REGULATION OF DGK1-ENCODED DIACYLGLYCEROL KINASE UPON RESUMPTION OF GROWTH FROM STATIONARY PHASE IN SACCHAROMYCES CEREVISIAE CHRYSANTHOS KONSTANTINOU

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1 REGULATION OF DGK1-ENCODED DIACYLGLYCEROL KINASE UPON RESUMPTION OF GROWTH FROM STATIONARY PHASE IN SACCHAROMYCES CEREVISIAE By CHRYSANTHOS KONSTANTINOU A thesis submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Master of Science Graduate Program in Food Science written under the direction of Dr George M. Carman and approved by New Brunswick, New Jersey May, 2011

2 ABSTRACT OF THE THESIS Regulation of DGK1-encoded diacylglycerol kinase upon resumption of growth from stationary phase in Saccharomyces cerevisiae by CHRYSANTHOS KONSTANTINOU Thesis Director: Dr George M. Carman Studies in our laboratory are focused on the mobilization of triacylglycerol for phospholipid synthesis when yeast resumes growth from the stationary phase. This metabolic process includes the hydrolysis of triacylglycerol by lipase enzymes to generate free fatty acid and diacylglycerol. Fatty acids can be incorporated via multiple steps into the phospholipid precursor phosphatidic acid. Recent studies in our laboratory have uncovered a novel enzyme that can convert diacylglycerol and CTP into phosphatidic acid. This enzyme, known as diacylglycerol kinase, is ii

3 encoded by the DGK1 gene. In this study, we addressed the hypothesis that diacylglycerol kinase plays an important role in growth resumption from stationary phase. Experimentally, wild type yeast and yeast without the DGK1 gene (e.g., dgk1δ mutant) were first grown to the stationary phase, and then cultured in fresh medium to allow for growth resumption. The fatty acid synthesis inhibitor cerulenin was included in the medium to accentuate the need for triacylglycerol mobilization. While wild type yeast resumed growth (measured spectrophotometrically) from the stationary phase, the dgk1δ mutant cells did not resume growth. If dgk1δ mutant cells expressed the wild type DGK1 gene on a plasmid, cells resumed growth from the stationary phase. However, if dgk1δ cells expressed the catalytic dead version of DGK1 (D177A), cells did not resume growth. These data indicated that growth resumption required diacylglycerol kinase activity. In wild type cells, diacylglycerol kinase protein (measured by immunoblotting with anti-diacylglycerol kinase antibodies) and activity (measured by following the incorporation of radioactive CTP into diacylglycerol to form phosphatidic acid) increased as cells resumed growth. This work advanced our understanding of the metabolic processes involved in the mobilization of triacylglycerol for membrane phospholipid synthesis and growth resumption of stationary phase yeast. iii

4 DEDICATION I beg Thee, my Jesus, my love that is sweetest! O life of my soul and my heart s delectation, My intellect s brightness, O love that is perfect! O fountain of Love and my hope and my faith Teach me how I must seek Thee in order to find Thee Just tell me the way, for I want nothing else. And let not any powers or dominions detach me, Nor Belial the rival with his unholy angels Nor temporal pleasures of this age which is passing, Nor all of the world with its fleeting enjoyments My God and Creator, my love and my Savior Together with all the Righteous and Prophets, Apostles and Martyrs And all hosts of the heavens: Archangels and Angels, With the Cherubim, Seraphim, Thrones, and the Powers, And our sweetest, The lady of all, our most pure Theotokos Amen. iv

5 ACKNOWLEDGEMENTS I would like to thank Dr. George M. Carman for serving as my advisor. I am deeply grateful to all of my laboratory colleagues: Stylianos Fakas, Hyeon-Son Choi, Zhi Xu, Minjung Chae, Anibal Soto, Florencia Pascual; special thanks to Wen-Min Su. I would like also to thank the members of my thesis committee Drs. Loredana Quadro and Gil-Soo Han. This work was supported by National Institute of Health grant GM v

6 TABLE OF CONTENTS PAGE ABSTRACT OF THE THESIS ii DEDICATION...iv ACKNOWLEDGEMENTS v TABLE OF CONTENTS...vi LIST OF TABLES.ix LIST OF ILLUSTRATIONS.x LIST OF ABBREVIATIONS xii 1. INTRODUCTION Yeast: a Model Eukaryotic Organism to Study Lipid Metabolism Phospholipids Phospholipid in Yeast DGK1-Encoded Diacylglycerol Kinase 7 vi

7 1.4. TAG metabolism EXPERIMENTAL PROCEDURES Materials Strains and Growth Conditions Plasmid Construction Preparation of Cell Extracts Protein Concentration Preparation of [γ 32 -P]CTP DAG Kinase Assay Immunoblot Analysis RESULTS DAG Kinase Activity is Linear with Time and Protein Concentration Regulation of DAG Kinase Activity upon Resumption of Growth from Stationary Phase in Wild Type Cells Regulation of DAG Kinase Activity upon Resumption of Growth from Stationary Phase in a Strain Overexpressing DGK1 gene 39 vii

8 3.4. Regulation of DAG Kinase Protein Expression upon Resumption of Growth from Stationary Phase SUMMARY Discussion..46 REFERENCES..52 viii

9 LIST OF TABLES PAGE I. Strains and plasmids used in this work 26 ix

10 LIST OF ILLUSTRATIONS PAGE 1. Phospholipid biosynthetic pathways in S. cerevisiae Domain structure of DGK1-encoded diacylglycerol kinase Pathways for the mobilization of triacylglycerol upon resumption of growth from stationary phase Time-dependence of DAG kinase activity Protein concentration- dependence of DAG kinase activity Regulation of DAG kinase activity upon resumption of growth from stationary phase in wild type cells Resumption of growth from stationary phase of wild type cells Regulation of DAG kinase activity upon resumption of growth from stationary phase in wild type cells transformed with plasmid YEp351-DGK x

11 9. Resumption of growth from stationary of wild type cells transformed with plasmid YEp351-DGK Regulation of DAG kinase expression upon resumption of growth from stationary phase in wild type cells transformed with plasmid YEp351-DGK xi

12 LIST OF ABBREVIATIONS ATP adenosine triphosphate CDP-DAG cytidine diphosphate diacylglycerol CTP cytidine triphosphate DAG diacylglycerol FA fatty acid IgG immunoglobulin G I inositol MOPS 3-(N-morpholino)propanesulfonic acid mrna messenger RNA P phosphate PA phosphatidic acid (also known as phosphatidate) PC phosphatidylcholine PE phosphatidylethanolamine PG phosphatidylglycerol xii

13 STE steryl ester ST sterol TAG triacylglycerol PI4,5P 2 phosphatidylinositol-4,5-bisphosphate UAS INO inositol sensitive upstream activation sequence xiii

14 1 CHAPTER 1 INTRODUCTION 1.1 Yeast: a Model Eukaryotic Organism to Study Lipid Metabolism Saccharomyces cerevisiae, widely known as baker s or brewer s yeast, is used in industry for the production of bread, wine and beer. The yeast S. cerevisiae is an ideal model eukaryote for biological studies. Its genome is highly conserved among higher eukaryotes. However, the similarity is not confined to homology of the individual genes encoding proteins that account for evolutionary conserved function, but extends to the architecture of the major metabolic and signaling pathways and other cellular processes, such as membrane trafficking and cell division (1). Furthermore, the ease of the genetic manipulation (highly versatile DNA transformation system) of S. cerevisiae has greatly enhanced its use as a model organism. Additional features that render S. cerevisiae a practical tool for biological studies are its rapid growth, easy of replica plating and mutant isolation, non-pathogenicity and cheap commercial availability. S. cerevisiae was the first eukaryote whose genome was totally sequenced. The overwhelming advantages of yeast is best illustrated by the fact that many mammalian genes can be inserted into yeast in order to study the biological functions of their equivalent gene products. Furthermore, the broad range of accessible yeast mutants allows the perturbation of lipid metabolism and examination of its effects on molecular and

15 2 cellular levels. To a high degree, the fundamental lipid metabolism of yeast is akin to that of mammalian cells. 1.2 Phospholipids Membranes form boundaries surrounding the cell (plasma membrane) and cellular organelles (e.g. nucleus, mitochondria). They serve as selectively permeable barriers (thereby enabling the internal environment of the cell or organelle to be different from the outside) and are engaged in signaling processes. Lipids are a wide class of biological molecules. The main types of lipids found in biological membranes are phospholipids, sphingolipids and sterols. Phospholipids are made up of a hydrophobic tail and a hydrophilic head. The tail is formed by the attachment of two fatty acyl chains to a glycerol backbone. The hydrophilic head is composed of a phosphate and polar group (e.g. choline). The tail and the head are combined through a phosphodiester bond. When exposed to an external water environment, phospholipids tend to associate with each other through hydrophobic interactions forming lipid bilayers, which are the structural components of biological membranes. The name of a phospholipid is dictated by the head group. The most commonly found phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG). The simplest phospholipid is phosphatidic acid (PA) in which only a phosphate group is esterified to carbon-3 of the glycerol

16 3 backbone. Although PA is of low abundance in membranes, it is the major precursor to membrane phospholipids Phospholipid Biosynthesis in Yeast The membrane phospholipids of S. cerevisiae in ascending order of abundance are PS, PI, PE and PC (2). Phospholipid synthesis in yeast is regulated by both genetic and biochemical mechanisms (3). On the one hand, the activity of phospholipid biosynthetic enzymes is regulated by gene expression at the level of transcription and mrna stability (3). On the other hand the activity of phospholipid biosynthetic enzymes is controlled biochemically by lipids, water soluble phospholipid precursors and products, and by the covalent modification of phosphorylation (3). The metabolic pathways of membrane phospholipid biosynthesis in yeast are best illustrated by Figure 1. PA is a key intermediate in lipid biosynthesis. Two major de novo biosynthetic pathways that synthesize PA utilize either glycerol-3-phosphate (G-3-P) or dihydroxyacetone phosphate (DHAP) as precursors. G-3-P is acylated by G-3-P acyltranferases (encoded by GAT1, GAT2) at the sn1 position to produce lysopa which is, in turn, acylated by 1- acyl-g-3-p acyltranferase (encoded by SLC1 and SLC4) in the sn2 position giving rise to PA (4). Alternatively, DHAP is acylated at the sn1 position by DHAP acyltranferase. The product, 1-acyl-DHAP is acylated by 1-acyl-G-3-P acyltranferase, which is then reduced to lysopa by NADPH-dependent 1-acyl DHAP reductase (encoded by AYR1). PA may also be synthesized from

17 4 phospholipids via the action of phospholipase D, or by phosphorylation of DAG by DAG kinase (4). PA is the major precursor for the synthesis of membrane phospholipids via the de novo CDP-DAG pathway, which is the only source of phospholipids in the absence of exogenous lipid precursors ethanolamine and/ or choline (Figure 1) (3). PA is the substrate for CDP-DAG synthase enzyme. The gene encoding this enzyme (CDS1) is essential for the vegetative growth and spore germination (5). This finding is not surprising since the liponucleotide intermediate CDP-DAG is the starting material for the synthesis of PI as well as PC. PC is made through PS and PE. PS is produced by the exchange of CMP from CDP-DAG for L-serine. PI is the product when inositol replaces CMP in CDP-DAG, which is catalyzed by PI synthase (encoded by the PIS1). PS can be decarboxylated by PS decarboxylases (encoded by PSD1 and PSD2) to PE, which in turn, can be methylated to produce PC. The conversion of PE to PC involves three methylation steps. The first methylation step is catalyzed by PE methyltranferase encoded by CHO2 gene. The second and third methylation steps are carried out by phospholipid methyltransferase, which is the product of OPI3/PEM2 gene. Inositol exists in different stereoisomers among which the most abundant in nature is myo-inositol. Myo-inositol is synthesized by the conversion of D-glucose 6-phosphate to L-myo-inositol 3-phosphate by the enzyme L-myoinositol 3- phosphate synthase (encoded by INO1), which is subsequently dephosphorylated to myo-inositol by a specific Mg 2+ -dependent myo-inositol-1- monophosphatase (encoded by INM1).

18 5 In the presence of choline and/or ethanolamine, DAG produced either by dephosphorylation of PA or the deacylation of TAG may be directly channeled into phospholipid synthesis via the Kennedy pathway (Figure 1) (6, 7). The ethanolamine and choline required for the Kennedy pathway is obtained from the growth medium or from the phospholipase D-mediated turnover of PC or PE made through the CDP-DAG pathway (3). The CDP-choline and CDPethanolamine pathways enable cells to make PC and PE, respectively, when the CDP-DAG pathway is blocked. PE is synthesized in the CDP-ethanolamine pathway through a sequence of steps catalyzed by ethanolamine kinase (encoded by EKI1), phosphoethanolamine cytidylyltransferase (encoded by ECT1) and ethanolamine phosphotransferase (encoded by EPT1). Choline kinase (encoded by CKI1), phosphocholine cytidylyltransferase (encoded by PCT1) and choline phosphotranferase (encoded by CPT1) catalyze the formation of PC in the CDPcholine pathway (8). In mammalian cells, PC is primarily synthesized through the Kennedy pathway, because most tissues lack the PE N-methyltranferase activity required for the synthesis of PC from PE (liver specific enzyme) (9). Genes encoding enzymes involved in CDP-DAG (CDS1, CHO1, PSD1, CHO2, OPI3) and Kennedy (EKI1, EPT1, CKI1, CPT1) pathways as well as in the synthesis of PI (INO1) contain a UAS INO element in their promoters (3, 10). These genes are regulated in a coordinated fashion and a model has been postulated explaining the coordinated regulation of genes containing the UAS INO element (11). The UAS INO element is the binding site (E-box-containing UAS INO

19 6 motif) for the Ino2-Ino4 heterodimer complex that activates transcription of the above mentioned genes. The Opi1 repressor serves as the transmitter of the signal produced by PA. It senses the levels of PA, and represses the UAS INO -containing gene expression and its function is controlled by its nuclear localization (3). Opi1 is tethered to the nuclear/er membrane by an integral membrane protein encoded by SCS2 gene (12). When levels of PA are reduced, Opi1 is released from the nuclear/er membrane and moves into the nucleus where it attenuates the transcription by binding to the INO2 gene product, which is a positive regulator of phospholipid biosynthesis (3). Hence, PA content at the nuclear/er membrane is a very critical factor, that regulates the Opi1-mediated regulation of UAS INO - containing gene expression (3). Conditions that trigger the regulation of UAS INO - containing genes include nutrient availability (e.g. inositol and zinc) and growth stage (3). PI is an essential phospholipid and mutants incapable of synthesizing inositol will die unless they are supplemented with inositol. The transcription of genes containing the UAS INO element is increased in the absence of inositol and choline due to elevated levels of PA (causing the levels of PA to be reduced due to the derepression of INO1 gene, which results in inositol production incorporated towards PI synthesis) (13). Derepression requires the binding of the Ino2-Ino4 heterodimer complex to UAS INO promoter element. In the absence of inositol, Opi1 is localized to the nuclear/er membrane (12). Addition of inositol to the medium induces repression of the system because it causes pools of PA to decrease due to the utilization of CDP-DAG and increased PI synthesis (elevated

20 7 production of PI at the expense of PC as well as its precursors PE and PS) (3). The presence of both inositol and choline in the medium causes maximal repression, because inactivation of the pathway that converts PE to PC, leads to synthesis of PC from DAG and choline (13). DAG is made by the dephosphorylation of PA, which causes PA levels to be decreased. A key post-translational mechanism for the regulation of membrane phospholipid biosynthesis is phosphorylation; the covalent attachment of a phosphate group to a target protein. The phosphorylation status of a protein (phosphorylated or unphosphorylated) impacts on enzyme activity, cellular localization, protein turnover or interaction with other proteins (12). The two major kinases that regulate membrane phospholipid biosynthetic enzymes are protein kinase A and protein kinase C. The activity of several enzymes involved in membrane phospholipid biosynthesis is regulated by phosphorylation. These include URA7-encoded CTP synthase (14-16), CHO1-encoded PS synthase (17), and PAH1-encoded PA phosphatase (18-20). The Opi1 is upregulated by phosphorylation as well (21-23). 1.3 DGK1-Encoded Diacylglycerol Kinase Kinases are a family of enzymes that transfer phosphate group from a high energy molecule, usually ATP, to a substrate. DAG kinase is the enzyme that phosphorylates DAG to make PA. DAG kinases are found in various living

21 8 Figure 1. Phospholipid biosynthetic pathways in S. cerevisiae. The PA structure shown with fatty acyl groups of 16:0 (sn1) and 18:1 (sn2) is highlighted by gray shading. The genes that are known to encode enzymes catalyzing individual steps are indicated. The UAS INO -containing genes that are subject to regulation by the Ino2-Ino4 protein activation complex and the Opi1p repressor are highlighted by gray shading. Gro, glycerol; DHAP, dihydroxyacetone phosphate; Glu, glucose; Ins, inositol; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; PDE, phosphatidyldimethylethanolamine; PC, phosphatidylcholine; Etn, ethanolamine; Cho, choline (24).

22 9 Gro-3-P GAT1 GAT2 LysoPA SLC1 SLC4 O O DHAP GAT1 GAT2 AYR1 acyl-dhap Glu-6-P INO1 CDS1 CTP Ins-3-P CDP-DAG Ins INM1 PI PIS1 CHO1 PSD1 PSD2 CHO2 UAS INO -containing gene PS PE PME CTP DAG Etn EKI1 P-Etn ECT1 CTP EPT1 CDP-Etn Cho OPI3 CKI1 P-Cho PDE PCT1 CTP OPI3 CPT1 CDP-Cho PC PA O O P OH O H HO O PAH1 DGK1 TAG DGA1 LRO1 SPO14

23 10 organisms such as Drosophilia melanogaster, Caenorhabditis elegans, Aradiposis thaliana, mammals, and plants (25). In particular, in mammals ten isoforms have been identified to date, whose major function is to block DAG signaling. Mammalian DAG kinases phosphorylate only sn1, 2-DAG. In mammals, agonists 1 induce the cleavage of PI4,5P 2 by PI-specific phospholipase C, leading to the formation of DAG (as response to the formation of DAG, PKC is translocated to membranes) and inositol trisphosphate (IP 3 ). An IP 3 phosphatase converts I1,4,5P 3 to I1,4P 2 and so inactivates IP 3, while DAG is either phosphorylated to PA by DAG kinase or dephosphorylated to MAG by a DAG lipase. Both DAG and IP 3 serve as second messengers to activate two independent but parallel pathways. DAG functions within the plane of the membrane to increase the protein phosphorylation by activating PKC. DAG kinase has been detected in both soluble and membrane-bound subcellular compartments including cytosol, microsomes, synaptosomes, and nuclear envelopes (26). IP 3 functions as a second messenger by mobilizing calcium from intracellular stores. These two signaling pathways appears to act in a synergistic way to stimulate a broad variety of cellular processes. Recently, many studies have focused on how the production of PA by DAG kinases regulates signaling events, indicating that PA binds to and activates proteins such as PI4P5 kinase (27). Moreover, PA is a crucial component for the re-synthesis of PI; part of the PI cycle (28, 29). DAG kinases have been also identified in unicellular organisms such as bacteria. However, bacterial DAG 1 An agonist is a chemical that binds to receptor of a cell and triggers a respond by that cell.

24 11 kinase, as opposed to its counterparts found in higher eukaryotes, is a small integral protein that phosphorylates not only DAG but also other lipids, such as ceramide (30). Because DAG signaling had been found in yeast, it was expected that DAG kinase activity was present in the organism (25). Furthermore, a previous study had showed that yeast membranes contain an activity that catalyzes the transfer of the γ-phosphate of CTP into both dolichol phosphate and PA (31). The fact that SEC59 was identified as the structural gene encoding for dolichol kinase enzyme verified that different enzymes account for the CTP-dependent formation of dolichol phosphate and PA in yeast membranes (32, 33). No homology of the yeast genome sequence was identified by making BLAST 2 searches with various parts of mammalian DAG kinase sequences (34, 35). As a matter of fact, it was assumed that the enzyme was present in yeast but had no homology to other DAG kinases (36). Therefore, the function of the DGK1 gene product was not known until recently. The function of the gene product was first identified in a genetic screen performed in our laboratory (36). This screen was done in order to identify high copy suppressors that could rescue the lethality caused by the overexpression of PAH1-7P or the NEM1-SPO7 phosphatase (high PA levels). Both screens identified the very same gene DGK1 (previously known 2 BLAST, stands for Basic Local Alignment Search tool, is an algorithm widely used for sequence similarity search

25 12 as HSD1). DGK1/HSD1 was first isolated in a genetic screen for multicopy suppressors of sly1δ mutant, which is involved in the trafficking between Golgi apparatus and ER (37). The DGK1 gene product is a 32.8 kda protein. It is comprised of a short N-terminal hydrophilic region (residues 1-73), followed by four transmembrane spanning domains containing a predicted cytidylyltransferase domain (residues ) (Figure 3), that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-DAG synthase (5). Unlike other DAG kinases previously described, DGK1-encoded DAG kinase utilizes CTP as the phosphate donor instead of ATP (36). That DGK1-encoded DAG kinase utilizes CTP instead of ATP accounts for the fact that yeast DAG kinase does not display any sequence similarity to DAG kinases found in other species. In addition, DAG kinases from other species, such as mammals, have a more complex domain structure, containing additional functional domains. DGK1-encoded DAG kinase utilizes only DAG as its substrate and does not catalyze other CTP-dependent reactions (24). DGK1-encoded DAG kinase is localized to ER (36, 37). DAG kinase activity is found at sites where its substrate DAG is present. Lipids are usually associated with membranes and so does DAG. DAG binds strongly to certain proteins through hydrophobic interactions, thereby causing alternations in the physical properties of the bilayer. In mammals, DAG is usually produced in the plasma membrane but can also be formed in the membrane of internal organelles, especially in response to a stimuli. In yeast, DAG is generated either at lipid droplets via the hydrolysis of TAG, or at the ER through the

26 13 Figure 3. Domain structure of DGK1-encoded DAG kinase. The diagram shows the positions of the CTP transferase domain, the transmembrane-spanning domains, and the conserved residues of DGK1-encoded DAG kinase that are found in the cytidylyltranferase domain of CDS1-encoded CDP-DAG synthetase and SEC59- encoded dolichol kinase.

27 14 Inhibitory Domain CTP Transferase Catalytic Domain N- -C RK Conserved Residues AA Transmembrane Domains D G A A

28 15 dephosphorylation of PA by PAH1-encoded PA phosphatase. DGK1-encoded DAG kinase is ER-associated enzyme, hence, there should exist a mechanism for the translocation of DAG, derived from the hydrolysis of TAG, for DAG kinase activity. Cells lacking DGK1 exhibit no significant phenotypes; no apparent growth defect or gross nuclear/er membrane abnormality (36). DAG kinase counterbalances the course of action of PA phosphatase, both in lipid metabolism and cell physiology. The overexpression of DGK1 gene brings about an elevation in PA levels and the abnormal nuclear/er membrane expansion (24) like the phenotypes displayed by pah1δ mutants. This phenotype is due to the DGK1- encoded DAG kinase activity since overexpression of a mutant allele that encodes a catalytically inactive DAG kinase does not exhibit this phenotype (24). Furthermore, the overexpression of DGK1 gene causes temperature-sensitivity (24) at 37 C like the phenotype exhibited by pah1δ mutants (36). The temperaturesensitivity phenotype of the cells overexpressing DKG1 is attributed to DGK1- encoded DAG kinase activity and not to some other function of the DGK1 gene product (24). In addition, the overexpression of DAG kinase activity complements the inositol auxotrophy caused by overexpression of PA phosphatase activity (36). Moreover, the loss of DAG kinase activity complements the phenotypes (nuclear expansion) caused by loss of PA phosphatase activity in pah1δ dgk1δ double mutants, by decreasing PA levels (36). However, pah1δ dgk1δ double mutants are

29 16 still temperature sensitive like the single pah1δ mutant (36). The levels of PE and PC are also returned back to normal in pah1δ dkg1δ double mutant (36). Nevertheless, the abnormal levels of TAG and PI did not change in pah1δ dkg1δ compared with pah1δ single mutant suggesting that TAG and PI may be involved in toxicity rather than in nuclear membrane structure (36). Lack of biosynthetic PA enzymes restores nuclear structure of pah1δ, which means that the decrease in PA levels is causing the restoration of normal nucleus in a pah1 dkg1 mutant (36). 1.4 TAG Metabolism in Yeast The upsurge in lipid-associated diseases such as type 2 diabetes and obesity has forced the scientific community to focus on gaining a deeper insight in TAG metabolism and its associated organelle, lipid droplets. The importance of using yeast as a model to study TAG metabolism is underscored by the fact that all mammalian enzymes identified to be involved in TAG formation, storage and metabolism have a yeast counterpart (4). Sterols (STs) and fatty acids (FAs) are the major energy substrates in eukaryotes and are both synthesized via acetyl-coa. TAG and steryl ester (STE) are the major energy storage lipids in eukaryotic cells. Storage of STs and FAs as STE and TAG, respectively, ensures effective conservation of the chemical energy in a biologically inactive form. TAG accumulation prevents against

30 17 lipotoxicity caused by elevated cellular levels of free FAs. In the absence of other carbon sources, hydrolysis of TAG provides energy substrates (FAs) to meet cellular energy needs (38). Moreover, breakdown of TAG also provides free FAs for the initiation of membrane phospholipid synthesis allowing cells to exit stationary phase and enter vegetative growth (38). Lastly, TAG also provides metabolites for sporulation (39), and cell cycle progression; lipolysis requirement coincide with bud emergence (40). Hence, the role of TAG metabolism in energy homeostasis is of utmost importance. Storage of neutral lipids is closely correlated with the formation of lipid droplets, where TAG depots are exclusively located in yeast. Lipid droplets consist of a hydrophobic core of neutral lipids (TAG and STE are not able to integrate into phospholipid bilayers and they cluster) and a membrane monolayer of phospholipids containing some proteins, which are involved in lipid metabolism (4). This structure is very similar to that of lipid droplets in mammals. The role of lipid droplets in lipid homeostasis and its interaction with other cellular compartments associated with neutral lipid dynamics, such as ER and plasma membrane, are under investigation (4). TAG are synthesized primarily from DAG via two different pathways. The first is through an acetyl-coa-dependent reaction that is catalyzed by DAG acyltranferase (DGAT2 gene family) which utilizes acyl-coa as the acyl donor. The corresponding gene that encodes DAG acyltranferase activity is DGA1. DAG

31 18 acyltransferase enzyme is localized to lipid droplets. However, in dga1δ mutant cells DAG acyltransferase activity is dramatically reduced in lipid droplets while the activity is only slightly affected in the microsomal fraction. This indicates the existence of DAG acyltransferase isoezyme in the microsomal fraction, which is formed from the ER (41). The second pathway is catalyzed by DAG acyltranferase (PDAT gene family), which mediates esterification of DAG using a phospholipid as the acyl donor (LRO1). LRO1-encoded DAG acyltranferase is an ER membrane-associated enzyme (42). The involvement of more than one biosynthetic system in neutral lipid synthesis seems to be the consequence of differential regulation (depending on growth phase regulation, availability of heme 3, substrate specificity, alternative localization). Surprisingly, dga1δ lro1δ double mutant exhibits approximately 5% of the DAG esterification activity. This is due to ST acyltranferase enzymes encoded by ARE1 and ARE2 genes, since synthesis of TAG is totally abolished in dga1δ lro1δ are1δ are2δ quadruple mutants (41, 43-45). TAG are synthesized during the exponential phase of growth and it accumulates during the stationary (quiescence) phase in order for the cell to be able to sustain extended periods of starvation (46-51). The ER contains the complete set of enzymes that are involved in TAG synthesis and is the main site 3 Heme is prosthetic group that consist of an iron atom contained in center of a large heterocyclic ring called porphyrin.

32 19 of TAG formation (4). Nevertheless, lipid droplets may synthesize TAG on their own. According to the most broadly accepted model for the membrane biogenesis, TAG and ST are concentrated in the hydrophobic region of the ER-membrane phospholipid bilayer forming microdroplets between the two leaflets of the ERmembrane bilayer (4). Finally, a lipid droplet bounded by phospholipid monolayer containing specific proteins is removed after reaching a certain size and released into the cytosol; this hypothetical model is reminiscent of bud formation (4). Proteins on the membrane, provided that they do not contain any transmembrane domain, may be incorporated in the nascent lipid particles (4). In yeast, mobilization of neutral lipid depots occurs in response to at least three stimuli. In stationary phase, when nutrient depletion occurs, FAs are produced as a result of the hydrolysis of TAG depots and subject to peroxisomal β-oxidation, providing the required energy for cellular maintenance (52). Furthermore, when cells exit stationary phase and enter vegetative growth rapid TAG degradation occurs, because de novo FA synthesis is inadequate to fulfill the cellular requirement for FAs. Due to the repression of peroxisomes under these condition, released FA are directed toward making membrane phospholipids meant to be used for membrane synthesis, which is essential for the initiation of cellular growth and division (53, 54). The mobilization of TAG due to transfer to fresh medium occurs during an initial lag phase; when cells enter vegetative growth, initiation of the de novo synthesis of FA causes lipogenesis to outweigh lipolysis and TAG pools are replenished. Lastly, lipolysis is necessary in the absence of

33 20 cellular growth in diploid cells undergoing meiosis and sporulation (54, 55). Three TAG lipases have been identified so far, encoded by TGL3, TGL4, and TGL5 genes (43, 56). They are all localized to lipid droplets. They also possess a consensus motif GXSXG, which is characteristic of hydrolytic enzymes. However, they lack additional motifs characteristic for lipases from other sources (43, 56). TGL4-encoded triaglyceride lipase is a functional ortholog 4 of adipose TAG lipase (ATGL) (40). Only TGL3-encoded TAG lipase substantially contributes to TAG lipolysis in growing cells (43, 46, 54, 56). The most direct route for membrane phospholipid synthesis upon breakdown of TAG is via the conversion of DAG to PA, which is catalyzed by the DAG kinase. Free FAs released upon hydrolysis of TAG can also be incorporated to synthesize membrane phospholipids. However, this pathway involves three sequential reactions Figure 1.2. FAs produced de novo may be incorporated into membrane phospholipid synthesis, too. It has been shown that dgk1δ mutant cells are unable to resume growth in the presence of cerulenin, which inhibits the de novo FA synthesis and creates condition of cellular FA depletion (57). The loss of growth phenotype was due to the DAG kinase activity of the DGK1 gene product (57). In order to elucidate the molecular basis behind 4 Orthologs are genes in different species that evolved from the same common ancestral gene by speciation; they normally retain the same function.

34 21 the requirement for the DGK1 gene, we hypothesized that DAG kinase activity and protein might be upregulated when cells resume growth from stationary phase.

35 22 Figure 2. Pathways for the mobilization of TAG upon resumption of growth from stationary phase. The pathways shown for the mobilization of TAG for the synthesis of phospholipids include the relevant steps discussed in this work. Although not shown in the figure, the fatty acid derived from TAG is converted to fatty acyl-coa before it is incorporated into PA. The reaction catalyzed by DAG kinase is highlighted in the figure.

36 23 Choline P ChAoline Kennedy Pathway PC CDP-DAG Pathway PI, PS, PE, PC CDP Choline Lipase DAG Kinase TAG DAG PA CTP CDP CDP DAG Acetyl CoA FA lysopa cerulenin Gro 3 P

37 24 CHAPTER 2 EXPERIMENTAL PROCEDURES 2.1 Materials All chemicals were reagent grade. Growth medium components were purchased from Difco. Cerulenin, nucleoside 5 -diphosphate kinase and protease inhibitors (phenylmethylsulfonyl fluoride, benzamidine, aprotinin, leupeptin, and pepstatin) were from Sigma. Radiochemicals were purchased from PerkinElmer Life Sciences. Dioleoyl DAG was from Avanti Polar Lipids. Protein assay reagents, electrophoretic reagents and protein size standards were purchased from BioRad. Polyethyleneimine-cellulose (PEI) plates were from EM Science. Polyvinylidene difluoride membranes and the enhanced chemifluorescence Western blot reagent were from GE Healthcare. Scintillation counting supplies were from National Diagnostics. 2.2 Strains and Growth Conditions The strains used in this work are shown in Table 1. Yeast cultures were grown at 30 C in synthetic complete (SC) medium as described previously (58, 59). For resumption of growth from stationary phase, cultures (three isolates per

38 25 strain) were grown for 48 h in SC medium to reach stationary phase, and diluted with fresh SC medium (with or without cerulenin) to an absorbance of 1. Then cells were harvested by centrifugation at various time intervals for the analysis of DAG kinase activity and protein. Dilution of cells to higher cell density enhanced enzyme analysis and allowed cells to enter exponential phase of growth within a short time span. The FA synthesis inhibitor cerulenin was added to the cultures from stock dilutions (10 mg/ml) in ethanol. Control experiments demonstrated that the relatively small amount of ethanol was not toxic since it did not affect growth. 2.3 Plasmid Construction -Yeast Transformation The 2.4-kb DNA fragment containing the entire DGK1 coding sequence (0.873 kb), the 5 -untranslated region (1kb), and the 3 -untranslated region (0.5 kb) was released from plasmid psf211 (57) after digestion with Xbal/HindIII and inserted into plasmid YEp351 at the same restriction sites to construct plasmid psf213. The multicopy E. coli/yeast shuttle vector YEp351 containing the LEU2 gene has been described previously (60). The DGK1-encoded DAG kinase was expressed under the control of the endogenous DGK1 promoter. The transformation of S. cerevisiae was performed by LiAc/SS carrier DNA/PEG method (61). To select yeast cells bearing plasmid, suitable amino acids were left out from the medium. Plasmid construction was conducted by Stylianos Fakas.

39 26 TABLE I Strains used in this study Strain or plasmid Relevant characteristics Source or Ref. S. cerevisiae RS453 ΜΑΤa ade2-1 his3-11, 15 leu2-3,112 trp1-1 ura3-52 (62) SS1144 RS453 dgk1::his3 (36) Plasmid YEp-GAL1/10-DGK1 DGK1 controlled by GAL1/10 promoter in 2μ/LEU2 vector (36) psf211 DGK1 inserted into prs416 (57) YEp351 Multicopy E. Coli/yeast shuttle vector containing LEU2 (60) 2.4 Preparation of Cell Extracts All steps were carried out at 4 C in order to avoid any enzymatic activity loss due to temperature elevation. At the indicated time intervals, cells where harvested by centrifugation at 1,500 x g for 5 min. The cell pellet was then washed once with distilled water and resuspended in cell lysis buffer. To break open the cells, glass bead vortexing was applied. The abrasive action of the vortexed beads disrupts the cell wall, releasing the cytoplasmic contents. Cell lysis

40 27 buffer contained 50 mm Tris-HCl (ph 7.5), 300 mm sucrose, 10 mm β- mercaptoethanol, 1 mm Na 2 -EDTA as well as a cocktail of protease inhibitors (0.5 mm phenylmethanelsulfonyl fluoride, 1 mm benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin and 5 μg/ml pepstantin). Bead beating tubes were vortexed for 10 cycles of 1-minute bursts followed by 2-minute rest stages (to dissipate the heat generated during shaking, which could degrade the enzyme) using a Mini- BeadBeater-16 (BioSpecProducts, Inc.). The samples were centrifuged at 1,500 x g for 5 min. The supernant was transferred to a microcentrifuge tube, leaving the glass beads and broken cells behind. Membrane fractions for Western blot analysis were prepared by ultracentrifugation of cell extracts at 47, 000 rpm for 70 min using a Beckman coulter optima TLX ultracentrifuge. Membrane fraction was used in preference to homogenate because the DGK1-encoded DAG kinase is an ER membrane-associated protein, and this enhanced epitope abundance for antibody detection. 2.5 Protein Concentration Protein concentration was measured by the Bradford method using bovine serum albumin as the standard (63). 2.6 Preparation of the [γ 32 -P]CTP The radiolabeled [γ- 32 P]CTP was not available commercially and, thus, it

41 28 was produced enzymatically from CDP and [γ- 32 P]ATP with nucleoside 5 - diphosphate kinase from baker s yeast. The reaction mixture contained 10 mm MOPS-NaOH ( ph 7.6), 0.01 mm CDP, 5 mm MgCl 2, 30 μci of [γ- 32 P]ATP, and 1 unit of nucleoside 5 - diphosphate kinase in a total volume of 30 μl. The reaction was terminated by adding 50 mm EDTA. After the termination of the reaction, 1 ml of 1 mm CTP was added to the mixture in order to be able to adjust the specific activity of the label. The conversion of [γ- 32 P]ATP to [γ- 32 P]CTP was verified by thin layer chromatography using a PEI-cellulose plate and 0.75 M KH 2 PO 4 -H 3 PO 4 as the solvent system. 2.7 DAG Kinase Assay The conditions under which the assay was performed were the optimum as described previously (24). The DAG kinase reaction was performed using [γ- P 32 ]CTP as the phosphate donor and dioleoyl-dag as the lipid acceptor. DAG kinase activity was measured by following the incorporation of the γ-phosphate of water soluble [γ- 32 P]CTP (30,000 cpm/nmol) into the chloroform-soluble PA. The reaction mixture contained 50 mm Tris-HCl (ph 7.5), 1 mm CTP, 1 mm Triton X-100, 0.1 mm dioleoyl DAG, 1 mm CaCl 2, 10 mm β-mercaptoethanol, and cell extract in a total volume of 0.1 ml. The reaction was terminated by adding 0.4 ml 0.1 N HCl. The radioactive chloroform-soluble PA was separated from the radioactive water-soluble CTP by a chloroform/methanol/water phase partition. The radioactive chloroform phase was then subject to liquid scintillation counting.

42 29 Since a crude source of enzyme was utilized, it was critical to determine the time course of the reaction to ensure that neither CTP nor PA were utilized by other enzymes. The length of the incubation time and the amount of protein can vary only as long as initial rates are constant; production of PA is proportional to both time and enzyme concentration. When time-dependent and dose-dependent assay were run, all the other conditions remained as described. Specific activity was obtained by normalizing the activity values to the total protein in cell lysates. The DAG kinase assay was performed in triplicates and it was linear with time and protein concentration. The average standard deviation of the DAG kinase activity assay was ±10 %. One unit of activity was defined as the amount of enzyme that catalyzed the formation of 1 pmol of product per minute, and the specific activity was defined as units/mg of total protein. 2.8 Immunoblot Analysis SDS-PAGE (64) and immunobloting (65) was conducted as described previously using anti-dag kinase antibody (36). Polyvinylidene difluoride membrane was used for the protein blotting. The membrane was probed with anti- DAG kinase antibodies at a diluton of 1 μg/ml, followed by alkaline phosphatase conjugated anti-rabbit IgG at a dilution of 1:5000. The immune complexes were detected using enhanced chemifluorescence reagents and the signal was processed with a Fluorimager. The immunoblot signals were in the linear range of detection.

43 30 CHAPTER 3 RESULTS 3.1 DAG Kinase Activity is Linear with Time and Protein Concentration Wild type cells were grown to stationary phase and then resuspended to fresh medium. Four hours following resuspension, cells were harvested and a cell extract was prepared. Under the conditions employed in this work, DAG kinase activity was proportional with time (Figure 4) and the amount of protein (Figure 5). 3.2 Regulation of DAG Kinase Activity upon Resumption of Growth from Stationary Phase in Wild Type Cells We examined the regulation of DAG kinase activity when wild type cells resume growth from stationary phase in the presence and absence of the de novo fatty acid synthesis inhibitor cerulenin. The reason for inclusion of cerulenin in the medium was to accentuate the requirement of TAG hydrolysis during growth resumption. DAG kinase activity was increased 3-fold in the absence of cerulenin (Figure 6). Addition of cerulenin to medium caused a marginally higher 3.7-fold induction in DAG kinase activity (Figure 6).

44 31 Figure 4. Time-dependence of DAG kinase activity. Wild type cells were grown to stationary phase in SC medium. The stationary phase cells were resuspended in fresh medium and incubated for four hours. Following the incubation, a cell extract was prepared and used for the measurement of DAG kinase activity.

45 32 DAG Kinase Activity, pmol/mg Time, min

46 33 Figure 5. Protein-dependence of DAG kinase activity. Wild type cells were grown to stationary phase in SC medium. The stationary phase cells were then resuspended in fresh growth medium and incubated for four hours. Following the incubation, a cell extract was prepared and used for the measurement of DAG kinase activity. This assay was conducted for the indicated protein concentrations.

47 34 DAG Kinase Activity, pmol/min Protein, μg

48 35 Figure 6. DAG kinase activity is induced during growth resumption from stationary phase. Wild type cells were grown to stationary phase in SC medium and then resuspended in fresh medium with and without 10 μg/ml cerulenin. At the indicated time points, cells were harvested, cell extracts were prepared, and DAG kinase activity was measured. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments.

49 36 DAG Kinase, Units/mg Cerulenin + Cerulenin Time, h

50 37 Figure 7. Induction of DAG kinase activity occurs during an initial lag phase following resumption of growth from stationary phase. Wild type cells were transformed with plasmid YEp351-DGK1. Cells were grown to stationary phase in SC medium and then diluted in fresh medium with and without 10 μg/ml of cerulenin. Growth following transfer to fresh medium was monitored with a spectrophotometer at an absorbance of 600 nm.

51 38 3 A 600 nm Cerulenin + Cerulenin Time, h

52 Regulation of DAG Kinase Activity upon Resumption of Growth in a Strain Overexpressing DGK1 Gene Wild type cells were transformed with a high copy plasmid YEp351 DGK1 in order to overexpress DGK1 gene. The regulation of the DAG kinase activity when cells resume growth from stationary phase in the presence and absence of cerulenin was examined. DAG kinase activity was induced both in the presence and absence of cerulenin, 3.5-fold and 3.8-fold (Figure 8), respectively, similar to that obtained with wild type cells without the plasmid. Resumption of growth from stationary phase was monitored spectrophotometrically to identify the phase during which DAG kinase activity is induced (Figure 9). 3.4 Regulation of DAG Kinase Protein Expression upon Resumption of Growth from Stationary Phase To address the question of whether the induction of DAG kinase activity was due to a mechanism that involved an increase in the enzyme expression, the membrane fraction was used for Western blot analysis. The position of the DAG kinase protein on the blot was confirmed by its absence on the blot for the dgk1. The regulation of DAG kinase protein expression when wild type cells transformed with plasmid YEp351 resume growth from stationary phase was examined. The DAG kinase protein expression was induced 3-fold upon resumption of growth from stationary phase.

53 40 Figure 8. DAG kinase activity increases upon resumption of growth from stationary phase in a strain overexpressing DGK1 gene. Wild type cells transformed with plasmid YEp351-DGK1 were grown to stationary phase in SC medium and then resuspended in fresh medium with and without cerulenin. At the indicated time points, cells were harvested, cell extracts were prepared, and DAG kinase activity was measured.

54 41 DAG Kinase, Units/mg Time, h - Cerulenin + Cerulenin

55 42 Figure 9. Induction of DAG kinase activity occurs during an initial lag phase following resumption of growth from stationary phase. Wild-type cells were transformed with plasmid YEp351-DGK1. Cells were grown to stationary phase in SC medium and then diluted in fresh medium with and without 10 μg/ml of cerulenin. Growth following transfer to fresh medium was monitored with a spectrophotometer at an absorbance of 600 nm.

56 43 4 A 600 nm Cerulenin + Cerulenin Time, h

57 44 Figure 10. DAG kinase protein expression was induced upon resumption of growth from stationary phase. A, wild type cells transformed with plasmid YEp351-DGK1 were grown to stationary phase and then resuspended to fresh medium. Following transfer, cells were harvested at the indicated time points. Cell extracts were prepared and ultracentrifuged to isolate the membrane fraction. Protein samples were resolved by SDS-PAGE followed by Western blotting using anti-dag kinase antibody. The identity of the DAG kinase protein on the blot was confirmed by the fact that it was absent in dgk1 mutant. The position of the DAG kinase protein is indicated by dotted line in the dgk1 sample. The solid line indicates the position of the protein in cells overexpressing the protein. Ponceau S staining of the blot indicated equal loading of lanes. B, the relative amount of DAG kinase in the membrane was measured by ImageQuant analysis of the immunoblot shown in panel A. The amount of DAG kinase at time zero was arbitrary set to 1.

58 45 A Time, h Dgk1p B Dgk1p, Arbitrary Unit Time, h

59 46 CHAPTER 4 SUMMARY 4.1 Discussion The recent dramatic outbreak of obesity and type 2 diabetes in western countries, has necessitated research on TAG metabolism because these diseases are associated with the excessive accumulation of TAG. Having knowledge of TAG metabolism is the prerequisite for the identification of potential therapeutic targets and drug design. That yeast TAG metabolism is very similar to that of higher eukaryotes renders yeast an ideal organism to study TAG metabolism. The gene encoding for DAG kinase in yeast remained unknown until recently. The DGK1 gene was identified in a genetic screen as a regulator of PA levels (36). The DGK1-encoded DAG kinase is a novel DAG kinase because it utilizes CTP as the phosphate donor, instead of ATP. When cells resume growth from the quiescence phase, hydrolysis of TAG occurs. Breakdown of TAG provides free FA for the initiation of membrane phospholipid synthesis. It has been demonstrated that yeast cells defective in TAG lipase activity were incapable of resuming growth from stationary and cell cycle arrested phase in the presence of the fatty acid synthesis inhibitor cerulenin (40). DAG kinase is the enzyme that catalyzes the conversion of DAG, formed via the hydrolysis of TAG

60 47 by TAG lipases, to PA. Considering the importance of PA, which is the major membrane phospholipid precursor, one could speculate that DAG kinase might be essential for the resumption of growth from stationary phase as well. The singlestep conversion of DAG to PA by DAG kinase is the most direct route to synthesize PA upon TAG hydrolysis. Incorporation of free FA to PA requires three sequential reactions. Indeed, a recent study (57) has showed that DGK1 gene is required for the growth resumption from stationary phase in the presence of cerulenin; dgk1 mutant cells were unable to exit quiescence and enter vegetative growth. This loss-of-growth phenotype was due to the abolishment of DAG kinase activity and not due to the loss of some other function of the DGK1 gene product. Given, the elevated substrate availability due to the hydrolysis of TAG and the requirement for DAG kinase activity, it was hypothesized that DAG kinase activity might be upregulated upon growth resumption from stationary phase. In this study, the regulation of DAG kinase activity when cells resume growth from stationary phase was examined. DAG kinase activity was induced upon resumption of growth from stationary phase, both in the presence and absence of cerulenin. The increase of DAG kinase activity was 3-fold in the absence of cerulenin and inclusion of cerulenin into the medium caused a marginally higher 3.7- fold increase. Based on the previous findings (57), it was expected that the inclusion of cerulenin in the medium would be the primary reason provoking DAG kinase activity induction, since dgk1 cells were able to