Heidi M. Blank

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.

The Unfolded Protein Response Is Not Necessary for the G1/S Transition, but It Is Required for Chromosome Maintenance in Saccharomyces cerevisiae

Background The unfolded protein response (UPR) is a eukaryotic signaling pathway, from the endoplasmic reticulum (ER) to the nucleus. Protein misfolding in the ER triggers the UPR. Accumulating evidence links the UPR in diverse aspects of cellular homeostasis. The UPR responds to the overall protein synthesis capacity and metabolic fluxes of the cell. Because the coupling of metabolism with cell division governs when cells start dividing, here we examined the role of UPR signaling in the timing of initiation of cell division and cell cycle progression, in the yeast Saccharomyces cerevisiae. Methodology/Principal Findings We report that cells lacking the ER-resident stress sensor Ire1p, which cannot trigger the UPR, nonetheless completed the G1/S transition on time. Furthermore, loss of UPR signaling neither affected the nutrient and growth rate dependence of the G1/S transition, nor the metabolic oscillations that yeast cells display in defined steady-state conditions. Remarkably, however, loss of UPR signaling led to hypersensitivity to genotoxic stress and a ten-fold increase in chromosome loss. Conclusions/Significance Taken together, our results strongly suggest that UPR signaling is not necessary for the normal coupling of metabolism with cell division, but it has a role in genome maintenance. These results add to previous work that linked the UPR with cytokinesis in yeast. UPR signaling is conserved in all eukaryotes, and it malfunctions in a variety of diseases, including cancer. Therefore, our findings may be relevant to other systems, including humans.

Sulfur metabolism actively promotes initiation of cell division in yeast.

Background Sulfur metabolism is required for initiation of cell division, but whether or not it can actively promote cell division remains unknown. Methodology/Principal Findings Here we show that yeast cells with more mtDNA have an expanded reductive phase of their metabolic cycle and an increased sulfur metabolic flux. We also show that in wild type cells manipulations of sulfur metabolic flux phenocopy the enhanced growth rate of cells with more mtDNA. Furthermore, introduction of a hyperactive cystathionine-β-synthase (CBS) allele in wild type cells accelerates initiation of DNA replication. Conclusions/Significance Our results reveal a novel connection between a key sulfur metabolic enzyme, CBS, and the cell cycle. Since the analogous hyperactive CBS allele in human CBS suppresses other disease-causing CBS mutations, our findings may be relevant for human pathology. Taken together, our results demonstrate the importance of sulfur metabolism in actively promoting initiation of cell division.

Translational control of lipogenic enzymes in the cell cycle of synchronous, growing yeast cells.

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate‐limiting enzyme acetyl‐CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.

CDK control of membrane-bound organelle homeostasis

In eukaryotes, the copy number and size of any given organelle compartment remain constant in dividing cells, underlying a tight coordination between cell division and organelle homeostasis. However, in most cases the mechanisms for this coordination remain mysterious. Here we outline a few cases where the cell cycle machinery directly impacts on organelle homeostasis, with emphasis on the control of vacuolar (lysosomal) copy number and size in budding yeast. We also discuss aspects of organelle biology that can profoundly affect certain cell cycle parameters, such as cell size.

Scaling of G1 Duration with Population Doubling Time by a Cyclin in Saccharomyces cerevisiae

The longer cells stay in particular phases of the cell cycle, the longer it will take these cell populations to increase. However, the above qualitative description has very little predictive value, unless it can be codified mathematically. A quantitative relation that defines the population doubling time (Td) as a function of the time eukaryotic cells spend in specific cell cycle phases would be instrumental for estimating rates of cell proliferation and for evaluating introduced perturbations. Here, we show that in human cells, the length of the G1 phase (TG1) regressed on Td with a slope of ≈0.75, while in the yeast Saccharomyces cerevisiae, the slope was slightly smaller, at ≈0.60. On the other hand, cell size was not strongly associated with Td or TG1 in cell cultures that were proliferating at different rates. Furthermore, we show that levels of the yeast G1 cyclin Cln3p were positively associated with rates of cell proliferation over a broad range, at least in part through translational control mediated by a short upstream ORF (uORF) in the CLN3 transcript. Cln3p was also necessary for the proper scaling between TG1 and Td. In contrast, yeast lacking the Whi5p transcriptional repressor maintained the scaling between TG1 and Td. These data reveal fundamental scaling relationships between the duration of eukaryotic cell cycle phases and rates of cell proliferation, point to the necessary role of Cln3p in these relationships in yeast, and provide a mechanistic basis linking Cln3p levels to proliferation rates and the scaling of G1 with doubling time.

Lipid biosynthesis: When the cell cycle meets protein synthesis?

Cells must tightly coordinate the levels of many of their proteins to navigate accurately and safely the transitions of the cell cycle. For cells to grow and divide, not only proteins but also lipids must be synthesized anew in every cell cycle. The amount, composition, and localization of the lipid repertoire are dynamic in dividing cells. However, little is known about how cells regulate their lipid content during a cell cycle. Here we highlight results from genome-wide studies in yeast and human cells that revealed surprising mechanisms of control, at the translational level, of lipid metabolism in the cell cycle. To find transcripts under translational control, ribosome profiling quantifies by deep sequencing all pieces of mRNAs in the cell bound to translating ribosomes. This technique has now been used to find cases of gene-specific, cell cycle-dependent, translational control. Different studies used different systems and methodologies to obtain synchronous samples. Blank and colleagues used budding yeast collected at specific sizes via centrifugal elutriation to examine by ribosome profiling a cell size series spanning the entire cell cycle. The synchrony achieved is free of possible arrest-induced artifacts and preserves as much as possible the normal coordination of growth and division. A striking result was that translation of mRNAs encoding the core enzymes of lipid biogenesis, acetylCoA carboxylase (ACC1) and fatty acid synthase (FAS1 and FAS2), was upregulated in mitosis. A short upstream open reading frame (uORF) adjusts the translation of ACC1, leading to >10-fold increase late in the cell cycle, and also represses translation of ACC1 in poor media. Human cells arrested at different points along the cell cycle were also subjected to ribosome profiling. The cells in the studies of Stumpf and colleagues were not released from their arrest, leaving open the possibility of artifacts. Nonetheless, translation of mRNAs encoding enzymes of lipid metabolism was regulated, with most of them peaking in mitosis. The human cells examined by Tanenbaum and colleagues were arrested in the G2 phase with a small-molecule inhibitor of the cyclin-dependent kinase CDK1. Washing the inhibitor away enabled the arrested cells to progress synchronously through mitosis, and enter the next G1. It is not clear if the cells in this experiment attained their normal degree of coupling between growth and division since the arrest period was 18 h and the cells were released for only 45 or 225 min. Despite these limitations, Tanenbaum and colleagues interrogated progress through a key cell cycle phase, mitosis, during which animal cells repress overall protein synthesis. Most (> 90%) of the mRNAs they identified were repressed translationally in mitosis. Under the same conditions, demonstrating the varying nature of transcript-specific translational control, some mRNAs had increased translational efficiency. Among them was FASN, encoding human fatty acid synthase, whose translational efficiency was increased by >2-fold in mitosis compared with the G2 phase. But mitotic upregulation of lipid metabolism need not come about only through translational control. De novo fatty acid synthesis and upregulation of human acetylCoA carboxylase (ACACA) through post-translational control were essential for completion of mitosis (Fig. 1). There could be many reasons why cells need new lipids late in the cell cycle. The most obvious need would arise from the sudden increase in the outer cell surface upon exit from mitosis, which approaches 40% for spherical cells. We note, however, that yeast cells with perturbed lipid homeostasis were still able to increase their exterior surface during mitosis, arguing for more specialized roles for lipids in the eukaryotic cell cycle. A comprehensive lipidomic study by Atilla-Gokcumen and colleagues demonstrated extensive changes in lipid composition and localization during the cell cycle in human cells, especially along the midbody before cell separation. They also found 23 lipid biosynthetic enzymes to be essential for cytokinesis, including enzymes of sphingolipid metabolism and fatty acid elongases. Finally, the nuclear membrane also goes through dramatic rearrangements during cell division, from complete breakdown and re-assembly in animal cells, to massive expansion during the closed mitosis that many fungi undergo. De novo lipid biogenesis is needed for the expansion of the nuclear membrane in yeast. Reduced function of Polo-like kinase, acetyl-CoA carboxylase or fatty acid synthase was proposed to lower phosphatidic acid levels, reducing the ability of cells to increase the area of their nuclear membrane. Overall, the studies we highlighted point to the emerging role of lipid metabolism in underpinning cell cycle landmarks

Correction: An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast.

Mary Bryk should also be listed as a corresponding author and should be contacted regarding chromatin aspects. Her e-mail address is ude.umat@kyrb.