Cell cycle-dependent localization of hexose transporter mRNA in Saccharomyces cerevisiae

Unicellular organisms like yeast face constantly changing environmental conditions. Especially fluctuating concentrations of glucose affect growth of S. cerevisiae. Thus yeast evolved several hexose transporters with different affinities and expression patterns. We observed that the high-affinity transporter Hxt2 is differentially localized during the cell cycle. Analyzing the localization of its mRNA, by Fluorescent In Situ Hybridization (FISH), unveiled a comparable distribution pattern. Under glucose-rich conditions, the mRNA is retained in the mother cell early in the cell cycle and only later, after Metaphase to Anaphase Transition (MAT), HXT2 mRNA is equally distributed. This is true for all four hexose transporters, we investigated.
Furthermore, we could show that the release from the mother cell to the bud after MAT, is dependent on active translation and that the binding of HXT2 mRNA to polysomes confers its stability. Upon deleting the RNA-binding protein Scp160, which is associated with translational control and binds to ribosomes, we found that HXT2 mRNA is enriched in the bud after MAT. Moreover, the deletion of ASC1, that mediates binding of Scp160/Bfr1 to ribosomes, also leads to the enrichment of HXT2 mRNA.
Interestingly, Asc1 was also described as being involved in the glucose responsiveness pathway of S. cerevisiae. Here, Asc1 is thought to inhibit the adenylyl cyclase. In fact, we could show that activation of the adenylyl cyclase and subsequently activating the cAMP-dependent protein kinase A, phenocopies the deletion of ASC1. Intriguingly, when we activated the glucose responsiveness pathway by re-feeding starved cells with glucose, we found that especially HXT2 mRNA is enriched in the bud after MAT. Moreover, this enrichment is transient and happens only during the first 1-2 cell divisions after coming from starvation. Next, we deleted the glucose receptor Gpr1 and its associated G-protein Gpa2 in order to investigate the involvement of upstream factors of the glucose responsiveness pathway. Whereas deletion of Gpr1/Gpa2 did not show an effect, a mutated version of Ras2, which does not elicits an increase of cAMP under glucose-shift conditions, inhibits the enrichment of HXT2 mRNA in the bud.
Apart from establishing the involvement of the Ras2/cAMP/PKA pathway, we demonstrated that transcription as well as nuclear segregation are necessary, but not sufficient for the enrichment of HXT2 mRNA in the bud. Furthermore, we identified Kar9, which is involved in the asymmetric spindle pole body segregation, as well as Mlp1, Mlp2 and Nup2, which are components of the nuclear basket, as important trans-acting factors for the asymmetric distribution of HXT2 mRNA under glucose shift conditions. Finally, we were wondering, whether there is an advantage for the daughter cell to be provided with high levels of HXT2 mRNA. We carried out growth tests and observed that cells that express HXT2 as their sole hexose transporter grow faster than cells expressing other HXTs. Taken together, the results presented in this thesis suggest that S. cerevisiae enriches specifically HXT2 mRNA in the daughter cell, when coming from quiescence or starvation. Expression of HXT2 presumably enables the daughter cell to rapidly accumulate glucose, switch from respiration to fermentation and to start re-growth ahead of cells, that do not enrich HXT2 mRNA.