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Systems biology of energy homeostasis in yeast
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Systems biology of energy homeostasis in yeast Jie Zhang, Goutham Vemuri and Jens Nielsen The yeast Saccharomyces cerevisiae attains energy homeostasis through complex regulatory events that are predominantly controlled by the Snf1 kinase. This master regulator senses the stress and energy starvation and activates the metabolic processes to produce ATP and inhibits biosynthesis. In doing so, Snf1 controls the switch between catabolism and anabolism accordingly, and regulates the cellular growth and development in coordination with other signaling pathways. Since its mammalian ortholog AMPK, a drug target for obesity and type II diabetes, also exerts analogous control of metabolism, there has been extensive interest recently to understand the chemical and biological aspects of Snf1 activation and regulation in yeast to expedite human disease studies as well as fundamental understanding of yeast. This review will focus on how Snf1 regulates lipid metabolism based on the cellular energy status in yeast and drawing parallels with the mammalian system. Address Systems Biology Group, Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemiva¨gen 10, SE-412 96 Go¨teborg, Sweden Corresponding authors: Nielsen, Jens ([email protected])
Current Opinion in Microbiology 2010, 13:382–388 This review comes from a themed issue on Systems Biology Edited by Jens Nielsen and Marc Vidal Available online 1st May 2010 1369-5274/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2010.04.004
Introduction Supply of energy, or more specifically molecular ATP, is essential in all living cells. Besides being involved in a substantial part of the cellular metabolism (e.g. about 200 reactions in the budding yeast), it also regulates a broad range of cellular processes. Energy is mainly produced by oxidative phosphorylation in connection with respiration, and to some extent in the glycolysis and the tricarboxylic acid (TCA) cycle. The b-oxidation of fatty acids also results in ATP formation, but this also requires respiration. Energy expenditure occurs during anabolism, during the biosynthesis of proteins, lipids, nucleotides and developmental processes. The delicate balance between energy production and expenditure is governed by the highly conserved AMP-activated protein kinase Current Opinion in Microbiology 2010, 13:382–388
(AMPK) in all eukaryotes, including yeast, plants and humans [1]. The modus operandi of AMPK is to sense the energetic status and control the production and utilization of ATP. Lipid synthesis is one of the most energetically expensive processes and therefore requires tight regulation, but once synthesized, lipids can also serve as energy reservoirs. The elegant operation of Snf1/AMPK in controlling the synthesis and metabolism of lipids has significant medical relevance in understanding the metabolic syndrome. In this review, we focus on recent progress in our understanding of the regulation of the energy homeostasis and lipid metabolism, mainly in yeast Saccharomyces cerevisiae, an excellent model organism for detailed molecular studies, and compare it to similar processes occurring in mammals.
Regulation of Snf1/AMPK activation Snf1 belongs to a group of remarkably conserved Serine/ Threonine kinase family that exists in all eukaryotes ranging from yeast, worm, fruit fly, plant and mammals. Snf1/AMPK is a heterotrimer, composed of a catalytic asubunit (Snf1 in yeast), a regulatory g-subunit (Snf4 in yeast), and a scaffolding b-subunit (one of Sip1, Sip2 or Gal83 in yeast) that secures the a- and g-subunits to form a functional complex. Despite many years of intense research, the exact molecular activation of Snf1/AMPK still remains obscure. Only AMPK (and not Snf1) is activated by high concentration of AMP. Structural studies of AMPK revealed three binding sites on the gsubunit, two of which can be competitively occupied by AMP or ATP [2,3]. The relative concentration of AMP and ATP decides the resident nucleotide on the g-subunit and hereby controls activation or inactivation of AMPK, respectively. There is a third nucleotide binding site, also shared by Snf1 that is constitutively bound by AMP [4,5], suggesting that AMP may not be the signal for Snf1 activation. Additionally, Snf1 and AMPK are activated by phosphorylation on the a-subunit (T210 in yeast and T172 in mammals) by upstream kinases (Tos3, Elm1 and Sak1 in yeast and LKB1 and CaMKKb in mammals). The phosphatases Glc7 or the PP2Ca inactivate Snf1 or AMPK, respectively. Since the upstream kinases themselves are not sensitive to energy status [6], and since AMPK cannot be activated in an LKB1-dependent manner in vitro [7], it seems like the upstream kinases play a lesser role for the activation of Snf1/AMPK. On the contrary, the phosphatase was shown to play an active role in governing Snf1 activity in yeast [8]. Due to its key role in regulating lipid metabolism, AMPK is an interesting drug target and therefore the mechanisms by which Snf1/AMPK is activated has substantial impact on the treatment of lipid related diseases www.sciencedirect.com
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such as obesity, type II diabetes, fatty liver and arteriosclerosis.
Processes regulated by Snf1/AMPK Upon glucose depletion, Snf1 is activated and controls many processes through positive or negative regulation of gene expression and phosphorylation of transcription factors and metabolic enzymes (Figure 1). For example, Snf1 regulates the metabolism of non-fermentable carbon sources and b-oxidation of fatty acids via Adr1 [9], regulates the histone acetyltransferase Gcn5 [10] and controls the transcriptional regulation through the Spt3–Spt15 complex [11]. A more comprehensive role for Snf1 in other aspects of metabolism and developmental processes was discovered using a systems biology approach [12]. AMPK also phosphorylates peroxisome proliferator-activated receptor-g coactivator 1a (PGC-1a) at Thr177 and Ser538 [13], leading to increased glucose uptake, mitochondrial biogenesis and b-oxidation. Another level of regulation on the aforementioned processes is that AMPK increases the cellular NAD+ concentration and activates NAD+-dependent deacetylase sirtuin-1 (SIRT1), which results in the deacetylation of
SIRT1 targets that include PGC-1a, FOX transcription factors FOXO1 and FOXO3a [14,15]. It may be worth mentioning that the general effect of Snf1/AMPK is to switch the cellular metabolism from fermentation, one of the characteristics of cancer cells, to respiration, and thereby the study of Snf1 in yeast may serve as a model for the role of AMPK in the metabolism of cancer cells. Apart from the regulation on cellular metabolism, AMPK also induces apoptosis by increasing p53 both at mRNA and protein levels under glucose depletion [16] and regulates the cell structure with the involvement of its upstream kinase LKB1 [17].
Interaction between Snf1/AMPK and other regulatory pathways Cellular metabolism and developmental processes are coordinated tightly in response to the environmental changes such as nutrient depletion or stress. For example, protein synthesis accounts for a fairly large portion of energy expenditure, and it is therefore important to inhibit this process during energy deficiency. The ribosomal elongation factor (eEF2) is directly phosphorylated by AMPK [18] as well as yeast translational elongation
Figure 1
Simplified scheme of the role of Snf1/AMPK in regulating various cellular processes. Upon activation by phosphorylation (on the Thr172 in mammals or Thr210 in yeast on the a-subunit) SNf1/AMPK regulate various processes. The activation and downstream effects of Snf1/AMPK are shown in dotted and solid arrows, respectively. The regulation that is prevalent only in mammals and not reported in yeast is indicated by pale arrows. Green lines indicate activation and red lines indicate repression of the downstream function. www.sciencedirect.com
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factors (Tef1 and Tef2) were identified as substrates of Snf1 by high throughput analysis [19]. Protein synthesis is also regulated through the target of rapamycin kinase complex 1 (Tor1), which is also highly conserved in eukaryotes. Tor1 senses nitrogen availability and regulates the cellular growth by controlling the expression of amino acid permeases, ribosome biogenesis, protein biosynthesis and cell cycle [20–22]. While it has been conclusively shown in mammals that AMPK inhibits the mammalian TOR complex-1 (mTOR1) via the TSC1– TSC2 complex [1], hierarchy of the regulation between Snf1 and Tor1 in yeast remains elusive, although there is clear evidence that Snf1 plays a role in nitrogen signaling [23,24,25]. Interestingly, activation of Snf1/AMPK or repression of Tor1/mTOR1 increases life span [15,26,27], probably through interaction with the sirtuins (Sir2 in yeast or SIRT1 in mammals). It has also been found that resveratrol, the anti-oxidant in red wine, activates the sirtuins and PGC-1a to extend the life span in yeast [28] and mammals [29]. The cAMP-dependant protein kinase A (PKA) also regulates cell growth through transcriptional regulation, which accounts for, together with Sch9 (PKB), around 90% of the high glucoseinduced transcriptional changes in yeast [24]. PKA and Snf1 share the regulation of many processes such as carboxylic acid metabolism, b-oxidation of fatty acids,
stress response and filamentous growth, but under different conditions as they are activated by glucose excess or depletion respectively (Figure 2). On the contrary, PKA and Tor1 are both active in response to the nutritional stimuli (glucose and nitrogen) thereby function in parallel, to promote the growth via ribosome biogenesis and repress meiosis. The apparent complexity and high connectivity of these interacting signaling pathway and regulatory networks makes systems biology a very well suited approach for such studies, where the biological systems can be described in the form of network models or even detailed mathematical models that describes the kinetics of the pathways, and these models can be applied to integrate the large-scale experimental datasets and extract new biological information, in particularly on the cross talk between the different pathways. However, still there needs to be detailed reconstruction of the networks, and here the systems biological study of the Snf1 network represents a good starting point [12].
Lipid metabolism and carbon storage in yeast In addition to playing an essential role in membrane integrity and cell signaling, lipids also serve as energy storage. While carbohydrates function as instant energy sources or energy sources that can be mobilized rapidly,
Figure 2
Interactions between Snf1, Tor1 and PKA pathways in yeast. The blue and red lines indicate activation or repression through regulation, respectively. The black arrows indicate either single or multiple metabolic pathways. AKG: a-ketoglutarate; Glu: glutamate; Gln: glutamine. Current Opinion in Microbiology 2010, 13:382–388
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lipids represent energy storage that can be mobilized upon starvation. The coordination of lipid metabolism with glucose availability and energy status is the first and one of the most studied roles of Snf1 [30,31]. AcetylCoA is the precursor for the synthesis of fatty acids (phospholipids and triacylglycerols) and sterols. AcetylCoA carboxylase (ACC) is the first committed step towards fatty acids and acetyl-CoA thiolase commits carbon to sterol biosynthesis. These two pathways from acetyl-CoA are controlled by Snf1/AMPK either by direct phosphorylation or via transcription factors that regulate the expression of the genes in these pathways (Figure 3). The ACC1 isoform of ACC is phosphorylated by Snf1/AMPK and the subsequent fatty acid synthesis is under the transcriptional regulators such as Spt23 and Ino2/4 in yeast [32,33] and sterol and carbohydrate response-elements binding proteins (SREBP-1 and ChRBP) in mammals [34]. More recently, an important regulatory role for the Tor1 kinase in regulating lipid biosynthesis in mammals was shown [35], providing yet another link for the cross talk between carbon and nitrogen metabolism. A major breakthrough in understanding lipid metabolism came from the discovery that
the oxidation of fatty acids is not just the reversal of fatty acid synthesis, and that these two processes have distinct pathways and are regulated differently. Fatty acid oxidation is predominantly controlled by transcription factors such as Oaf1, Pip2, etc in yeast (Figure 3), which stimulate peroxisome biogenesis and are analogous to the peroxisome proliferator-activated receptor-a (PPAR-a) in mammals. While the kinase that activates the yeast transcription factors is not identified, PPAR-a was shown to be under AMPK control [36]. The other isoform of ACC, ACC2 is mitochondrial and is important for regulation of fatty acid oxidation, which also occurs in the mitochondria of mammals. Fatty acids are converted into acylcarnitines before they enter the mitochondria for b-oxidation. The conversion is mediated by carnitine palmitoyltransferase (CPT1), which is inhibited by malonyl-CoA, the product of ACC2. Although yeast has an ortholog for ACC2 [37], this regulation does not appear to be conserved because fatty acid oxidation is restricted to the peroxisomes in yeast. The sterol pathway is also conserved between yeast and mammals and the main rate-controlling step in the path-
Figure 3
The regulation of lipid metabolism in yeast. The blue and red lines indicate activation or repression through regulation, respectively. The black arrows indicate either single or multiple metabolic pathways. C16:0 – palmitic acid; C16:1 – palmitoleic acid; C18:0 – stearic acid; C18:1 – oleic acid; DAG – diacylglycerol; TAG – triacylglycerol; PL – phospholipid; PA – phosphatidic acid; SE – sterolester. www.sciencedirect.com
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way is the HMG-CoA reductase (HMGR), which is directly controlled by AMPK in humans [38]. Although there are no reports of any interaction between Snf1 and Hmg1, Snf1 appears to regulate the sterol pathway via repressing the transcription factors Upc2 and Ecm22 (Zhang et al., unpublished). This regulation at the transcriptional level also appears to be conserved in between yeast and mammals, with SREBP-2 regulating sterol biosynthetic genes in mammals [39]. In addition to lipids, glycogen and trehalose are also conventionally considered as energy reserves, since they are a quick source of carbohydrates. Several lines of evidence suggest that trehalose does not fit this role and mainly functions as a highly efficient protecting agent to maintain structural integrity of the cytoplasm during stress [40]. The link between storage carbohydrate metabolism and Snf1/AMPK is evident from the glycogen-binding domains on the b-subunit of the complex. High level of glycogen is associated with low AMPK activity [41], supporting the idea that glycogen could inhibit the activity of AMPK. Intuitively, it seems logical that AMPK activity would not be needed when the level of storage carbohydrates is high. More recently, AMPK is also attributed the role of being a glycogen sensor [42]. Thus, point mutations in the residues of the g-subunit domain, that interacts with the glycogen-binding domain of the b-subunit, result in significant changes in activation in yeast and mammals [43,44]. Conversely, Snf1 was also shown to control glycogen accumulation by phosphorylating glycogen synthase [45]. Given the functional conservation of Snf1/AMPK in glycogen synthesis and metabolism, it is likely that Snf1 also serves as a sensor of glycogen in yeast.
Perspectives in using yeast as a human model organism The budding yeast has served as a good model organism owing to several inherent properties, such as the well-developed molecular tools, easy to culture, extensive annotation of the genome, the existence of many different databases, and the availability of substantial collections of dataset. The fact that yeast and mammals share many homologues or orthologs makes it possible to express the proteins from mammals in yeast to study the role of these proteins in their respective regulatory networks. An example of this is the identification of LKB1 as the upstream kinases of AMPK based on the study of Snf1 pathway in yeast [46]. In addition to the obvious proteins that share sequence similarity, there are many proteins that are remarkably similar in their function between yeast and mammals, despite the low sequence homology. For example, the transcription factors Oaf1/Pip2 promote the biogenesis of peroxisome in yeast and therefore activate the boxidation. In mammals, peroxisome formation is Current Opinion in Microbiology 2010, 13:382–388
Table 1 Functional similarity between yeast and human Cellular processes
Yeast
Human
Glycolysis Fatty acid biosynthesis
Pfk1/2 Acc1, Mga2/Spt23
b-Oxidation Sterol metabolism
Adr1, Oaf1/Pip2 Hmg1, Upc2/Ecm22
Mitochondria biogenesis Amino acid biosynthesis Protein synthesis Ageing Heat shot response Cell cycle
? Gcn4 Tor1 Sir2 Hsf1 Cdc28
PFKM ACC1, SREBP-1 PPARs HMGR, SREBP-2 PGC-1a ATF4 mTOR1 SIRT1 HSF1 CDC2
SREBP: sterol regulatory element binding protein; PPARs: peroxisome proliferator-activated receptors; HMGR: HMG-CoA reductase; PGC-1a: peroxisome proliferator-activated receptor-g coactivator 1a.
regulated by peroxisome proliferator-activated receptors (PPARs), a group of nuclear receptor proteins that act as transcription factors [39], and there are no (or very low) sequence homology between these proteins. A small sample of the proteins involved in important processes that are conserved between yeast and humans is listed in Table 1. Thus, despite the lack of direct homology many processes are often conserved and this opens for performing detailed molecular studies in yeast, and based on this build general models or reaction networks that can then be evaluated in mammalian cells.
Concluding remarks Eukaryotes control the energy homeostasis by integrating a series of nutrients sensing and signaling cascades, as well as coordinating many metabolic pathways and developmental processes tightly. Many of these pathways/networks, such as Snf1 and Tor1, are very complex and highly connected to each other, making it very difficult to analyze the high throughput data from largescale experiments and extract concise biological information. Systems biology, which is characterized by mathematical models, can be applied as a scaffold to integrate omics datasets and this approach is therefore well suited for study of large and complex regulatory networks. A recently reconstructed network for Snf1 may serve as a template for study of AMPK in mammals, given the high degree of conservation between Snf1 and AMPK pathways, as well as it may serve as a starting point for establishing a larger interaction network for different protein kinases (Nandy et al., in press).
Acknowledgements Research activities on nutrient sensing in yeast in our laboratory are supported by the Chalmers Foundation, the Knut and Alice Wallenberg Foundation and the EU-funded project UNICELLSYS. www.sciencedirect.com
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Systems biology of energy homeostasis in yeast Jie Zhang, Goutham Vemuri and Jens Nielsen The yeast Saccharomyces cerevisiae attains energy homeostasis through complex regulatory events that are predominantly controlled by the Snf1 kinase. This master regulator senses the stress and energy starvation and activates the metabolic processes to produce ATP and inhibits biosynthesis. In doing so, Snf1 controls the switch between catabolism and anabolism accordingly, and regulates the cellular growth and development in coordination with other signaling pathways. Since its mammalian ortholog AMPK, a drug target for obesity and type II diabetes, also exerts analogous control of metabolism, there has been extensive interest recently to understand the chemical and biological aspects of Snf1 activation and regulation in yeast to expedite human disease studies as well as fundamental understanding of yeast. This review will focus on how Snf1 regulates lipid metabolism based on the cellular energy status in yeast and drawing parallels with the mammalian system. Address Systems Biology Group, Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemiva¨gen 10, SE-412 96 Go¨teborg, Sweden Corresponding authors: Nielsen, Jens ([email protected])
Current Opinion in Microbiology 2010, 13:382–388 This review comes from a themed issue on Systems Biology Edited by Jens Nielsen and Marc Vidal Available online 1st May 2010 1369-5274/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2010.04.004
Introduction Supply of energy, or more specifically molecular ATP, is essential in all living cells. Besides being involved in a substantial part of the cellular metabolism (e.g. about 200 reactions in the budding yeast), it also regulates a broad range of cellular processes. Energy is mainly produced by oxidative phosphorylation in connection with respiration, and to some extent in the glycolysis and the tricarboxylic acid (TCA) cycle. The b-oxidation of fatty acids also results in ATP formation, but this also requires respiration. Energy expenditure occurs during anabolism, during the biosynthesis of proteins, lipids, nucleotides and developmental processes. The delicate balance between energy production and expenditure is governed by the highly conserved AMP-activated protein kinase Current Opinion in Microbiology 2010, 13:382–388
(AMPK) in all eukaryotes, including yeast, plants and humans [1]. The modus operandi of AMPK is to sense the energetic status and control the production and utilization of ATP. Lipid synthesis is one of the most energetically expensive processes and therefore requires tight regulation, but once synthesized, lipids can also serve as energy reservoirs. The elegant operation of Snf1/AMPK in controlling the synthesis and metabolism of lipids has significant medical relevance in understanding the metabolic syndrome. In this review, we focus on recent progress in our understanding of the regulation of the energy homeostasis and lipid metabolism, mainly in yeast Saccharomyces cerevisiae, an excellent model organism for detailed molecular studies, and compare it to similar processes occurring in mammals.
Regulation of Snf1/AMPK activation Snf1 belongs to a group of remarkably conserved Serine/ Threonine kinase family that exists in all eukaryotes ranging from yeast, worm, fruit fly, plant and mammals. Snf1/AMPK is a heterotrimer, composed of a catalytic asubunit (Snf1 in yeast), a regulatory g-subunit (Snf4 in yeast), and a scaffolding b-subunit (one of Sip1, Sip2 or Gal83 in yeast) that secures the a- and g-subunits to form a functional complex. Despite many years of intense research, the exact molecular activation of Snf1/AMPK still remains obscure. Only AMPK (and not Snf1) is activated by high concentration of AMP. Structural studies of AMPK revealed three binding sites on the gsubunit, two of which can be competitively occupied by AMP or ATP [2,3]. The relative concentration of AMP and ATP decides the resident nucleotide on the g-subunit and hereby controls activation or inactivation of AMPK, respectively. There is a third nucleotide binding site, also shared by Snf1 that is constitutively bound by AMP [4,5], suggesting that AMP may not be the signal for Snf1 activation. Additionally, Snf1 and AMPK are activated by phosphorylation on the a-subunit (T210 in yeast and T172 in mammals) by upstream kinases (Tos3, Elm1 and Sak1 in yeast and LKB1 and CaMKKb in mammals). The phosphatases Glc7 or the PP2Ca inactivate Snf1 or AMPK, respectively. Since the upstream kinases themselves are not sensitive to energy status [6], and since AMPK cannot be activated in an LKB1-dependent manner in vitro [7], it seems like the upstream kinases play a lesser role for the activation of Snf1/AMPK. On the contrary, the phosphatase was shown to play an active role in governing Snf1 activity in yeast [8]. Due to its key role in regulating lipid metabolism, AMPK is an interesting drug target and therefore the mechanisms by which Snf1/AMPK is activated has substantial impact on the treatment of lipid related diseases www.sciencedirect.com
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such as obesity, type II diabetes, fatty liver and arteriosclerosis.
Processes regulated by Snf1/AMPK Upon glucose depletion, Snf1 is activated and controls many processes through positive or negative regulation of gene expression and phosphorylation of transcription factors and metabolic enzymes (Figure 1). For example, Snf1 regulates the metabolism of non-fermentable carbon sources and b-oxidation of fatty acids via Adr1 [9], regulates the histone acetyltransferase Gcn5 [10] and controls the transcriptional regulation through the Spt3–Spt15 complex [11]. A more comprehensive role for Snf1 in other aspects of metabolism and developmental processes was discovered using a systems biology approach [12]. AMPK also phosphorylates peroxisome proliferator-activated receptor-g coactivator 1a (PGC-1a) at Thr177 and Ser538 [13], leading to increased glucose uptake, mitochondrial biogenesis and b-oxidation. Another level of regulation on the aforementioned processes is that AMPK increases the cellular NAD+ concentration and activates NAD+-dependent deacetylase sirtuin-1 (SIRT1), which results in the deacetylation of
SIRT1 targets that include PGC-1a, FOX transcription factors FOXO1 and FOXO3a [14,15]. It may be worth mentioning that the general effect of Snf1/AMPK is to switch the cellular metabolism from fermentation, one of the characteristics of cancer cells, to respiration, and thereby the study of Snf1 in yeast may serve as a model for the role of AMPK in the metabolism of cancer cells. Apart from the regulation on cellular metabolism, AMPK also induces apoptosis by increasing p53 both at mRNA and protein levels under glucose depletion [16] and regulates the cell structure with the involvement of its upstream kinase LKB1 [17].
Interaction between Snf1/AMPK and other regulatory pathways Cellular metabolism and developmental processes are coordinated tightly in response to the environmental changes such as nutrient depletion or stress. For example, protein synthesis accounts for a fairly large portion of energy expenditure, and it is therefore important to inhibit this process during energy deficiency. The ribosomal elongation factor (eEF2) is directly phosphorylated by AMPK [18] as well as yeast translational elongation
Figure 1
Simplified scheme of the role of Snf1/AMPK in regulating various cellular processes. Upon activation by phosphorylation (on the Thr172 in mammals or Thr210 in yeast on the a-subunit) SNf1/AMPK regulate various processes. The activation and downstream effects of Snf1/AMPK are shown in dotted and solid arrows, respectively. The regulation that is prevalent only in mammals and not reported in yeast is indicated by pale arrows. Green lines indicate activation and red lines indicate repression of the downstream function. www.sciencedirect.com
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factors (Tef1 and Tef2) were identified as substrates of Snf1 by high throughput analysis [19]. Protein synthesis is also regulated through the target of rapamycin kinase complex 1 (Tor1), which is also highly conserved in eukaryotes. Tor1 senses nitrogen availability and regulates the cellular growth by controlling the expression of amino acid permeases, ribosome biogenesis, protein biosynthesis and cell cycle [20–22]. While it has been conclusively shown in mammals that AMPK inhibits the mammalian TOR complex-1 (mTOR1) via the TSC1– TSC2 complex [1], hierarchy of the regulation between Snf1 and Tor1 in yeast remains elusive, although there is clear evidence that Snf1 plays a role in nitrogen signaling [23,24,25]. Interestingly, activation of Snf1/AMPK or repression of Tor1/mTOR1 increases life span [15,26,27], probably through interaction with the sirtuins (Sir2 in yeast or SIRT1 in mammals). It has also been found that resveratrol, the anti-oxidant in red wine, activates the sirtuins and PGC-1a to extend the life span in yeast [28] and mammals [29]. The cAMP-dependant protein kinase A (PKA) also regulates cell growth through transcriptional regulation, which accounts for, together with Sch9 (PKB), around 90% of the high glucoseinduced transcriptional changes in yeast [24]. PKA and Snf1 share the regulation of many processes such as carboxylic acid metabolism, b-oxidation of fatty acids,
stress response and filamentous growth, but under different conditions as they are activated by glucose excess or depletion respectively (Figure 2). On the contrary, PKA and Tor1 are both active in response to the nutritional stimuli (glucose and nitrogen) thereby function in parallel, to promote the growth via ribosome biogenesis and repress meiosis. The apparent complexity and high connectivity of these interacting signaling pathway and regulatory networks makes systems biology a very well suited approach for such studies, where the biological systems can be described in the form of network models or even detailed mathematical models that describes the kinetics of the pathways, and these models can be applied to integrate the large-scale experimental datasets and extract new biological information, in particularly on the cross talk between the different pathways. However, still there needs to be detailed reconstruction of the networks, and here the systems biological study of the Snf1 network represents a good starting point [12].
Lipid metabolism and carbon storage in yeast In addition to playing an essential role in membrane integrity and cell signaling, lipids also serve as energy storage. While carbohydrates function as instant energy sources or energy sources that can be mobilized rapidly,
Figure 2
Interactions between Snf1, Tor1 and PKA pathways in yeast. The blue and red lines indicate activation or repression through regulation, respectively. The black arrows indicate either single or multiple metabolic pathways. AKG: a-ketoglutarate; Glu: glutamate; Gln: glutamine. Current Opinion in Microbiology 2010, 13:382–388
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lipids represent energy storage that can be mobilized upon starvation. The coordination of lipid metabolism with glucose availability and energy status is the first and one of the most studied roles of Snf1 [30,31]. AcetylCoA is the precursor for the synthesis of fatty acids (phospholipids and triacylglycerols) and sterols. AcetylCoA carboxylase (ACC) is the first committed step towards fatty acids and acetyl-CoA thiolase commits carbon to sterol biosynthesis. These two pathways from acetyl-CoA are controlled by Snf1/AMPK either by direct phosphorylation or via transcription factors that regulate the expression of the genes in these pathways (Figure 3). The ACC1 isoform of ACC is phosphorylated by Snf1/AMPK and the subsequent fatty acid synthesis is under the transcriptional regulators such as Spt23 and Ino2/4 in yeast [32,33] and sterol and carbohydrate response-elements binding proteins (SREBP-1 and ChRBP) in mammals [34]. More recently, an important regulatory role for the Tor1 kinase in regulating lipid biosynthesis in mammals was shown [35], providing yet another link for the cross talk between carbon and nitrogen metabolism. A major breakthrough in understanding lipid metabolism came from the discovery that
the oxidation of fatty acids is not just the reversal of fatty acid synthesis, and that these two processes have distinct pathways and are regulated differently. Fatty acid oxidation is predominantly controlled by transcription factors such as Oaf1, Pip2, etc in yeast (Figure 3), which stimulate peroxisome biogenesis and are analogous to the peroxisome proliferator-activated receptor-a (PPAR-a) in mammals. While the kinase that activates the yeast transcription factors is not identified, PPAR-a was shown to be under AMPK control [36]. The other isoform of ACC, ACC2 is mitochondrial and is important for regulation of fatty acid oxidation, which also occurs in the mitochondria of mammals. Fatty acids are converted into acylcarnitines before they enter the mitochondria for b-oxidation. The conversion is mediated by carnitine palmitoyltransferase (CPT1), which is inhibited by malonyl-CoA, the product of ACC2. Although yeast has an ortholog for ACC2 [37], this regulation does not appear to be conserved because fatty acid oxidation is restricted to the peroxisomes in yeast. The sterol pathway is also conserved between yeast and mammals and the main rate-controlling step in the path-
Figure 3
The regulation of lipid metabolism in yeast. The blue and red lines indicate activation or repression through regulation, respectively. The black arrows indicate either single or multiple metabolic pathways. C16:0 – palmitic acid; C16:1 – palmitoleic acid; C18:0 – stearic acid; C18:1 – oleic acid; DAG – diacylglycerol; TAG – triacylglycerol; PL – phospholipid; PA – phosphatidic acid; SE – sterolester. www.sciencedirect.com
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way is the HMG-CoA reductase (HMGR), which is directly controlled by AMPK in humans [38]. Although there are no reports of any interaction between Snf1 and Hmg1, Snf1 appears to regulate the sterol pathway via repressing the transcription factors Upc2 and Ecm22 (Zhang et al., unpublished). This regulation at the transcriptional level also appears to be conserved in between yeast and mammals, with SREBP-2 regulating sterol biosynthetic genes in mammals [39]. In addition to lipids, glycogen and trehalose are also conventionally considered as energy reserves, since they are a quick source of carbohydrates. Several lines of evidence suggest that trehalose does not fit this role and mainly functions as a highly efficient protecting agent to maintain structural integrity of the cytoplasm during stress [40]. The link between storage carbohydrate metabolism and Snf1/AMPK is evident from the glycogen-binding domains on the b-subunit of the complex. High level of glycogen is associated with low AMPK activity [41], supporting the idea that glycogen could inhibit the activity of AMPK. Intuitively, it seems logical that AMPK activity would not be needed when the level of storage carbohydrates is high. More recently, AMPK is also attributed the role of being a glycogen sensor [42]. Thus, point mutations in the residues of the g-subunit domain, that interacts with the glycogen-binding domain of the b-subunit, result in significant changes in activation in yeast and mammals [43,44]. Conversely, Snf1 was also shown to control glycogen accumulation by phosphorylating glycogen synthase [45]. Given the functional conservation of Snf1/AMPK in glycogen synthesis and metabolism, it is likely that Snf1 also serves as a sensor of glycogen in yeast.
Perspectives in using yeast as a human model organism The budding yeast has served as a good model organism owing to several inherent properties, such as the well-developed molecular tools, easy to culture, extensive annotation of the genome, the existence of many different databases, and the availability of substantial collections of dataset. The fact that yeast and mammals share many homologues or orthologs makes it possible to express the proteins from mammals in yeast to study the role of these proteins in their respective regulatory networks. An example of this is the identification of LKB1 as the upstream kinases of AMPK based on the study of Snf1 pathway in yeast [46]. In addition to the obvious proteins that share sequence similarity, there are many proteins that are remarkably similar in their function between yeast and mammals, despite the low sequence homology. For example, the transcription factors Oaf1/Pip2 promote the biogenesis of peroxisome in yeast and therefore activate the boxidation. In mammals, peroxisome formation is Current Opinion in Microbiology 2010, 13:382–388
Table 1 Functional similarity between yeast and human Cellular processes
Yeast
Human
Glycolysis Fatty acid biosynthesis
Pfk1/2 Acc1, Mga2/Spt23
b-Oxidation Sterol metabolism
Adr1, Oaf1/Pip2 Hmg1, Upc2/Ecm22
Mitochondria biogenesis Amino acid biosynthesis Protein synthesis Ageing Heat shot response Cell cycle
? Gcn4 Tor1 Sir2 Hsf1 Cdc28
PFKM ACC1, SREBP-1 PPARs HMGR, SREBP-2 PGC-1a ATF4 mTOR1 SIRT1 HSF1 CDC2
SREBP: sterol regulatory element binding protein; PPARs: peroxisome proliferator-activated receptors; HMGR: HMG-CoA reductase; PGC-1a: peroxisome proliferator-activated receptor-g coactivator 1a.
regulated by peroxisome proliferator-activated receptors (PPARs), a group of nuclear receptor proteins that act as transcription factors [39], and there are no (or very low) sequence homology between these proteins. A small sample of the proteins involved in important processes that are conserved between yeast and humans is listed in Table 1. Thus, despite the lack of direct homology many processes are often conserved and this opens for performing detailed molecular studies in yeast, and based on this build general models or reaction networks that can then be evaluated in mammalian cells.
Concluding remarks Eukaryotes control the energy homeostasis by integrating a series of nutrients sensing and signaling cascades, as well as coordinating many metabolic pathways and developmental processes tightly. Many of these pathways/networks, such as Snf1 and Tor1, are very complex and highly connected to each other, making it very difficult to analyze the high throughput data from largescale experiments and extract concise biological information. Systems biology, which is characterized by mathematical models, can be applied as a scaffold to integrate omics datasets and this approach is therefore well suited for study of large and complex regulatory networks. A recently reconstructed network for Snf1 may serve as a template for study of AMPK in mammals, given the high degree of conservation between Snf1 and AMPK pathways, as well as it may serve as a starting point for establishing a larger interaction network for different protein kinases (Nandy et al., in press).
Acknowledgements Research activities on nutrient sensing in yeast in our laboratory are supported by the Chalmers Foundation, the Knut and Alice Wallenberg Foundation and the EU-funded project UNICELLSYS. www.sciencedirect.com
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