Multiple layers of phospho-regulation coordinate metabolism and the cell cycle in budding yeast

Abstract

19The coordination of metabolism and growth with cell division is crucial for proliferation. While it 20 has long been known that cell metabolism regulates the cell division cycle, it is becoming 21 increasingly clear that the cell division cycle also regulates metabolism. In budding yeast, we 22 previously showed that over half of all measured metabolites change concentration through the 23 cell cycle indicating that metabolic fluxes are extensively regulated during cell cycle progression. 24 However, how this regulation is achieved still remains poorly understood. Since both the cell cycle 25 and metabolism are regulated to a large extent by protein phosphorylation, we here decided to 26 measure the phosphoproteome through the budding yeast cell cycle. Specifically, we chose a cell 27 cycle synchronisation strategy that avoids stress and nutrient-related perturbations of metabolism, 28 and we grew the yeast on ethanol minimal medium to force cells to utilize their full biosynthetic 29 repertoire. Using a tandem-mass-tagging approach, we found over 200 sites on metabolic enzymes 30 and transporters to be phospho-regulated. These sites were distributed among many pathways 31 including carbohydrate catabolism, lipid metabolism and amino acid synthesis and therefore likely 32 contribute to changing metabolic fluxes through the cell cycle. Among all one thousand sites 33 whose phosphorylation increases through the cell cycle, the CDK consensus motif and an arginine-34 directed motif were highly enriched. This arginine-directed R-R-x-S motif is associated with 35 protein-kinase A, which regulates metabolism and promotes growth. Finally, we also found over 36 one thousand sites that are dephosphorylated through the G1/S transition. We speculate that the 37 phosphatase Glc7/ PP1, known to regulate both the cell cycle and carbon metabolism, may play 38 an important role because its regulatory subunits are phospho-regulated in our data. In summary, 39 our results identify extensive cell cycle dependent phosphorylation and dephosphorylation of 40 metabolic enzymes and suggest multiple mechanisms through which the cell division cycle 41 regulates metabolic signalling pathways to temporally coordinate biosynthesis with distinct phases 42 of the cell division cycle.43 45division cycle, which ensures that DNA and other crucial cellular components are duplicated and 46 divided between two daughter cells. In budding yeast, it was viewed that cell metabolism and 47 growth proceed largely independently of the cell cycle. This assumption comes from the 48 observation that mutants arrested in distinct phases of the cell cycle continued to grow and became 49 extremely large and irregularly shaped (Hartwell et al., 1974;Johnston et al., 1977; Pringle and 50 Hartwell, 1981). This showed clearly that a cell cycle arrest does not stop metabolism and mass 51 accumulation, which led to the text book model that in budding yeast growth controls division, but 52 not vice versa (Morgan, 2007). 53While the hierarchy of metaboli...

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets

“…Early dephosphorylated sites were enriched in Arg-directed residues, which are characteristic of PKA and other metabolic kinases, while at later time Pro-directed residues, typical for Cdc28 kinase were dephosphorylated. It must be noted that these motifs are almost identical to those identified by Zhang et al 59 as being phosphorylated in cells resuming cell cycle after G 1 arrest. This similarity remarks the massive effect of Ppz1 overexpression in modifying cell cycle-regulated phosphoproteins.…”

“…Furthermore, the non-phosphorylatable Snf1-T210A mutant shows a slow growth phenotype and a delayed G 1 /S transition, and these phenotypes can be fully rescued by expression of the phosphomimetic (T210E) form of Snf1. Very recently, it has been reported that Snf1 becomes dephosphorylated at T210 during G 1 to S phase transition but becomes phosphorylated again at the onset of DNA synthesis 59,68 . Therefore, it is plausible to assume that continuous dephosphorylation of Snf1 at Thr210 forced by overexpression of Ppz1 might contribute to the blockage at the G 1 -S transition in these cells (Fig.…”

Building blocks are synthesized on demand during the yeast cell cycle

“…Both metabolite abundance data and morphological changes in the mitochondria similarly highlight that metabolic activity increases as cells begin to replicate. In particular, phase-dependent differences in biosynthetic precursors, seen both at the enzyme level and, less intuitively, at the metabolite level, highlight a complex coordination between metabolism and the cell cycle (2,7).…”

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

“…We next sought to identify if the increase of higher phosphorylated isoforms is triggered solely by the periodically increasing Cdk1 activity (16,19) or whether, PKA phosphorylation increases additionally, as PKA activity has been suggested to increase at the G1/S transition (17,39). The Nth1 ∆PKA/S66 revealed no phosphorylation in G1-arrested cells, showing that the bands which are present in G1 are only the four known PKA sites.…”