Dual control of NAD+ synthesis by purine metabolites in yeast.

Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD+ biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD+. This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network.

Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation Drug AICAR Are Conserved in Yeast and Humans.

Identifying synthetic lethal interactions has emerged as a promising new therapeutic approach aimed at targeting cancer cells directly. Here, we used the yeast Saccharomyces cerevisiae as a simple eukaryotic model to screen for mutations resulting in a synthetic lethality with 5-amino-4-imidazole carboxamide ribonucleoside (AICAR) treatment. Indeed, AICAR has been reported to inhibit the proliferation of multiple cancer cell lines. Here, we found that loss of several histone-modifying enzymes, including Bre1 (histone H2B ubiquitination) and Set1 (histone H3 lysine 4 methylation), greatly enhanced AICAR inhibition on growth via the combined effects of both the drug and mutations on G1 cyclins. Our results point to AICAR impacting on Cln3 subcellular localization and at the Cln1 protein level, while the bre1 or set1 deletion affected CLN1 and CLN2 expression. As a consequence, AICAR and bre1/set1 deletions jointly affected all three G1 cyclins (Cln1, Cln2, and Cln3), leading to a condition known to result in synthetic lethality. Significantly, these chemo-genetic synthetic interactions were conserved in human HCT116 cells. Indeed, knock-down of RNF40, ASH2L, and KMT2D/MLL2 induced a highly significant increase in AICAR sensitivity. Given that KMT2D/MLL2 is mutated at high frequency in a variety of cancers, this synthetic lethal interaction has an interesting therapeutic potential.

Identification of yeast and human 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAr) transporters.

5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation.

An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence.

The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit.

Metabolic status rather than cell cycle signals control quiescence entry and exit.

Quiescence is defined as a temporary arrest of proliferation, yet it likely encompasses various cellular situations. Our knowledge about this widespread cellular state remains limited. In particular, little is known about the molecular determinants that orchestrate quiescence establishment and exit. Here we show that upon carbon source exhaustion, budding yeast can enter quiescence from all cell cycle phases. Moreover, using cellular structures that are candidate markers for quiescence, we found that the first steps of quiescence exit can be triggered independently of cell growth and proliferation by the sole addition of glucose in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Importantly, glucose needs to be internalized and catabolized all the way down to glycolysis to mobilize quiescent cell specific structures, but, strikingly, ATP replenishment is apparently not the key signal. Altogether, these findings strongly suggest that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.