Yeast cell size control: an interplay among ribosome biogenesis, protein synthesis and MAPK routes

Cell-size homeostasis requires that proliferating cells coordinate growth and cell cycle, such that each division is matched by a doubling of mass. Size homeostasis is a universal but poorly understood feature of the cell cycle control. In the unicellular budding yeast Saccharomyces cerevisiae, the coordination of division with growth occurs at Start, a short interval during late G1 phase, after which cells are committed to division. A prerequisite for the passage through Start is the attainment of a critical cell size, whose value is set by ploidy and growth conditions. The critical-size threshold maintains uniform the cell size over many generations, and under minimal nutrient conditions forces cells to accumulate the energy stores required to complete the division cycle. Nutrients modulate the critical cell-size threshold according to the proliferation rate. Generally, cells growing slowly on a poor medium pass Start at a smaller size than fast-growing cells on a rich medium. In S. cerevisiae mutants that subvert the size control process have two phenotypes: small (whi) and large (lge). The former undergo Start a smaller cell size and the latter at a larger. Moreover, a systematic determination of cell-size distributions for all yeast deletion strains identified many new potential Start regulators. Many of the genes encoding potential Start repressors are implicated in ribosome biogenesis, suggesting the existence of a link between these two seemingly disparate processes. One of the smallest whi mutant is linket to SFP1 gene deletion. sfp1Δ cells display a disproportionate effect on size relative to the change in growth rate. SFP1 gene encodes a zinc-finger protein that is a key transcriptional regulator of ribosome biogenesis whose function is required for normal yeast growth. Nuclear localization of Sfp1, requires active TORC1 and it is highly stress sensitive. In addition, Sfp1 interacts directly with and is phosphorylated by TORC1. In contrast to Sch9 kinase, a major downstream target of TORC1, TORC1 phosphorylation of Sfp1 is unaffected by either osmotic or nutritional stresses, suggesting a different mode of regulation. Significantly, Sfp1, through its transcriptional activation function, exerts a negative feedback control on TORC1 activity toward the Sch9 kinase. Sfp1 also interacts with Mrs6, a conserved Rab escort protein that in turn regulates Sfp1 nuclear localization. The Mrs6 interaction with Sfp1 and TORC1 is related to a still poorly understood connection between TOR signaling and vesicle transport. The aim of this work has been to better characterized the relationship among Sfp1, the cell size control and some signalling pathways involved in the coordination of division with growth. In order to better elucidate the role of Sfp1 as a negative regulator of Start, we analyzed the level of some of the key players of the G1 to S transition, the G1 cyclins (Cln1-3) and the Cki Sic1, in a sfp1 mutant. sfp1Δ cells are characterized by a whi phenotype, slow growth, decreased budding index elongation of G1 phase and reduction of G2/M transition. Accordingly with some aspects of this phenotype, the Cln1-2 level resulted decreased while Cln3 levels were unaffected in agreement with data reporting that the mechanism through which Sfp1 couples ribosome biogenesis to Start is independent of Cln3. Interestingly, the main effect of the SFP1 deletion is on Sic1 that resulted entirely nuclear, all linked to Clb5 and stabilized by phosphorylation on threonine 173 (Thr173). Phosphorylation that is well known to induce Sic1 accumulation by preventing its degradation. In the sfp1 mutant, Sic1 stabilization is required for both the elongation of the G1 phase and the reduction of the G2/M transition. A similar situation that involves Sic1 stabilization by phosphorylation on Thr173 but leading to a G1 arrest is observed after inhibition of TORC1 by rapamycin where Sic1 accumulates in the nucleus to avoid improper Clb5/6-Cdc28-driven DNA replication under conditions of poor nutrient availability. This parallelism is in line with the fact that Sfp1 associated with Tor1 kinase and that this binding is essential for a correct localization of TORC1 together with Sfp1 at the RP promoters. A condition of poor nutrient availability can be considered as a stress condition for a cell. Similarly, the activation of the Hog1 MAP kinase after osmotic stress also induces a cellular response where the stabilization of Sic1, always via Thr173 phosphorylation is involved. Moreover, in this context, the cellular response to SFP1 inactivation appears more similar to the response to osmotic stress than that to rapamycin. In fact, the latter induces a G1 arrest linked to a Sic1 stabilization but subsequently a decrease of Cln3 accumulation takes place; such a decrease is essential for maintaining a prolonged G1 arrest. On the contrary, after the osmotic stress Sic1 is stabilized, Cln1 and Cln2 are low, Cln3 levels are unaffected as in the mutant. In addition, the stress response do not always provoke a cell cycle arrest, but is often a slowdown of cell cycle progression, necessary for cell adaptation to new conditions. Only if the stress is too intense, cells arrest growth. Yeast cells modulate stress response via the activation of mitogen-activated protein kinases (MAPKs) which respond to different conditions such as pheromone signals (mediated by the MAPK Fus3), osmolarity (mediated by the MAPK Hog1), nutrient deprivation (mediated by the MAPK Kss1) and cell wall stress (mediated by the MAPK Slt2). Since the first three MAPKs pathways use basically the same signaling machinery, when one of the three pathways is activated, the others are suppressed (cross-talk). The stress response linked to SFP1 inactivation involves a complex cross-talk between the Hog1 and Kiss1 pathways. Both pathways are activated but only Kiss1 is phosphorylated. Kss1 is the MAP kinase that primarily functions under conditions of nutrient deprivation such as the lack of nitrogen and/or glucose in the growth medium. Under these conditions the signal mucin Msb2 regulates the activation of the filamentous growth (FG) pathway that induces the phosphorylation of Kss1, necessary to guarantee cell survival. The lack of Sfp1 is sensed by the cell as a condition of nutrient scarcity. In fact, under optimal growth conditions, Sfp1 localizes to the nucleus, where it promotes the RP and RiBi genes expression. In response to changes in nutrient availability, Sfp1 is released from RP and RiBi gene promoters and exits from the nucleus; thus, the ribosome biogenesis is down-regulated. Moreover, since Msb2 is also required for activation of the Hog1 pathway a reciprocal inhibitory loop takes place between the Hog1 and Kss1 pathways allowing stable activation of the latter. We found that once activated Kiss1 is able to stabilized Sic1. We hypothesize that the activation of the FG pathway following Sfp1 lost of function involves a glycosylation defective-like response. In fact, activation of FG pathway by inhibition of N-glycosylation combined with a specific O-glycosylation defect induces activation of both Hog1 and Kss1 pathways and only Kss1 is phosphorylated. We found that SFP1 inactivation induces some defects that are also observed following the inhibition of glycosylation such as alterations in cell wall permeability, activation of the cell wall integrity pathway and alteration in the secretory pathway. All our data indicate that not only Sfp1 is regulated by stress and nutrients (both affecting its localization), but that Sfp1 can, in turn, regulate the stress response. The linker between Sfp1 and stress response pathway is the secretory pathway. We can hypothesize that a reduction of ribosome biogenesis may induce a defect in the secretory pathway leading to the activation of Msb2 and thus of the FG pathway. The exit from the nucleus of Sfp1, necessary for the reduction of ribosome biogenesis, allows the release of the Rab GTPase that is essential to switch off the defect in the secretory pathway. Consequently, the inactivation of SFP1 induces a complex activation of the MAPKs pathway that is responsible of the regulation of different aspects that characterized the mutant. The main of these is the regulation of the G1-S transitions by the stabilization of Sic1. Finally we showed that consequently to the SFP1 inactivation (probably due to the alteration in the secretory pathway), the mutant cells are characterized by an alteration of Cytoplasmic volume/ Protein content linked to an increase in the cytoplasmic volume. This let us to speculate that growth might be composed of two elements: the Size that is the growth in cell volume and the Mass that is the increase in the protein content. Consequently, alterations of cell growth in response to changes in the environmental conditions imply a coordinate regulation of Size and Mass with the aim of maintaining their ratio constant. One of the key elements necessary to maintain this balance is Sfp1.