Impact of nutrients and stress, including antifungal drugs, on growth and ribosomal content in Saccharomyces cerevisiae
Manzanaro Moreno, Nahuel
Ribosomes are the machines that produce proteins in the cell. They are, with histones, the most expressed proteins, accounting together for almost half of the total gene expression. Ribosome number varies with growth rate, the energy state of the cell, and with the cell cycle. Bacterial cells express more ribosomes the higher their growth rate; the number will also increase when there is an inhibitor of translation present. In contrast to ribosomal genes, constitutive genes show a decrease in expression with growth rate, as cells prioritize ribosome and histone genes. Although these results are well established in bacteria, they are much less so in eukaryotic cells. The aim of this thesis was to determine if the eukaryote budding yeast behaved similarly to Escherichia coli and obeyed the same phenomenological relationships. First, a reliable way to modulate growth rate without the use of drugs is by changing the concentration of carbon source, but yeast grows slowly only in prohibitively low concentrations of glucose, the typical carbon source. Raffinose is a poorly fermented trisaccharide. Here I show that it follows a gentle Monod curve, increasing growth rate steadily with the increase in concentration and plateauing only at high concentrations. I argue that it is the sugar of choice for tuning the growth rate in yeast. Second, I explore orthogonal induction systems in yeast with the goal of causing competition for translational resources. I conclude that the inducer doxycycline is deleterious in respiring yeast likely because of adverse effects on the mitochondria. Third, I wanted to determine the behaviour of constitutively expressed genes as a function of growth rate as a benchmark for the measurement of ribosomal expression. Using a set of reported constitutive promoters controlling the expression of fluorescent proteins, cells were grown at different rates to measure expression at the point of maximal growth rate. I conclude that constitutive expression typically shows a moderate decrease in expression at higher growth rates. Fourth, I developed a new, straightforward protocol to estimate the concentration of ribosomes. I used a series of strains with GFP ribosomal tags in plate readers to estimate ribosomal expression and demonstrated that the fluorescence was directly correlated with growth rate, following quantitatively the relationship obtained by others using mass spectrometry. My results show that ribosomes can be quantified simply, avoiding biochemical extractions and allowing real-time measurements. Fifth, to determine the interrelationships between translation-poisoning drugs, ribosomal concentrations, and growth rate in yeast, I selected a strain with the RPL3 tag and exposed cells to different concentrations of cycloheximide and raffinose. I proved that the concentrations of ribosomes had the same behaviours as a function of growth rate as E. coli does to chloramphenicol: the correlation between ribosomes and maximal growth rate increases steepness at higher drug dosages. A further observation was that plotting time-series data, there is an almost linear decrease in fluorescence with growth rate after the point of maximal fluorescence that follows closely the ribosome to growth relationship at maximal growth rate. It may suggest that cells maintain the maximal possible number of ribosomes during the deceleration phase of the growth curve. Finally, some translation-poisoning antibiotics are more effective on slow growing bacteria and others are more effective on fast growing bacteria. This behaviour is a result of the dependence of growth rate on ribosomes and, given my results, it should hold too in yeast. I developed and quantitively compared different ways to characterise cellular growth with the aim of finding the best method to compare the effects of drugs at different growth rates. I then demonstrated that for yeast too the efficacy of some drugs increases with growth rate and that of others decreases. In summary, I show that bacterial growth laws extend to eukaryotic cells and that, like E. coli, budding yeast is constrained in how it allocates intracellular resources to synthesise proteins.