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Review in Advance first posted online on May 11, 2012. (Changes may still occur before final publication online and in print.)

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Driving the Cell Cycle Through Metabolism
Ling Cai and Benjamin P. Tu
Annu. Rev. Cell Dev. Biol. 2012.28. Downloaded from www.annualreviews.org by Ecole Polytechnique Federal Lausanne on 06/20/12. For personal use only.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038; email: benjamin.tu@utsouthwestern.edu

Annu. Rev. Cell Dev. Biol. 2012. 28:3.1–3.29 The Annual Review of Cell and Developmental Biology is online at cellbio.annualreviews.org This article’s doi: 10.1146/annurev-cellbio-092910-154010 Copyright c 2012 by Annual Reviews. All rights reserved 1081-0706/12/1110-0001$20.00

Keywords cell growth, cell proliferation, metabolic cycle, growth control, nutrients, yeast

Abstract
For unicellular organisms, the decision to enter the cell cycle can be viewed most fundamentally as a metabolic problem. A cell must assess its nutritional and metabolic status to ensure it can synthesize sufficient biomass to produce a new daughter cell. The cell must then direct the appropriate metabolic outputs to ensure completion of the division process. Herein, we discuss the changes in metabolism that accompany entry to, and exit from, the cell cycle for the unicellular eukaryote Saccharomyces cerevisiae. Studies of budding yeast under continuous, slow-growth conditions have provided insights into the essence of these metabolic changes at unprecedented temporal resolution. Some of these mechanisms by which cell growth and proliferation are coordinated with metabolism are likely to be conserved in multicellular organisms. An improved understanding of the metabolic basis of cell cycle control promises to reveal fundamental principles governing tumorigenesis, metazoan development, niche expansion, and many additional aspects of cell and organismal growth control.

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Contents
INTRODUCTION . . . . . . . . . . . . . . . . . 3.2 A BRIEF OVERVIEW OF THE CELL CYCLE IN THE BUDDING YEAST SACCHAROMYCES CEREVISIAE . . . . . . . . . . . . . . . . . . . . 3.3 Cellular Events . . . . . . . . . . . . . . . . . . . 3.3 Regulatory Pathways . . . . . . . . . . . . . 3.3 BUILDING BLOCKS OF THE CELL . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Carbon . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Sulfur . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Phosphorus . . . . . . . . . . . . . . . . . . . . . . 3.7 Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 LINKS BETWEEN CELL CYCLE REGULATION AND METABOLISM REVEALED THROUGH GENOME- AND PROTEOME-WIDE STUDIES . . . . . . . . . . . . . . . . . . . . . . . 3.8 Genetic Screens . . . . . . . . . . . . . . . . . . 3.8 Transcript Profiling . . . . . . . . . . . . . . 3.9 ChIP-Chip . . . . . . . . . . . . . . . . . . . . . . . 3.10 Proteomics . . . . . . . . . . . . . . . . . . . . . . . 3.10 DYNAMICS OF METABOLISM OVER THE CELL CYCLE . . . . . 3.11 Two Modes of Investigating Cell Growth and Proliferation: The Transition From Quiescence to

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Growth Versus Continuous Rapid Growth . . . . . . . . . . . . . . . . . 3.11 The Yeast Metabolic Cycle: A Useful Platform for Cell Cycle and Metabolism Research . . . . . . 3.11 What Happens During Growth (the OX Phase of the Yeast Metabolic Cycle)? . . . . . . . . . . . . . 3.12 Metabolism During the Cell Division Process (The RB Phase of the Yeast Metabolic Cycle) . . 3.13 Exit from Growth and Entry into Quiescence (the RC Phase of the Yeast Metabolic Cycle) . . . . . 3.14 KEY FEATURES OF THE CELLULAR GROWTH PROGRAM . . . . . . . . . . . . . . . . . . . . . 3.14 Nutrient-Sensing Pathways and Ribosome Biogenesis . . . . . . . . . . 3.14 Acetyl-Coenzyme A . . . . . . . . . . . . . . 3.15 NADPH . . . . . . . . . . . . . . . . . . . . . . . . . 3.16 Sulfur Metabolism . . . . . . . . . . . . . . . . 3.17 NAD+ . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.18 Amino Acid and Nucleotide Metabolism . . . . . . . . . . . . . . . . . . . 3.18 Phosphate Metabolism . . . . . . . . . . . 3.19 Ion Homeostasis . . . . . . . . . . . . . . . . . 3.19 METABOLIC CHANGES ASSOCIATED WITH G0 AND QUIESCENCE . . . . . . . . . . . . 3.19 SUMMARY AND PERSPECTIVE . . . . . . . . . . . . . . . . . 3.21

INTRODUCTION
All proliferating cells must be capable of executing a program devoted to cell growth and cell division. In response to appropriate cues, cells may either enter the cell cycle or persist in a quiescent (G0 ) or quiescent-like state. Over the past few decades, genetic and cell biological studies have provided remarkable insights into the core cell cycle machinery and the workings of the cell division process itself. However, many of these studies have largely overlooked
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and ignored fundamental yet critical contributions of metabolism to the cell cycle. Specifically, the building of a new cell is an extraordinary task that depends on a myriad of metabolic and biosynthetic reactions, many of which are important for accumulating biomass. It has become increasingly evident that solving cell cycle control might be most fundamentally a metabolic problem (Deberardinis et al. 2008, Vander Heiden et al. 2009). What metabolic changes accompany and are required for entry

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into the cell cycle? How do cells assess whether conditions are appropriate for entering a round of growth and division? In turn, which aspects of cellular metabolism might be regulated by the cell cycle machinery? In this review, we discuss some of these important questions by touching on past and present studies using the single-cell eukaryote Saccharomyces cerevisiae.

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A BRIEF OVERVIEW OF THE CELL CYCLE IN THE BUDDING YEAST SACCHAROMYCES CEREVISIAE Cellular Events
The eukaryotic cell division cycle can be divided into the sequential G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitotic) phases. In the case of budding yeast, in G1 the cell increases in size until it reaches the G1 checkpoint upon which it needs to make a decision. Under nutrientrestrictive or starvation conditions, the cell typically arrests growth and enters a quiescent state called G0 . In the case of a diploid cell, it may also initiate sporulation or pseudohyphal differentiation programs. Haploids, if exposed to pheromone, arrest at G1 and commit to the mating program. Otherwise, the cell may undergo cell division (Lew et al. 1997). Upon entering the cell cycle, a yeast cell starts duplicating its spindle pole body (SPB) in late G1 , which is the microtubule organizing center embedded in the nuclear membrane ( Jaspersen & Winey 2004). It also undergoes polarization with the assembly of actin filaments from the bud site, which serve as the supply chain for building the daughter cell (Pruyne & Bretscher 2000). Post-Golgi complex secretory vesicles containing materials for membrane and cell wall synthesis are delivered to the bud in preparation for surface expansion. Septins are assembled at the bud site and a ring of chitin is deposited on the cell wall where the bud will emerge (Cabib et al. 2001). Cargo containing membrane-bound organelles also travel along the actin cable to the bud to inherit organelles such as vacuoles, mitochondria,

late Golgi complexes, and peroxisomes. These organelles continue to segregate throughout the budding process (Barr 2002, Boldogh et al. 2001, Fagarasanu et al. 2010, Pruyne et al. 2004, Weisman 2003). The next stage, S phase, is devoted to DNA synthesis. The fidelity of DNA replication is under the surveillance of checkpoint mechanisms that become activated when replication forks stall (Nyberg et al. 2002). Chromatin proteins such as histones are also synthesized and assembled into the new DNA. Cohesin molecules are deposited along the sister chromatids to hold them together, and kinetochores are assembled at the centromeres in preparation for their subsequent separation (Hirano 2000, Tanaka et al. 2005). S. cerevisiae does not exhibit a distinct G2 phase (Forsburg & Nurse 1991). Mitotic spindle assembly begins in late S phase after the duplicated SPBs separate and the cell transitions to M phase. During mitosis, chromosomal DNA condenses. Spindle checkpoints are imposed to ensure proper attachment of the kinetochore and correct spindle alignment before sister chromatids are segregated to the two poles of the spindle (Musacchio & Hardwick 2002). Cytokinesis and septation occur to seal the bud neck and promote release of the daughter cell (Figure 1).

Regulatory Pathways
Cyclin-dependent kinases (CDKs) are the central drivers of cell cycle progression (Morgan 1997). There are six CDKs in budding yeast: Kin28p, Sgv1p, Ssn3p, Ctk1p, Pho85p, and Cdc28p. Four of these (Kin28p, Sgv1p, Ssn3p, and Ctk1p) harbor one cyclin partner. These cyclin/CDK pairs are involved in transcriptional regulation through phosphorylation of RNA polymerase II (Morgan 1997). Pho85, activated by as many as 10 cyclins, has roles in both metabolic regulation and cell cycle control (Carroll & O’Shea 2002). Among these 10 cyclin genes, transcripts of PCL1, PCL2, and PCL9 fluctuate significantly across the cell cycle (Measday et al. 1997). They are www.annualreviews.org • Metabolism and Cell Cycle 3.3

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hase RC p

se pha RC

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Autophagy Heat shock proteins Peroxisome Ubiquitin/proteasome Vacuole

Ribosome biogenesis Amino acid synthesis Sulfur metabolism Translation

DNA replication Histones Spindle pole

YMC gene expression

OX phase

Trehalose Glycogen

Acetyl-CoA NADPH

Nucleotides SAM

YMC metabolites

SBF
Swi4

Swi6

MBF
Mbp1 Cdc28

Swi6

Mcm1

Ndd1 Fkh1/2 Cdc28 Clb5

Swi5 Ace2 Clb3 Cdc28 Cdc28 Clb4 C Clb2 C Mcm1 Cdc28 Clb1 Pho85 Pcl9

Cell cycle transcription factors

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Cdc28 Cln3

Cdc28 Cln1 Cdc28 Cln2 C Clb6 C

Pho85 Pcl1 Cdc28

CDK machinery Cell cycle stages

G0

G1
Ribosome biogenesis Bud initiation SPB duplication Actin cytoskeleton polarization

S
DNA replication DNA repair Chromatin assembly

M
Chromosome segregation Cell wall synthesis Cytokinesis Septation

Cell cycle events

Storage accumulation
Trehalose Glycogen Polyphosphate

Nutrient consumption
Glycolysis Respiration Sulfur utilization

Metabolic events

TOR Snf1 Nitrogen

PKA Pho85 Carbon Phosphate

Growth entry

Nutrient sensing pathways

Figure 1 Snapshot of the changes in metabolism that occur as a function of the cell cycle in budding yeast. Depiction of when key gene expression programs, metabolites, transcription factors, cyclin/CDK pairs, cell cycle events, and metabolic events are thought to be active or upregulated with respect to cell cycle stage (G0 , G1 , S, and M) and metabolic cycle phase [OX (growth), RB (division), and RC (survival/quiescence)]. The YMC trace represents absolute dissolved oxygen concentrations in the growth medium—a drop in dO2 is indicative of oxygen consumption. Abbreviations: CDK, cyclin-dependent kinase; CoA, coenzyme A; dO2 , dissolved oxygen; OX, oxidative, respiratory; PKA, protein kinase A; RB, reductive, building; RC, reductive, charging; SAM, S-adenosylmethionine; SPB, spindle pole body; TOR, target of rapamycin; YMC, yeast metabolic cycle.

regulators of the cell cycle (Espinoza et al. 1994, Measday et al. 1994, Tennyson et al. 1998). The remaining Pcl-Pho85 complexes are implicated in various aspects of metabolism (Carroll & O’Shea 2002, Huang et al. 2007). The remaining CDK, Cdc28p (Cdk1p), is the best-studied CDK that plays a major role in controlling the cell cycle (Morgan 1997).
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It has three G1 cyclins, including Cln3p and Cln1p/Cln2p, and six B-type cyclins, including Clb5p/Clb6p, Clb3p/Clb4p, and Clb1p/Clb2p, which are activated sequentially in the cell cycle. The CDK machinery intersects with three main transcription networks across the cell cycle, including the MluI Cell-Cycle Box Binding Factor (MBF) and Swi4/6 Cell-Cycle Box

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Binding Factor (SBF) complexes for G1 /S, the Mcm1p-Fkh1/2p-Ndd1p complex for G2 /M, and the Ace2p and Swi5p transcription factors for M/G1 (Bahler 2005, Breeden 2003), to regulate various events including bud morphogenesis, DNA replication, SPB duplication, mitotic spindle assembly, and exit from mitosis (Bloom & Cross 2007). For more detailed information on these core cell cycle events and pathways, we refer the reader to existing reviews (Bahler 2005, Bloom & Cross 2007, Breeden 2003).

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BUILDING BLOCKS OF THE CELL
Observations from a wide spectrum of organisms suggest that attainment of a critical cell size is a prerequisite for the onset of the cell cycle (Hartwell & Unger 1977). In yeast, entry into the cell cycle typically depends on a period of growth and expansion to increase cell size, in preparation for the division process ( Jorgensen & Tyers 2004). To ensure it can execute the many components of the growth program successfully, the cell must obtain sufficient biosynthetic precursors, or “building blocks,” in the form of carbon, nitrogen, sulfur, phosphate, and oxygen sources. Over the years, studies have shown that when these key nutrient sources are deficient, yeast cells arrest growth and division and then enter alternative states that promote persistence and survival (e.g., G0 /quiescence, pseudohyphal growth, sporulation) (Boer et al. 2008, Gray et al. 2004). The general functions of these basic building blocks in metabolism and biosynthesis are discussed briefly below.

for growth and proliferation. Glucose not only fuels glycolysis and the tricarboxylic acid (TCA) cycle for the production of ATP but also enters the pentose phosphate pathway to produce ribose sugars that are needed for DNA and RNA synthesis (Figure 2). The pentose phosphate pathway also produces reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), which is required for fatty acid and sterol biosynthesis as well as other biosynthetic processes (Figure 2). Metabolic intermediates from glycolysis and the TCA cycle also branch into many other biosynthetic and metabolic pathways (Deberardinis et al. 2008). Glucose is also the precursor for many sugars involved in numerous glycosylation modifications that function along the secretory pathway or at the cell surface (Helenius & Aebi 2001) (Figure 2). Importantly, yeast can synthesize glucose from products of glycolysis such as ethanol and acetate via the glyoxylate pathway. Glucose can also be stored in the form of the disaccharide trehalose and the polymer glycogen (Francois & Parrou 2001).

Nitrogen
Nitrogen is required for the synthesis of amino acids, for the nitrogenous bases (purines and pyrimidines) that make up DNA and RNA, and for a variety of essential vitamins and cofactors (Figure 3). Yeast cells can utilize nitrogen from many sources including amino acids, nucleic acids and their derivatives, and ammonia (Cooper 1982). Among these, ammonia is of special importance, as it is also the main product of nitrogen catabolic pathways. To recycle nitrogen from ammonia, ammonia can be converted to glutamate or glutamine as an entry point back into anabolic pathways (ter Schure et al. 2000) (Figure 3).

Carbon
Carbon forms the backbone of virtually all organic molecules required for life. Carbohydrates produced by photosynthetic organisms provide both energy and structural components for the organisms that consume them. Glucose is a focal point of carbon metabolism (Figure 2). Many sugars and carbohydrates can be metabolized to glucose, which then drives the synthesis of many metabolites required

Sulfur
Sulfur sources are required for the biosynthesis of the amino acids cysteine and methionine (Thomas & Surdin-Kerjan 1997) (Figure 4). In addition to its role as an amino acid for www.annualreviews.org • Metabolism and Cell Cycle 3.5

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Glucose Trehalose/glycogen (polysaccharides) NADP+

Antioxidant Lipid biosynthesis Sulfate assimilation NADPH Ribose-5-P

Nucleotide synthesis

Glucose UDP-glucose Glycoproteins Glycolipids Glucans (cell wall) G6P F6P FBP G3P Glycolysis BPG Fatty acid 3PG 2PG PEP Pyruvate DHAP

Pentose phosphate pathway

Lipid biosynthesis

β-

Ethanol Acetaldehyde Acetate Acetyl-CoA Citrate (Metazoans) Isocitrate Lipid biosynthesis Protein acetylation

i ox n tio da

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Acetyl-CoA Oxaloacetate Malate Fumarate Succinate Succinyl-CoA TCA cycle

α-ketoglutarate

Energy ATP

FADH2

NADH Oxidative phosphorylation

Figure 2 Major metabolic pathways of glucose and carbon utilization in budding yeast. Glucose is transported into the cell from external sources or released from storage carbohydrates such as trehalose and glycogen. Glucose can be used for glycosylation of lipids and proteins and can be turned into components of the cell wall. Catabolism of glucose through the pentose phosphate pathway, glycolysis, and tricarboxylic acid (TCA) cycle provides essential metabolites that are building blocks of lipids and nucleic acids. Glucose catabolism also provides reducing power for biosynthetic reactions and production of ATP through oxidative phosphorylation. Abbreviations: 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; BPG, 2,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; F6P, fructose-6-phosphate; FADH2 , reduced flavin adenine dinucleotide; FBP, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvate; UDP-glucose, uridine diphosphate glucose.

protein synthesis, methionine is the immediate precursor of S-adenosylmethionine (SAM or AdoMet), which is the biological methyl donor required for methylation modifications that occur within the cell. SAM is also required as a substrate for the biosynthesis of particular lipids and polyamines (Figure 4). Cysteine fulfills additional critical roles as a precursor for the biosynthesis of glutathione, which is the major cellular reducing agent typically
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present in millimolar concentrations (Hwang et al. 1992), and redox-active iron-sulfur (Fe-S) clusters, which are essential for the function of particular proteins (Lill 2009). Cysteine is also the sulfur donor for the thiolation of particular bases within tRNAs (Pedrioli et al. 2008). Sulfur is also present in several important vitamins, including thiamine, biotin, and coenzyme A (CoA) (Figure 4).

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Histidine Glucose G6P F6P FBP G3P Glycolysis BPG Glycine Cysteine Isoleucine Asparagine Methionine Serine 3PG 2PG Threonine Aspartate PEP Pyruvate Acetyl-CoA Oxaloacetate Malate Fumarate Succinate Succinyl-CoA α-ketoglutarate Glutamate Lysine TCA cycle Citrate Isocitrate DHAP 6PG Ru5P R5P + Xu5P G3P + S7P

Phenylalanine Tyrosine Tryptophan F6P E4P Xu5P G3P + F6P

Pentose phosphate pathway

Nitrogen utilization
NH4+ α-ketoglutarate NADPH Alanine Valine Leucine NADP+ NH4+ ATP Glutamate Amino acids Purines Pyrimidines Amino acids

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ADP + Pi

Glutamine

Glutamine Arginine Proline

Figure 3 Major metabolic pathways of nitrogen utilization in budding yeast. Glycolysis and tricarboxylic acid (TCA) cycle intermediates provide the backbones for amino acid biosynthesis. Nitrogen assimilation and donation to amino acids and nitrogenous bases purines and pyrimidines are primarily mediated through the interconversion of α-ketoglutarate, glutamate, and glutamine. Abbreviations: 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; 6PG, 6-phosphogluconate; BPG, 2,3-bisphosphoglycerate; CoA, coenzyme A; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvate; R5P, ribose5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; Xu5P, xylulose-5-phosphate.

Phosphorus
Phosphate is a key component of nucleotides within the cell, including ATP. The high energy provided by phosphoanhydride bonds in nucleotide phosphates play numerous roles in biology, including functioning as donors in phosphorylation modifications and enabling polymerization reactions (Westheimer 1987). Importantly, phosphate comprises the backbone of nucleic acids such as DNA and RNA because the bases are stitched together via phosphodiester linkages. Phosphate is also present in phospholipids, which are major components of cell membranes and messengers in signaling transduction pathways (Martin 1998), and in a variety of metabolic intermediates.

Oxygen
Molecular oxygen fuels the synthesis of ATP via oxidative phosphorylation by functioning as the terminal electron acceptor. During aerobic respiration, by-products of oxygen reduction such as superoxide and hydrogen peroxide can be generated. These reactive oxygen species, if not scavenged by cellular antioxidant systems, can cause damage to DNA, amino acids, and lipids (Figure 5). In addition to its role in fueling respiration, molecular oxygen is used as a direct substrate by many cellular enzymes, including those required for hydroxylation reactions, unsaturated fatty acid synthesis, sterol and heme biosynthesis, and fatty acid oxidation. It also functions as the preferred terminal electron www.annualreviews.org • Metabolism and Cell Cycle 3.7

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SO42– 4 NADPH 4 NADP+ S2– Fe-S clusters tRNA thiolation CoA Biotin Thiamine

Homoserine O-acetyl-L-homoserine

Unsaturated fatty acid synthesis Ergosterol synthesis Heme synthesis Disulfide bond formation Fatty acid oxidation

O2
Homocysteine Cystathionine Cysteine Glutathione (GSH) Methionine Protein methylation Metabolite methylation Polyamine biosynthesis

H2O

FADH2

NADH Oxidative phosphorylation

S-adenosylmethionine (SAM)

ROS
S-adenosylhomocysteine (SAH)

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Figure 4 Major metabolic pathways of sulfur utilization in budding yeast. Budding yeast use NADPH for the reduction of sulfate to sulfide, which is then used to synthesize the sulfur-containing amino acids cysteine and methionine. Cysteine is a sulfur donor for iron-sulfur cluster synthesis, tRNA thiolation, and several vitamins. It is also a component of the antioxidant glutathione and accounts for its reducing power. Methionine is a precursor of S-adenosylmethionine (SAM), which is a methyl donor for methylation reactions. SAM is also used in the biosynthesis of polyamines. Abbreviations: CoA, coenzyme A; Fe-S clusters, iron-sulfur clusters; NADPH, reduced nicotinamide adenine dinucleotide phosphate. Figure 5

DNA, amino acids, and lipids

acceptor for the formation of disulfide bonds in newly folding secretory proteins, which occurs in the endoplasmic reticulum (Tu & Weissman 2002, 2004) (Figure 5). Budding yeast can grow quite well under anaerobic conditions using a strictly fermentative metabolism if supplemented with oleic acid and ergosterol, two key metabolites that require molecular oxygen to be synthesized. Thus, cellular physiology and metabolic state can be profoundly influenced by the availability of molecular oxygen.

Oxygen metabolism in budding yeast. Oxygen is used for the biosynthesis of ergosterol, unsaturated fatty acids, and heme, as well as during fatty acid oxidation and disulfide bond formation. Oxygen is also the final electron acceptor for the electron transport chain, which oxidizes reduced equivalents of reduced flavin adenine dinucleotide (FADH2 ) and reduced nicotinamide adenine dinucleotide (NADH) for the synthesis of ATP. However, reactive oxygen species (ROS) are produced as a by-product during some of these processes. ROS can cause damage to DNA, amino acids, and lipids.

LINKS BETWEEN CELL CYCLE REGULATION AND METABOLISM REVEALED THROUGH GENOME- AND PROTEOME-WIDE STUDIES
The enormous challenge of building a new cell depends on access to sufficient quantities of these aforementioned metabolic building blocks. Myriad metabolic, biosynthetic, and catabolic events are critical for the timely and accurate completion of the cell division
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process. Thus, intimate connections between the cell cycle and metabolism must exist. In fact, it can be argued that metabolism is the key driver of cell cycle initiation. Studies over the years have provided clues to the many strategies that couple the cell cycle to metabolism in various organisms. With the advent of methods to monitor gene transcription, transcription factor binding, and kinase substrates on a global scale, such studies targeting the budding yeast cell cycle have begun to reveal its connections to various cellular processes including metabolism (Tyers 2004). Such connections in bacteria and mammalian cells have been reviewed recently (Buchakjian & Kornbluth 2010, Wang & Levin 2009).

Genetic Screens
In the early 1970s, the classic screens for genes involved in the cell division cycle (CDC)

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Table 1 Gene CDC1 CDC8 CDC10 CDC19 CDC21 CDC25 CDC30

Budding yeast CDC genes with links to metabolism Function Putative lipid phosphatase of the endoplasmic reticulum Thymidylate and uridylate kinase Component of the septin ring Pyruvate kinase Thymidylate synthase Guanine nucleotide exchange factor Phosphoglucose isomerase Ubiquitin-conjugating enzyme (E2) Adenylate cyclase Cullin, structural protein of SCF complexes Leucyl tRNA synthetase Alanyl-tRNA synthetase Cell cycle phenotype Arrest at G1 Arrest at S Defective cytokinesis Arrest at G1 Arrest at S Arrest at G1 Arrest at S Arrest at G1 Arrest at G1 Arrest at G1 Arrest at G1 Arrest at G1 Link to metabolism Regulates Mn2+ homeostasis (Paidhungat & Garrett (1998) Nucleotide metabolism ( Jong et al. 1984, Sclafani & Fangman 1984) Regulates carbohydrate metabolism (Voronkova et al. 2006) Glycolytic enzyme (Maitra & Lobo 1977, Sprague 1977) Nucleotide metabolism (Game 1976) Involved in PKA nutrient-sensing pathway (Broek et al. 1987) Glycolytic enzyme (Dickinson & Williams 1987) Involved in methionine biosynthesis (Patton et al. 1998) Involved in PKA nutrient-sensing pathway (Boutelet et al. 1985) Involved in methionine biosynthesis (Patton et al. 1998) Amino acid metabolism (Hohmann & Thevelein 1992) Amino acid metabolism (Wrobel et al. 1999)

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CDC34 CDC35 CDC53 CDC60 CDC64

Abbreviations: PKA, protein kinase A; SCF, Skp1/Cullin/F-box.

were conducted (Hartwell et al. 1974). Among these CDC genes, many turned out to have interesting links to metabolism (Table 1). The mechanisms by which some of these metabolic pathways induce cell cycle arrest remain poorly understood. More recently, genome-wide, reverse genetic approaches have been adopted to identify genes important for cell cycle progression. A survey of essential genes identified a large number of genes involved in protein synthesis that induce cell cycle arrest at G1 when depleted (Yu et al. 2006). A prior genome-wide deletion screen identified many ribosome biogenesis genes as determinants of the critical cell size at START ( Jorgensen et al. 2002). These studies reaffirm the concept that increasing translation capacity is a prerequisite for cell growth and thus entry into the cell cycle (Rudra & Warner 2004). The study also identified several genes (CDC8, CDC21, DFR1, FOL2, DUT1) involved in nucleotide biosynthetic pathways, including known CDC genes required for progression through S phase, highlighting the reliance of

DNA synthesis on upregulation of nucleotide metabolism. Overexpression screens also identified hundreds of cell cycle–associated genes with potential links to metabolism (Niu et al. 2008, Stevenson et al. 2001).

Transcript Profiling
To investigate the cell cycle regulation of transcription, microarray studies were conducted to obtain gene expression profiles of cells set up to proceed through the cell cycle synchronously (Cho et al. 1998, Spellman et al. 1998). Genes involved in glycolysis, respiration, and membrane and cell wall synthesis, as well as a significant number of nutritional genes, were cyclically expressed as a function of the cell cycle (Cho et al. 1998, Spellman et al. 1998). In addition, several methionine biosynthesis genes were “unexpectedly” periodic (Spellman et al. 1998). Given the importance of ribosome biogenesis for cell growth and proliferation ( Jorgensen & Tyers 2004) (Figure 6), it is perhaps surprising that www.annualreviews.org • Metabolism and Cell Cycle 3.9

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Nutrients

ChIP-Chip
Transcription of cell cycle–regulated genes is orchestrated by major transcription factors at different stages of the cell cycle. ChIP (chromatin immunoprecipitation) studies of the key cell cycle transcription factors MBF, SBF, Mcm1p-Fkh1/2p-Ndd1p, Ace2p, and Swi5p have unveiled some aspects of cell cycle control over metabolism (Iyer et al. 2001, Simon et al. 2001). These data indicate that genes involved in cell wall and membrane synthesis were among the most significantly enriched classes of SBF targets (Iyer et al. 2001). In vegetative yeast cells, the cell wall comprises 15–30% of the dry weight and its major components are mannoproteins (40%) and glucans (50– 60%), which are composed of polysaccharides (Orlean 1997). These demanding biosynthetic processes may be subject to regulation by nutrient or carbohydrate availability. In addition to direct targets of SBF, a subsequent study revealed downstream transcription networks regulated by nine transcription factors (Hcm1p, Plm2p, Pog1p, Tos4p, Tos8p, Tye7p, Yap5p, Yhp1p, and Yox1p) as targets of SBF (Horak et al. 2002). These transcription factors bind to specific functional classes of genes. For example, a large number of Hcm1p targets have functions in cell wall synthesis and carbohydrate metabolism, and many of the Tye7p and Yap5p targets are involved in energy metabolism (Horak et al. 2002).

TOR

PKA

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ribi genes

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RP genes

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rRNA

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Increase translation capacity

Increase in cell mass

G1/S transition

Cell division

Figure 6 Ribosome biogenesis is critical for cell growth and proliferation. Ribosome biogenesis is controlled by the target of rapamycin (TOR) and protein kinase A (PKA) nutrient-sensing pathways. Transcription of cohorts of ribosomal protein (RP) genes and ribosome biogenesis (ribi) genes, as well as rRNA, are concurrently regulated by specific transcription factors downstream of TOR and PKA signaling. As a result of the increase in translation capacity, the cell increases in mass and size until it is ready to go through division. The acetylation of histones at RP and ribi genes is crucial for their induction (see Figure 7).

Proteomics
As Cdk1p has been established as a master regulator of the cell cycle, proteomic approaches have been undertaken to comprehensively identify its targets (Archambault et al. 2004, Ubersax et al. 2003). Although not significantly enriched, many genes involved in various aspects of metabolism were identified to be putative substrates of the kinase. Among these, Smp2p, an enzyme in phospholipid biosynthesis, was confirmed to be a Cdk1p substrate (Santos-Rosa et al. 2005). Another enzyme in lipid metabolism, Tgl4p, is also phosphorylated by Cdk1 (Kurat et al. 2009).

ribosomal protein (RP) genes and ribosome biogenesis (ribi) genes which encode rRNA processing and ribosome assembly factors were not definitively identified as cell cycle regulated in these studies. A potential explanation for this conundrum is provided in a later section.
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Phosphorylation of Tgl4p triggers lipolysis of the storage triacylglycerols to release fatty acids for membrane synthesis in late G1 , and Cdk1p phosphorylation of Smp2p at the onset of mitosis promotes phospholipid synthesis (Kurat et al. 2009, Santos-Rosa et al. 2005). Although these two enzymes are the only characterized metabolic targets of Cdk1p thus far, it is conceivable that more will be confirmed in the future. Cdk1p also phosphorylates the transcription factor Pho2p (Liu et al. 2000), which is involved in transcriptional activation of many metabolic genes.
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bypassed under such rapid growth conditions. Therefore, it is important to recognize the distinction between these two different modes of growth: one in which the cell transitions from a quiescent-like, starved state to growth, and the other in which a cell enters the next cell cycle with almost no delay under nutrient-rich conditions. The predominant mechanisms governing cell cycle regulation and control may differ depending on the growth state (i.e., exit from quiescence and entry into growth versus continuous rapid growth).

DYNAMICS OF METABOLISM OVER THE CELL CYCLE Two Modes of Investigating Cell Growth and Proliferation: The Transition From Quiescence to Growth Versus Continuous Rapid Growth
Many microorganisms must persist in quiescent or quiescent-like states for long periods in the absence of favorable nutritional conditions. Because nutrient availability is often unpredictable and sporadic in the wild, these cells must be primed to quickly respond to nutritional cues in order to readily commit to growth and proliferation upon return to favorable conditions (Sillje et al. 1997, 1999). Thus, the life of a microbial cell in the wild may often be composed of phases of rapid proliferation that alternate with potentially long periods of quiescence (G0 ) when nutrient availability becomes scarce. In contrast, typical growth conditions utilized in the laboratory are quite different, because cells are exposed to highly nutrientrich growth media containing an excess of these basic building blocks. Under such conditions, cells enter an exponential phase of rapid growth that is not limited by nutrient availability. Cell divisions initiate one after another with virtually no delay. In essence, there are virtually no G0 -like phases during exponential growth under nutrient-rich conditions. Certain nutritional checkpoints for cell cycle entry might be

The Yeast Metabolic Cycle: A Useful Platform for Cell Cycle and Metabolism Research
The chemostat was invented to slow down the growth of a microbial cell population to facilitate the study of numerous aspects of growth and metabolism (Novick & Szilard 1950). The growth rate of the cell population can be set by the dilution rate, which is the rate at which new medium is introduced into the growth vessel. Moreover, the chemostat enables the maintenance of constant pH, aeration, temperature, and nutrient levels, thereby creating a continuous, steady-state growth environment. By growing yeast in such a manner, it becomes possible to observe aspects of cell growth and behavior that are not possible using traditional batch cultures. Under such conditions, budding yeast cells become highly synchronized and exhibit robust oscillations of oxygen consumption called yeast metabolic cycles (YMC). Although such oscillatory behavior in budding yeast has been documented since the 1960s (Kaspar von Meyenburg 1969), only recently has the logic underlying such oscillations emerged through their systematic characterization. The gene expression and metabolite changes that occur as a function of long-period metabolic cycles with a period on the order of ∼4–5 h were determined (Tu et al. 2005, Tu et al. 2007). With respect to gene expression, over half of yeast genes (∼57%) are expressed periodically as a precise function of the YMC. Genes that encode proteins with a common function often display www.annualreviews.org • Metabolism and Cell Cycle 3.11

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similar temporal expression patterns, and different classes of genes are upregulated during entirely different temporal windows of the YMC (Figure 1). Analysis of the temporal gene expression profiles revealed three superclusters of gene expression, thereby defining three phases of the YMC (Figure 1): OX (oxidative, respiratory), RB (reductive, building), and RC (reductive, charging) (Tu et al. 2005). Subsequent analysis and interpretation of these data have revealed that the OX phase can be likened to growth (G1 ), the RB phase to cell division (S/M), and RC phase to survival/quiescence (G0 ). In dividing cells, mitosis persists throughout most of the RC phase (Figure 1). In short, under the continuous culture conditions used to observe the YMC, the growth rate of the cell population is reduced significantly, so that it becomes possible to observe what happens when cells exit quiescent-like phases and transition to growth-like phases. Moreover, such temporal orchestration of cellular processes should be accompanied by dynamic changes in the metabolic state of cells, which was subsequently shown by comprehensive metabolite profiling studies using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and twodimensional gas chromatography with time of flight mass spectrometry (GC x GC-TOFMS) methods (Mohler et al. 2008, Tu et al. 2007). These cyclic changes in metabolic state that occur as a function of these metabolic cycles, which are coupled to the cell cycle, have provided insights into the metabolic basis of cell cycle control. We now briefly summarize some of the metabolic changes that accompany entry into growth and the cell cycle as gleaned from studies of the YMC.

What Happens During Growth (the OX Phase of the Yeast Metabolic Cycle)?
When cells enter the OX phase of the YMC, which can be likened to G1 , the rate of oxygen consumption increases substantially, signifying a burst of mitochondrial respiration (Figure 1).
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The rapid transcriptional activation of virtually all genes required for growth or growthassociated functions is tightly coupled to this increase in mitochondrial activity (Cai et al. 2011, Tu et al. 2005). This includes genes encoding proteins involved in protein translation and amino acid biosynthesis, as well as virtually every gene important for ribosome biogenesis. Moreover, sulfur metabolism, nucleolar genes, and genes involved in the processing of rRNA and tRNAs are also activated during growth. The genes with unknown function that are induced during the OX phase can be predicted to serve some growth-associated function. As viewed in the YMC, this represents a collection of ∼1,100 genes, or ∼18% of the known open reading frames. The induction of this class of transcripts coincident with a burst of mitochondrial respiration may signify the need to couple these energetically costly growth processes to increased rates of ATP synthesis. A separate study aimed to identify those transcripts in budding yeast that are either positively or negatively correlated with growth rate (Brauer et al. 2008). Strikingly, the set of genes positively correlated with increasing growth rate displayed a substantial overlap with genes induced during the OX phase of the YMC. Moreover, a number of previous studies identified genes induced in response to glucose addition in yeast (Radonjic et al. 2005, Slattery & Heideman 2007, Wang et al. 2004). Many of these genes are found in the set of OX phase genes of the YMC and in the set of genes that are positively correlated with growth rate. Notably, the induction of these genes can be triggered by glucose through a poorly understood mechanism. Collectively, these growth genes appear devoted to the upregulation of translational capacity required for the building of a new cell, highlighting the importance of ribosome biogenesis for cell growth and entry into the cell cycle ( Jorgensen & Tyers 2004) (Figure 6). An analysis of the extracellular medium of cycling cells revealed the presence of ethanol and acetate upon entry into the OX growth phase (Tu et al. 2005). Ethanol and acetate are products of glycolytic metabolism in yeast and

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are indicative of a high glycolytic flux. However, it may seem paradoxical that cells growing under such a nutrient-challenging environment would upregulate glycolysis only to secrete ethanol as the end product, which yields only 2 ATP per molecule of glucose. By contrast, the complete oxidation of glucose via the TCA cycle and oxidative phosphorylation would produce on the order of 30+ ATP per glucose. This phenomenon of aerobic glycolysis was originally observed in S. cerevisiae by Crabtree (1928) and has been termed the Crabtree effect. Intriguingly, the secretion of ethanol as a product of aerobic glycolysis during yeast growth is highly reminiscent of the Warburg effect, in which cancer cells are frequently observed to secrete lactate despite their consumption of copious amounts of glucose (Koppenol et al. 2011). The seemingly inefficient metabolism of glucose by both yeast and cancer cells suggests that there may be an important reason for secreting these end products of glycolysis for rapidly proliferating cell populations. Coupled with the observation that dissolved oxygen is consumed rapidly upon entry into growth, it appears that rates of both glycolysis and mitochondrial oxidative phosphorylation increase upon entry into growth. Many metabolites were identified to increase in abundance upon entry into the OX growth phase (Tu et al. 2007). The upregulated production of each of these metabolites might be critical for some aspect of growth or entry into the cell cycle (Figure 1). These growth metabolites include glucose, numerous amino acid precursors and amino acids, nucleotide precursors, and sulfur metabolites. Most notably, the levels of two key metabolites, acetyl-CoA and NADPH, increase substantially upon entry into growth (Tu et al. 2005, 2007) and are discussed further below. For some of these metabolites, the increase in intracellular concentration may be required to support the increased rates of protein or DNA synthesis that accompany cell growth and division. Moreover, some of these metabolites may play important regulatory roles, in addition to functioning as substrates in biosynthetic

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reactions. Such sentinel metabolites may be directly sensed by particular enzymes or proteins within the cell and used as a barometer of cellular metabolic state to regulate a variety of cellular outputs (Cai & Tu 2011, Cai et al. 2011). An independent study identified cellular metabolites that are either positively or negatively correlated with growth rate (Boer et al. 2010). Metabolites that increase in abundance with respect to increasing growth rate may be considered as growth metabolites. In contrast, those that are negatively correlated with growth rate may have some function associated with starvation or quiescence.

Metabolism During the Cell Division Process (The RB Phase of the Yeast Metabolic Cycle)
The majority of the changes in metabolism discussed so far appear to be features of the G1 /OX growth phase, prior to the actual cell division process itself. The metabolic changes in G1 can be viewed as a growth period whose events occur in preparation for building a new cell. Because of its extraordinary metabolic synchrony, the YMC has enabled the precise timing of cell cycle gene expression at a previously unachievable resolution of ∼2–3 min (Rowicka et al. 2007). Most of the genes involved in growth and metabolism peak in expression during the OX growth phase, prior to the G1 /S transition and START. Many of the core cell cycle genes subsequently become activated during the RB phase. During M phase (which coincides with RB-RC phases), many fewer genes are induced at the transcriptional level (Rowicka et al. 2007). These observations are consistent with a general repression of transcription upon chromosome condensation during mitosis in mammalian cells (Gottesfeld & Forbes 1997). Though the degree of compaction of yeast chromosomes is far less than metazoan chromosomes during mitosis (Guacci et al. 1994), the reduction of transcriptional activity may be a strategy to avoid topological disturbances to the process of DNA segregation during mitosis. www.annualreviews.org • Metabolism and Cell Cycle 3.13

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As for metabolite fluctuations, upregulation of purine and thymidine nucleotide biosynthesis is observed. There is also increased utilization of SAM as seen by increased levels of S-adenosylhomocysteine (SAH), presumably for methylation of histones and biosynthetic substrates (Tu et al. 2007). Generally fewer changes in metabolite abundance seem to occur during mitosis compared to the period prior to the G1 /S transition. However, additional metabolic, biosynthetic, and catabolic events in terms of transcript and metabolite fluctuation may occur during cell division itself that simply cannot be detected at the gross level. For example, such events could be involved in lipid metabolism and membrane remodeling. It is also unclear which core cytological processes of mitosis might be coordinated with cellular metabolism.

Exit from Growth and Entry into Quiescence (the RC Phase of the Yeast Metabolic Cycle)
In the YMC, typically only a fraction of the cell population (