Guanylic nucleotides are not only "building blocks" for nucleic acids but are also crucial for the regulation of many cellular processes such as G-proteins based signaling pathways. Therefore, cells must maintain their concentrations at a critical level. However, the molecular relationships between intracellular guanylic nucleotide levels and cell proliferation crucial events remain poorly understood.
Here, we have demonstrated that MPA treatment does not cause a firm cell cycle arrest in yeast. Treated cells continue to proliferate, although at a reduced rate (this study and ). Further, we have shown that conditional mutants unable to synthesize guanylic nucleotides do not arrest in a particular stage of the cell cycle. Therefore, our results establish that there is no guanylic nucleotide checkpoint is S. cerevisiae. By contrast, in mammalian cells, it was previously shown that MPA treatment cause an arrest of cellular proliferation but no guanylic nucleotide specific checkpoint has clearly been identified. Several reports have described that MPA treatment affects mammalian cells ability to commit into division by blocking the transition from G0 to the S phase of the cell cycle [13, 17, 22]. However, if MPA is added when cells have already entered the S phase, the cell cycle arrest occurs in G2/M . Besides, the MPA-induced arrest is not fully reversed by the replenishment of guanylic nucleotide pools [13, 17, 22]. Here, we have shown that even if MPA treatment slows all stages of the yeast cell cycle progression, the most affected step is the mother-daughter cell wall separation, giving rise to "bibudded" cells. This result supports our previous observation that MPA treatment leads to the appearance of many cells with 3N DNA content . Importantly, MPA treatment has the same effects on both non-synchronized and synchronized cells treated with the drug upon release from G1 (Fig. 2) or upon release from G2/M after nocodazole synchronization (I. S., B. D.-F. unpublished results). Therefore, unlike in mammals, in yeast, MPA treatment causes the same effects whatever the cell cycle stage of the cells at the time of drug addition. Further, our analysis of mutants demonstrates that the mother-daughter separation defect results solely from guanylic nucleotide pools depletion and is independent of potential MPA secondary targets.
In yeast, the fact that a guanylic nucleotides starvation causes a mother-daughter separation defect was unexpected. Indeed, one could have intuitively supposed that this depletion would rather provoke a drastic defect during DNA replication upon S phase or disturb cell cycle steps for which GTPase driven molecular processes are essential. In fact, upon MPA treatment, cells can still build a daughter cell and cell polarity was found even less affected in this study than in our earlier work . Daughter cell appears to grow normally, mitosis proceeds unperturbed, and mother-daughter closure is properly achieved. Thus, guanylic nucleotides starvation does not critically affect the functions of key GTP binding proteins, such as Cdc42p, Tem1p, tubulin and septins. In this study we observed that in the "bibudded" cells, placement of the second daughter cell properly follows the axial budding pattern of haploid cells, suggesting that the bud-site selection machinery is properly located. In contrast, prolonged MPA treatment (48 hours) leads to a random budding pattern . Thus, long-term guanylic nucleotides starvation may have more drastic effects. Most importantly, our experiments show that complete mother-daughter separation is not required for the mother cell to pass through START and to generate a second daughter cell (Fig. 2E). Therefore, our data confirm that no additional checkpoint blocks the cell cycle progression when anaphase is properly achieved.
What molecular targets trigger the mother-daughter separation defect upon guanylic nucleotide starvation? Observation of "bibudded" cells by electron microscopy revealed no obvious defect in the overall septum architecture of abnormally unseparated daughter cell. Nevertheless, MPA treatment increases cells sensitivity to SDS , zymolyase or sonication (I. S., B. D.-F. unpublished results), strongly suggesting cell wall defects. In fact, previous works have illustrated links between guanylic nucleotides metabolism and cell wall integrity, particularly through the synthesis of mannoproteins, essential components of the fungal cell wall. GDP-mannose is the common substrate for mannosyltransferases, enzymes catalyzing the addition of mannose residues on mannoproteins core oligosaccharides. In budding yeast, the GDP-mannose pyrophosphorylase Psa1p is an essential enzyme that synthesizes GDP-mannose, using GTP as a substrate. It was previously shown that MPA treatment affects Psa1p expression  and that Psa1p depletion leads to cell separation failure . Further, Shimma et al have demonstrated that the major defect of a guk1 conditional mutant strain, that is impaired for GDP biosynthesis (Fig. 1), was a decrease in GDP-mannose level (to about 25% of the wild type levels) that leads to mannose outer chain elongation defects . Accordingly, we have observed cell separation defect in guk1 cells (I. S., B. D.-F., unpublished results). In addition, yeast lacking mannosyltransferase encoding genes, such as OCH1, MNN10 or ANP1 display both hypersensitivity to MPA  and a cell separation defect similar to the one observed for guanylic nucleotide starved cells [25–27]. Therefore, one major consequence of guanylic nucleotide starvation could be a significant decrease in the GDP-mannose pool that in turn leads to a mother-daughter separation defect.
The most intriguing aspect of the regulation of intracellular guanylic nucleotide pools is the correlation between the IMPDH activity and cellular proliferation in mammalians models. Interestingly, the transcription of the IMD2 gene is actively shut off via regulatory sequences when yeast cells enter stationary phase upon nutrients limitation . Transcriptome analyses have demonstrated that AAH1, HPT1 and GUA1 are among the most promptly down regulated genes when nutrients become limiting. Thus, it appears that an entire process is devoted to rapidly decrease the intracellular guanylic nucleotide pools when cells enter stationary phase. Here, we have demonstrated that the typical "bibudded" phenotype obtained during guanylic nucleotide starvation also occurs in untreated wild type cells achieving their last divisions upon nutrients limitation. Thus, an identical morphology is observed for both guanylic nucleotide starvation and entry into quiescence.
Finally, the GTP/GDP ratio is very sensitive to growth conditions, rapidly decreasing during the diauxic shift and drastically dropping upon nutrients starvation. Further, this ratio may regulate RAS GTPases activity by influencing its guanylic nucleotide loading equilibrium . RAS and TOR pathways are key regulators that coordinate yeast proliferation with nutrients availability. Recent work has suggested that the TOR protein acts as ATP sensor in mammals . Thus it is appealing to speculate that in parallel to the TOR pathway, intracellular guanylic nucleotides levels are part of a signal that regulate cell proliferation via the modulation of RAS GTPases activity.