Discovery of lipid enzymes and their modulators using metabolite profiling of yeast (saccharomyces cerevisiae) mutants

2.5.1 Mass spectrometry of lipid extracts 20 2.5.3 Precursor ion scan mass spectrometry 21 Mass Spectrometry 2.5.5 Computational analysis of mass spectral data 22 2.6 Sub cloning YBR042

DISCOVERY OF LIPID HYDROLYSING ENZYMES AND THEIR MODULATORS USING METABOLITE PROFILING OF YEAST

(SACCHAROMYCES CEREVISIAE) MUTANTS

PRADEEP GOPALAKRISHNAN

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2007

ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr Markus Wenk for his guidance and support I would like to thank everybody at the lab for their advice and friendship It’s the best lab I have been in and everybody has been great They are past and present Guang Hou, Anne, Aaron, Asif, Con, Leroy, Gek Huey, Joyce, Wei Fun, Robin Chan, Xueli, Tommy, Hong Sang, Yoke Yin, Sravan, Wei Kiang, Kai Leng, Mee Kian, Ignascius, Huimin

I have received considerable help and advice from Guang Hou, to whom I am particularly grateful Aaron, who has never refused my requests of help Leroy and Con for helping me with the corrections Gek Huey for her expertise with the computational work Robin for being a good friend and helping me ever so often Xue Li for her expert assistance and comments Sravan for his advice and suggestions

I am also indebted to Dr Robert Yang and all his lab members for his kind help with his lab space, resources and expertise Dr.Yang’s lab has been my second home and

I am very glad to have had you all as colleagues Jaspal, Choon Pei, Li Phing, Kelly, Siva I would also like to thank all the level 3 people especially Wei Hua who has helped

me with many experiments Without the friendly help of all the people at level 3 my experiments would have been very difficult to carry out I would like to thank my friend Lee for his support and encouragement

The friendly office staff at DBS and Biochemistry and so many more people who have all gone out of their way to help me Thank you all

Last but not the least; I thank my family and friends for their support

TABLE OF CONTENTS

2.5.1 Mass spectrometry of lipid extracts 20

2.5.3 Precursor ion scan mass spectrometry 21

Mass Spectrometry 2.5.5 Computational analysis of mass spectral data 22

2.6 Sub cloning YBR042C and expression in

products and vector

2.6.6 Preparation of competent yeast cells 26

2.7 Generation of double knock out mutant of

3.1 Validation of glycerophospholipid profiling-SLC1

3.2 Tandem mass spectrometric identification of

3.3 Lipid profiling of single deletion mutant strains 33

3.3.1 Lipid profiling of strains in a random manner 33 3.3.2 Lipid profiling of strains in a domain based manner 37 3.4 Identification of a putative novel acyltransferase 42

3.5 Rescue of a strain deleted of YBR042C 44

3.5.2 Restriction analysis of selected plasmid 44 3.5.3 Fluorescent imaging of transformed YBR042C 46

mutant cells 3.5.4 Mass spectrometric profile of transformed 47

YBR042C cells

3.6 Generation of double knockout mutants of

3.8 Lipid droplet staining and triacylglyceride level

3.9 Phenotypic changes observed in mutant strains 53

4.2 Lipid profiles of single gene deletion mutant 54

of a single gene deletion mutant and comparing it to the mass spectrum derived from the wild type This reverse genetics approach is particularly suited for

Saccharomyces cerevisiae as it has both its genome completely sequenced and the availability of a single gene deletion library The screening of mutants in one such single gene deletion library was done in both a random and a targeted fashion The presence of phospholipid related domains was used as the basis of targeted selection of ORFs

One hundred and twenty yeast strains were profiled Based on this screening regime we have identified a putative acyltransferase YBR042C, which was found to be involved in maintaining the levels of Phosphoinositols Further characterization of this ORF was carried out to better understand its molecular function The localization of YBR042C to the lipid droplets was independently

confirmed The substrate specificity of YBR042C was investigated via deletion of

YBR042C along with SLC1, which encodes another lipid droplet localizing

acyltransferase Preliminary data suggests that YBR042C transfers 18 carbon fatty acids to lysophosphatidic acid Quantitative measurements of glycerophospholipid and Triacylglyceride levels were carried out The decrease in Phosphoinositols was quantified Deletion of these two genes resulted in a several fold decrease of the major phosphoinositol species

This study shows that mass spectrometry based lipid profiling is a useful tool for studying gene function Strategies to scale up the profiling process are discussed

LIST OF TABLES

Table

Number

2 Yeast strains used in the random screen 11

3 Yeast strains used in the domain-based

screen

13

15 Primers used for amplification of His

20 Lipid profiling results for ORFs selected in

a domain based manner

38

LIST OF FIGURES

S.No Figure

ID

2 1.2 Phosphatidic acid and classes of phospholipids 4

6 2.3 PCR based gene targeting to knockout slc1 in a

YBR042C knockout

28

7 3.1 Phospholipid profile of a slc1 deletion mutant 31

8 3.2 Tandem mass spectrometric (MS-MS) identification

of lipid species with m/z value 835

32

9 3.3 Representative example of a comparative

phospholipid profiling strategy for single gene deletion mutants

34

10 3.4 The domain information of YBR042C shows that it

possesses an Acyltransferase domain

13 3.7 Gel photograph showing the restriction digestion of

plasmid isolated from positive E.coli colonies

45

15 3.9 Mass spectrometric profile of a YBR042C mutant 47

16 3.10 Quantitative measurement of lipid levels in the

rescued mutant

48

17 3.11 Gel Photograph showing PCR products to confirm 49

the replacement of SLC1 gene with his fragment

18 3.12 Phospholipid profile of a mutant deleted of both

21 3.15 Measurement of OD of Wildtype, YBR042C and a

double deletion mutant of YBR042C and SLC1

53

22 4.1 Possible mechanism by which alterations in PA

levels influence phosphoinositol levels

57

LIST OF ABBREVIATIONS AND SYMBOLS

DMPA

E coli Escherichia coli

EUROFAN European Functional Analysis Network

EUROSCARF European Saccharomyces cerevisiae Archive for Functional

Analysis

GPnEtn Glycerophosphono ethanolamines

HPLC High Performance Liquid Chromatography

MALDI Matrix Assisted Laser Desorption and Ionization

PCR Polymerase Chain Reaction

ss-DNA Single stranded Deoxyribo Nucleic acid

1 I N T R O D U C T I O N

Genome sequence information has revealed the presence of several ORFs (Open Reading Frames) whose function remains elusive To annotate the functions of such ORFs and to characterize their cellular roles remains a major task in this post

genome sequencing era (Kanehisa et al., 2003, Oliver 1997)

Several genes with a role in lipid metabolism or regulation have been functionally characterized, but our understanding of lipid metabolism especially regulation remains incomplete The relative lack of information on lipids is possibly due to an incomplete understanding of these molecules and has led to an under appreciation of the cellular roles of lipids (Chong, 2001) Absence of powerful analytical techniques has further hampered the study of lipids The techniques used to study these molecules in the past include Thin layer chromatography (TLC), Gas chromatography (GC) and biochemical assays These techniques often require large amounts of sample and are unable to resolve individual lipid species The adaptation

of mass spectrometry to study lipids and an increasing interest in these molecules has

dramatically changed the field of lipid science (Kerwin et al., 1994, Kim et al., 1994)

Mass spectrometry allows for the profiling of lipids, and helps in obtaining a relative ratio of the various species present in a sample The lipid profile of a sample, such as that of a gene-deletion mutant can be compared to the control condition (the wild type) to yield insights into the effects of gene disruption, and provides information on the metabolic pathways involved (Welti and Wang, 2004) This reverse genetics approach can help identify genes involved in lipid metabolism and regulation The major requirements for this approach include a completely sequenced genome and

genetic libraries, which are present for the baker’s yeast Saccharomyces cerevisiae

(Wallis and Browse, 2002; Guan and Wenk, 2006) These factors along with a

similarity in lipid pathways make the baker’s yeast Saccharomyces cerevisiae an ideal

organism for identifying genes involved in lipid metabolism

1 1 L i p i d s

Lipids as a class of biomolecules represent several subgroups which are chemically diverse with one common feature being their insolubility in water, though lipids such as phosphoinositols exhibit solubility in water The various subclasses of lipids include fatty acids, glycerophospholipids, Sphingolipids and sterols (Figure 1.1) The number of carbon atoms in fatty acids, the presence and position of double bonds and head groups result in structural diversity amongst lipids Specific classes of lipids such as the Glycerophospholipids have a common backbone but the presence of different functional groups results in new subclasses of lipids (Fig 1.2) The functional implications of this structural diversity are only beginning to be understood The discovery that lipids can give rise to signaling molecules such as diacylglycerol (DG, inositol 1, 4, 5-trisphosphate is one such example (Berridge, 2003) lipids are also involved in numerous lipid-lipid and lipid-protein interactions The discovery of an increasing number of domains on proteins that can bind to lipids underscores the importance of protein lipid interactions (Lemmon, 2003)

Figure 1.1 lipid classes and representatives

Figure 1.2 Phosphatidic acid and classes of Phospholipids

it is well established and relatively easy to carry out The major disadvantages are that

it requires a lot of sample and it cannot separate out lipids belonging to the same class High Performance Liquid Chromatography (HPLC) is another chromatographic technique that is well established to study lipids This technique is amenable to automation and is more sensitive than TLC A third major chromatographic technique

is Gas chromatography (GC) which is particularly suitable for the determination of Fatty acid composition GC requires the derivatization of lipids, which needs additional sample processing prior to analysis (Pulfer and Murphy, 2003)

Another possible method is Nuclear Magnetic Resonance (NMR), which is employed using radioactive Phosphorous and Hydrogen NMR allows for direct measurement of lipids in a non destructive manner Disadvantages include low

sensitivity and the spectra being dominated by abundant ions (Gawriach et al., 2002)

Various biochemical approaches such as the use of lipid assays, lipid antibodies are employed to study these molecules In addition imaging using fluorescent lipids allows for the study of some phospholipids and sterols

The adaptation of mass spectrometry to the study of lipids has helped overcome some of the difficulties encountered in above mentioned techniques used in lipids

1 3 M a s s s p e c t r o m e t r y a n d l i p i d o m i c s

Mass spectrometry involves the ionization of molecules followed by the determination of their mass to charge (m/z) ratio The representation of the mass to charge ratio (m/z) on the X-axis and the relative intensities of the various ions on the

Y axis is termed a mass spectrum Mass spectrometry of lipids was revolutionized with the development of soft ionization methods of Electro Spray Ionization (ESI) and Matrix Assisted Laser Desorption and Ionization (MALDI) These techniques allow

for the direct, quantitative and sensitive analysis of lipids (Kim HY et al., 1994; Kerwin JL et al., 1994; Brugger B et al., 1997) The major disadvantage of mass

spectrometry is the suppression of ionization, which affects ions that are present in lower abundance Even this disadvantage can be offset by the use of LC/MS which separates out different classes of lipids prior to mass spectrometry

The use of mass spectrometry and other techniques to study these molecules at systems levels has led to the development of a new field called lipidomics

Techniques such as mass spectrometry, chromatography, NMR and biochemical approaches such as lipid antibodies are used in lipidomics experiments The resolution and sensitivity of a mass spectrometer make it particularly suitable for lipidomics (Kitano, 2002; Wenk, 2005)

The general experimental approach in lipidomics is to extract lipids from the system under study and obtain a lipid profile (Figure 1.3) A lipid profile refers to the composition and relative amounts of the various lipids The lipid profile for a test condition is then compared to a reference condition, which could yield valuable clues about the system under study Crude lipid extracts can be directly used, which allows for a higher throughput Addition of internal standards can provide for semi

quantitative data (Welti et al., 2004) The amount of sample required to obtain a lipid

profile using Mass spectrometry is also minimal For example the profiling of

Phospholipids in Erythrocyte membranes required as little as 1µL of blood (Han et al., 1994)

Figure 1.3 A typical lipidomics Experiment

Homogenization + Organic solvent extraction

lipid extracts

Biological material (cells, tissues)

Mass spectrometry

Acylcarnitine species

Anionic lipid species

Positive-ion mode ESI/MS

Negative-ion mode ESI/MS

1 4 G l y c e r o p h o s p h o l i p i d s p r o f i l i n g

Glycerophospholipids perform a wide array of biological functions in the cell They constitute a significant portion of the plasma membrane, are involved in protein-lipid interactions, function as signaling molecules regulating many cellular processes such as membrane trafficking, cell growth and cytoskeletal rearrangements (Carman

and Henry, 1999; Dowhan, 1997; Odorizzi et al., 2000) Considering their multi

functional roles in the cells, a deeper understanding of glycerophospholipids would enable a better understanding of cellular mechanisms Glycerophospholipid profiling involves the extraction of these molecules preferentially This can be carried out using acid phase extraction Comparison of mutant profiles to wild type glycerophospholipid profiles can provide clues on genes involved in glycerophospholipid metabolism and regulation

1.4 1 S el ection of ca ndidate O R Fs f or gl ycerophosph olipid p rof ilin g

The yeast genome has 6604 ORFs broadly categorized as verified, uncharacterized and hypothetical For the functionally verified ORFs, in addition to their annotated functions other function may exist The yeast genome has 6604 ORFs,

of which 4538 have been annotated The remainder falls under the category of uncharacterized as well as dubious A comprehensive lipid profiling of the yeast would require profiling of all viable gene deletion mutants for all lipid classes Such a comprehensive effort would require a large scale initiative

A representative lipid profiling of the yeast would require the selection of candidate ORFs to be profiled In order to account for the possibility that annotated genes may have unknown functions, a random selection of ORFs is required An additional set of genes or gene functions to be screened may be selected based on the

presence of domains known to be involved in lipid metabolism The domains that can

be chosen include those that are involved in lipid binding and domains involved in lipid metabolism (Table 1)

Table 1 Lipid binding domains

Pleckstrin Homology(PH) domain Bind to phosphoinositols

however there are certain differences For example, the synthesis of phosphatidyl Serine involves an alternate pathway with a CDP-DAG intermediate which is not found in other eukaryotes Yeast has Ergosterol whereas other eukaryotes have Cholesterol Yeast sphingolipids have an inositol moiety, not found in the

spingolipids of mammals (Daum et al., 1999)

A systematic profiling of selected yeast strains with suspected defects in lipid metabolism has been carried out using TLC as a primary analytical technique (Daum

et al., 1999; Oliver, 1996) The suitability of yeast for mass spectrometry based profiling approach has been established by the development of methods to study

Phospholipids and Sphingolipids in yeast (Guan et al., 2006) The presence of a

completely sequenced genome and well characterized methods allow for the phospholipid analysis offering an opportunity to discover genes involved in their metabolism and/or regulation One primary requirement for such a study is a collection of gene deletion mutants Several functional analyses initiatives have created yeast single deletion libraries, providing a valuable resource for gene function

discovery One such gene deletion library collection in Saccharomyces has been

created by EUROFAN (European Functional Analysis Network) (Oliver, 1997; Oliver

et al., 1998) The presence of these resources allows for a reverse genetics approach

to identify gene function

1 6 O b j e c t i v e s o f t h e s t u d y

The objective of this study was to use glycerophospholipid profiling to discover gene functions involved in Phospholipid metabolism or regulation ORFs were profiled in both a random and domain based manner to identify candidate genes involved in metabolism or regulation of glycerophospholipids Additionally the project aims to serve as a pilot scale profiling project that can be scaled up for the subsequent analysis of the entire genome Towards this goal, strategies to reduce the time consuming process of lipid profiling were explored

The ORFs for analysis were chosen both randomly as well as on the basis of presence of domains involved in Phospholipid metabolism and recognition The list of strains used is displayed in Table 2

Table 2: Yeast gene-deletion strains used in the random screen

YML009C Mitochondrial ribosomal protein of the large subunit

YPL078C Subunit b of the stator stalk of mitochondrial F1F0 ATP

synthase

YML081C-A Subunit of the mitochondrial F1F0 ATP synthase

ORF DESCRIPTION

YDR512C Protein of unknown function, involved in transcriptional

induction and sporulation

YJR055W Protein of unknown function, required for growth at high

temperature

YDR500C Protein component of the large (60S) ribosomal subunit

YKL053C-A Mitochondrial intermembrane space cysteine motif protein

YLL013C Protein of the mitochondrial outer surface, links the Arp2/3

complex with the mitochore

YLL016W Non-essential Ras guanine nucleotide exchange factor (GEF)

YNR067C Daughter cell-specific secreted protein with similarity to

glucanases

ORF DESCRIPTION

YER161C Protein involved in negative regulation of transcription;

YER149C protein required for polarized morphogenesis, cell fusion, and

low affinity Ca2+ influx

YER150W GPI-anchored, serine/threonine rich cell wall protein of

unknown function

YER153C Specific translational activator for the COX3

YPR093C Protein involved in a putative alcohol-responsive signaling

pathway

YDL113C Protein required for transport of amino peptidase

Table 3 Yeast gene-deletion strains used in a domain based screen

YJR125C Protein containing an Epsin like domain involved

in Clathrin recruitment and traffic

ENTH/VHS

YDR150W Protein required for nuclear migration, localizes

to the mother cell cortex and the bud tip

ORF DESCRIPTION DOMAIN

YHR108W Golgi-localized protein interacts with and

regulates Arf1p and Arf2p in a GTP-dependent

manner in order to facilitate traffic through the

late Golgi

ENTH

YHR155W Mitochondrial protein with a potential role in

promoting mitochondrial fragmentation during

programmed cell death

PH domain like

PH pleckstrin like

YHR161C Protein involved in clathrin cage assembly Phosphoinositide

binding clathrin adaptor

transferase

YGR157W Phosphatidylethanolamine methyltransferases PEMT

YIL155C Mitochondrial glycerol-3-phosphate

kinase Sphingosine Kinase

YIL124W NADPH-dependent 1-acyl dihydroxyacetone

phosphate reductase

Short chain dehydrogenase

component

PH domain like

YPL057C Probable catalytic subunit of a mannosylinositol

phosphorylceramide (MIPC) synthase

mannosylinositol phosphorylceramide (MIPC) synthase

YDL022W NAD-dependent glycerol-3-phosphate

dehydrogenase,

Dehydrogenase

YLR260W Minor sphingoid long-chain base kinase Sphingosine kinase

YBR177C acyltransferase that plays a minor role in Hydrolase

medium-chain fatty acid ethyl ester biosynthesis

27

YIL002C Phosphatidylinositol 4,bisphosphate

5-phosphatase

phosphatase

Inositol-5-YOR109W Phosphatidylinositol 4,bisphosphate

5-phosphatase

phosphatase

Inositol-5-YDL113C Protein required for transport of amino peptidase PX domain

YBR200W Involved in establishing cell polarity and

morphogenesis

SH3 domains

decarboxylase

YBR129C Protein of unknown function, overproduction

blocks cell cycle arrest in the presence of mating

pheromone

PH domain like Pleckstrin like

YDR313C RING-type ubiquitin ligase of the endosomal and

vacuolar membranes

FYVE

YDR104C Meiosis-specific protein of unknown function PH

decayboxylase C2

YDR284C Diacylglycerol pyrophosphate (DGPP)

YDL161W Epsin-like protein involved in endocytosis ENTH/VHS domain

YDR326C Protein involved in programmed cell death

Shares domains with many proteins having

a PH domain

many proteins involved in Phosphate group transfer

2 2 P l a s m i d s

The plasmid vector used in this study was a E coli / Saccharomyces cerevisiae

shuttle vector YCplac111 This vector has a Leucine prototrophy and an Ampicillin resistance as selection markers (Figure 2.1)

Fig 2.1 Plasmid map of YCplac111

2 3 G r o w t h m e d i a a n d b u f f e r s

All media were heat sterilized at 121° C for 20 min under pressure of 120 Pa For solid media 20g/L of agarose was used In the case of heat sensitive compounds, filter sterilization was employed The media used and their composition are as listed below

2.3.1 Y e a s t c u l t u r e m e d i a

- YPD (yeast extract, peptone, dextrose) All media components were obtained

from Becton Dickinson and company Composition of YPD is shown in Table 4

Table 4 : YPD composition

1% Yeast extract 10g/L 2% Peptone 20g/L 2% Dextrose 20g/L

- Composition of the minimal media was as follows:

Table 5 : Minimal Media Composition

Yeast Nitrogen base 7 g/L

2.3.2 Bacterial culture media

- Luria-Bertani (LB) media

Table 6 : LB Composition

1% Bacto Tryptone 10g/L 0.5%Yeast extract 5g/L

- LB media with Ampicillin (LBA)

Table 7 : LBA composition

0.01% Ampicilin 100mg/L

2 4 G l y c e r o p h o s p h o l i p i d e x t r a c t i o n

To achieve complete cell lysis, two successive approaches were adopted First the cells were digested with the enzyme Lyticase (Sigma) The cell pellet was suspended in 100 µL of Lyticase This was followed by incubation for 30 min at 37o C and refreezing at -80o C for 30 min Again the cells were incubated at 37o C for 30 min This method of thawing and freezing helps to break the yeast cell wall

This was followed by the addition of 100 microlitre equivalents of glass beads and vortexed several times in 30 sec bursts followed by incubation in ice for 30 sec

At this stage, 2 micro gram equivalents of the internal standard DMPA was added The internal standard helps to ensure consistency in lipid extraction and also allows for semi quantitative data to be obtained To extract lipids organic solvents were

employed; 500 µL of Chlorofom : Methanol (1:1) mix was added and vortexed for 30 sec followed by incubation for 30 sec This step was followed by the addition of 400

µL of Chloroform and 300 µL of 1M HCL and vortexed for 30 sec The extraction of lipids at acid phase helps in the enhanced extraction of acidic Phospholipids The eppendorf tube was then spun at 8500 rpm for 5 min This results in the separation of aqueous and organic phases A volume of 300 µL of organic phase was removed and transferred to a new tube The organic phase was then dried using a Speedvac The dried lipid film was reconstituted in 400 µL of Chloroform : Methanol (1:1) A five fold dilution using Chloroform : Methanol (1:1) was carried out prior to mass spectrometric analysis

2 5 A n a l y s i s o f l i p i d s

2 5 1 M a s s S p e c t r o m e t r y o f l i p i d e x t r a c t s

The samples were ionized using ESI in a Q-Tof micro (Waters Corp., Milford, MA) in the negative ion mode The parameters of the Mass spectrometer are shown in Table 10 The HPLC (High Performance Liquid Chromatography) system comprised

of Waters CapLC autosampler and a Waters CapLC pump The mobile phase was Chloroform : Methanol (1:1) at a flow rate of 15 µL A total volume of 2 µL of sample was introduced into the mass spectrometer for lipid profiling

Table 10 : Mass Spectrometer Parameters

Capillary Voltage 3000V Sample cone voltage 50V Source Temperature 80°C Desolvation temperature 250°C Desolvation gas flow rate 400 L/hr Sample cone gas flow rate 50 L/hr Mass acquisition range 400-1200m/z Acquisition time 3 minutes

2 5 2 T a n d e m m a s s s p e c t r o m e t r y

Tandem mass spectrometry was performed on a Waters Micro mass Q-Tof micro (Waters Corp., Milford, MA) The sample was infused using an inbuilt syringe pump at a flow rate of 10 µl per minute A collision voltage of between 25 – 80 eV was employed to fragment the parent ions The fragment ions were used for structure elucidation

2 5 3 P r e c u r s o r i o n s c a n m a s s s p e c t r o m e t r y

Precursor ion scan mass spectrometry can be used to quantitatively measure

ions when used with requisite internal standards (Ekroos et al., 2002) For analysis of

phospholipids, precursor scans of ions at m/z 241 (negative mode), 196 (negative mode), 184 (positive mode) were used for phosphoinositols, phosphoethanolamines and phosphocholines respectively

The mass spectrometer used was an Agilent 1100 high-performance liquid chromatography (HPLC) system and a 4000 Q-Trap mass spectrometer (Applied Biosystems, Foster City, CA)

An injection volume of 15-30 µL of samples was introduced into the mass spectrometer for precursor scan HPLC auto sampler was used to pick up the samples and to carry samples directly into mass spectrometer without via a column Mobile phase was chloroform: methanol (1:1) at a flow rate of 0.25 mL min-1

2.5 4 TAG measu rement usin g mass sp ect ro met ry

Measurement of TAG was performed using an Agilent 1100 high-performance liquid chromatography (HPLC) system and a 4000 Q-Trap mass spectrometer (Applied Biosystems, Foster City, CA) The HPLC system is made up of an Agilent

1100 binary pump, an Agilent 1100 thermo sampler and an Agilent 1100 column oven A sensitive in-house method was developed using an Agilent Zorbax Eclipse

XDB-C18 column (Shui G et al., in preparation) The HPLC conditions are (1)

chloroform: methanol: 0.1M Ammonium Acetate (100:100:4) as mobile phase at a flow rate of 0.25 mL min-1; (2) column temperature: 25°C; (3) injection volume: 20

µL Mass spectrometry was recorded under both positive and negative ESI modes with EMS scan type, and ESI conditions are: Turbo Spray source voltage, 5000 and -

4500 volts for positive and negative, respectively; source temperature, 250 °C; scan rate:1000 amu/s; GS1: 30.00, GS2: 30.00, curtain gas: 25; DP 30.00 volts; scan range, 300-1100 da Dried extracts were resuspended in HPLC mobile phase A total run time of 30 min was utilized to elute both polar lipids and non-polar TAGs from the column, and the elution period of TAGs were averaged for comparison of TAG profiles

2 5 5 C o m p u t a t i o n a l A n a l y s e s o f M a s s S p e c t r a l D a t a

Mass spectrometric data was acquired using MasLynx 4.0 software (Waters Corp., Milford, MA) The text files were processed using a program developed within

the lab (Huey et al., manuscript in preparation) The first step is to align the spectra to

each other and this is done using a method called Co-relation optimized warping

(COW) (Nielsen et al., 1998) COW uses the principle of warping (piecewise linear

stretching and compression) the time axis of one of the profiles using the other profile

as the reference One of the replicate spectra from the control condition is chosen as the reference to which the other spectra are warped The same is done for the test condition A comparison of the warped average replicates is then carried out The difference in the profiles is expressed as a logarithmic ratio The statistical relevance

of the data was taken into account by using three replicates per sample and accounting for inter replicate differences

The incubated tubes were then centrifuged at full speed for 5 min The supernatant was removed and the pellet resuspended in 70% Ethanol After incubation

at - 80oC and centrifugation, the supernatant was discarded The residual after drying, was resuspended in 50 µL of sterile water

The plasmid YCplac111-scGFP was used in the construction of a GFP tagged YBR042C The plasmid was amplified using primers carrying requisite restriction

enzyme sites for BamH1 and HindIII Two different restriction sites were set up to

ensure directional cloning

PCR amplification of the YBR042C gene was done using primers with an overhang containing recognition sites for BamH1 and HindIII (Fig 2.2)

The primers used are shown in Table 11

Table 11 : Oligo nucleotide primer sequences

2 6 2 R e s t r i c t i o n d i g e s t i o n o f P C R p r o d u c t a n d v e c t o r

The PCR product and the vector YCplac111 were double digested with Hind III and BamH I (Promega, USA) The reaction mix composition was as described in Table 12 The reaction mix employed was the same for the plasmid and insert

Table 12 : Reaction mix composition

PCR

YBR042C

YBR042C

Figure 2.2 The gene of interest

YBR042C is amplified with primers carrying restriction

enzyme sites HindIII and

BamH1

2 6 3 L i g a t i o n o f v e c t o r a n d i n s e r t

A vector : insert ratio of 7:1 was used in ligation reaction as shown in Table

13 The reaction mixture was incubated at 16°C overnight

Table 13 Ligation reaction mix composition

2 6 4 T r a n s f o r m a t i o n o f B a c t e r i a l C e l l s

Ligated products were transformed into E coli For each transformation, 10

µL of ligated product was added to DH5α competent cells (Invitrogen, USA) The cells were mixed gently and incubated on ice for 30 min Following which the cells were heat shocked at 37° C for 20 sec and incubated on ice for 2 min About 200 µL

of LB media was added and the transformation mix was incubated at 37°C for 1 hour

A volume of 200 µL of transformation mix was plated on LBA plates and incubated

at 37° C overnight to select for ampicillin resistant colonies

2 6 5 A n a l y s i s o f i s o l a t e d p l a s m i d s

Plasmid DNA was isolated from 5ml overnight cultures using the QIA miniprep kit (Qiagen, USA) The isolated plasmids were analysed by restriction mapping to determine whether the correct insert was present The restriction digestion product was run on a 1% agarose gel along with a molecular marker to determine the correct plasmid

2 6 6 P r e p a r a t i o n o f c o m p e t e n t y e a s t c e l l s

The yeast strain of interest was incubated overnight in 5 ml of YPD medium From the overnight culture, 400 µL was inoculated into 100 mL YPD broth and incubated at 30o C/ 200 rpm The culture was incubated till an OD of ~0.4 was reached The culture was harvested in a sterile 50 mL centrifuge tubes at 5000 rpm for

5 minutes The cell pellet was resuspended in 25 mL of sterile water The tubes were centrifuged again at 5000 rpm for 5 minutes and the supernatant poured off The pellet was resuspended in 1 mL of 100 mM Lithium acetate and transferred to a 1.5

mL microfuge tube The cells were pelleted down at 8000 rpm for 15 sec and supernatant discarded The cells were resuspended in 400 µL of 100 mM LiAc and vortexed gently The cells were kept on ice, in preparation for transformation

2 6 7 T r a n s f o r m a t i o n o f y e a s t c e l l s

A Transformation mix was prepared as follows

Table 14: Transformation mix composition

The transformation mix was vortexed until it was homogenized and incubated

in ice for 30 min The cells were then heat shocked at 42o C for 30 min The cells were then spun down and supernatant removed A volume of 100 µL of sterile water was added and resuspended gently This mixture was then plated onto medium lacking Histidine and kept for incubation at 30o C

2 6 8 F l u o r e s c e n t i m a g i n g o f y e a s t c e l l s

Fluorescence imaging was performed on a Leica DMLB microscope (Wetzlar, Germany) with a Curtis 100 fluorescent lamp GFP signal was visualized with a 470/40-nm bandpass excitation filter, a 500-nm dichromatic mirror, and a 525/50-nm bandpass emission filter (Leica filter cube GFP) As for the observation of Nile red fluorescence, the same UV-filter set was used Images were processed with a Leica FW4000 software

2 7 G e n e r a t i o n o f d o u b l e k n o c k o u t s o f S L C 1 a n d Y B R 0 4 2 C

The generation of a double knockout was based on the replacement of the

gene of interest with a selectable marker gene The single knockout mutant, ybr042c was chosen as the reference strain to delete SLC1 The marker gene chosen was HIS3

The his3 fragment was amplified along with slc1 flanking sequences, using the

following primers (Table 15) and PCR reaction mix (Table 16) and conditions (Table 17)

Table 15 Primers used for amplification of his3 template DNA