethanol-grown-cells. The observation that FAR1 overexpression has different effects in sfp1Δ cells grown in ethanol and glucose media was not entirely unexpected. First, in untransformed cells, the Far1 level of glucose-grown cells is larger than in ethanol-grown cells, while ectopically expressed Far1 accumulates to a similar level regardless of the carbon source: as a result, Far1 overexpression is more dramatic in ethanol-grown cells than in glucose-grown cells (Alberghina et al., 2004). Accordingly, the effect of Far1 overexpression on cell size are minor on cells grown in glucose-supplemented media and much more dramatic in ethanol-grown cells. In the second part of this study we try to determine whether (and possibly, to which extent) the regulatory function of glucose can be separated from its nutrient function. To this aim, we characterized yeast strains in which glucose sensing is strongly reduced. An essential requisite for the survival of free living microorganism like the budding yeast Saccharomyces cerevisiae is the capacity to regulate growth and cell cycle progression according to the frequent changes in the nutrient status, so that proliferation is rapid when large supplies of nutrients are available and ceases when these becomes exhausted. Nutrients like glucose must therefore generate signals that are somehow received and elaborated by the complex machinery governing growth and cell cycle progression. Besides being the favorite carbon and energy source for S. cerevisiae, glucose can act as a signaling molecule (“hormone”) to regulate multiple aspects of yeast physiology: addition of glucose to quiescent or ethanol growing cells triggers a fast and massive reconfiguration of the transcriptional program, which enables the switch to fermentative metabolism and promotes an outstanding increase of the cell biosynthetic capacity. Yeast cells evolved several mechanisms for monitoring glucose level in their habitat: the cAMP-PKA pathways (with its two branches comprising Ras and the Gpr1-Gpa2 module), the Rgt2/Snf3-Rgt1 pathway and the main repression pathway involving the kinase Snf1. In order to investigate whether the glucose effect on cell size was due to its function as nutrient, that require metabolism of the sugar, or to sensing of extracellular glucose levels, yeast strains in which one or more of the glucose sensing pathway was impaired, due to gene deletion of glucose receptors (GPR1, SNF3, RGT2), were analyzed. These mutants show only a partial nutritional modulation of cell size and/or of duplication time. The gpa2Δ gpr1Δ strain does not show substantial changes in duplication time compared to its isogenic wild type grown in the same conditions, while its protein content is consistently lower. In the snf3Δ rgt2Δ and in the snf3Δ rgt2Δ gpa2Δ gpr1Δ strains a lower protein content is accompanied by an increase in duplication time, when compared to wild type strain. Furthermore, in all glucose sensing mutants the variation of protein content, as function of glucose levels, is less than the wild type. In the presence of ethanol, the kinetic parameters of mutants strain analyzed are comparable to wild type: there is only a strong increase in the duplication time, while there isn’t a further decrease in protein content compared to 0.05% glucose concentration. Data obtained show that the Gpa2-Gpr1 pathway specifically modulates Ps setting, while the Snf3-Rgt2 pathway plays an important role in Ps and growth rate setting of the cells. In conclusion, this work highlighted that the elements involved in the cell size determination are multiple and interconnected. A strong alteration in cell size and protein content could originate not only from alteration in the dosage of genes involved in the molecular mechanism of the threshold which controls Ps, but also from environmental conditions. Of particular relevance seems to be that glucose effect is largely acting as a signaling molecule, rather than as an energy source. Further studies are necessary in order to clarify the molecular mechanism that link the glucose sensing to the molecular machinery responsible of G1-S transition.