Name: analysis of neutral lipid synthesis in saccharomyces cerevisiae by metabolic labeling and thin layer chromatography
Uploaded: 2021-02-02T13:30:00
Description: Watch this Scientific Journal Video about Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography at JoVE.com

The authors would like to thank the members of the Henne lab for help and conceptual advice in the completion of this study. W.M.H. is supported by funds from the Welch Foundation (I-1873), the NIH NIGMS (GM119768), the Ara Paresghian Medical Research Fund, and the UT Southwestern Endowed Scholars Program. S.R has been supported by a T32 program grant (5T32GM008297).

1. Growth and labeling of yeast cells with 14C-acetic acid
<ol>
	<li>Inoculate a yeast culture by picking a colony from a plate and dispensing it into 20 mL of synthetic complete (SC) media containing 2% dextrose (see Supplementary File for the recipe of SC media). Incubate at 30 &#730;C for overnight with shaking at 200 rpm. 
	NOTE: Growth condition, sample volume, and treatment will differ based on the lipid(s) of interest. Prior to running full experiments, optimal growth conditions ...

<ol>
	<li>Konige, M., Wang, H., Sztalryd, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+adipose+specific+lipid+droplet+proteins+in+maintaining+whole+body+energy+homeostasis.">Role of adipose specific lipid droplet proteins in maintaining whole body energy homeostasis.</a> Biochimica Et Biophysica Acta. 1842 (3), 393-401 (2014).</li><li>Arrese, E. L., Saudale, F. Z., Soulages, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+as+signaling+platforms+linking+metabolic+and+cellular+functions.">Lipid droplets as signaling platforms linking metabolic and cellular functions.</a> Lipid Insights. 7, 7-16 (2014).</li><li>Walther, T. C., Chung, J., Farese, R. 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K., Bates, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+phase+HPLC+method+for+combined+separation+of+both+polar+and+neutral+lipid+classes+with+application+to+lipid+metabolic+flux.">Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux.</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 1145, 122099 (2020).</li><li>Knittelfelder, O. L., Weberhofer, B. P., Eichmann, T. O., Kohlwein, S. D., Rechberger, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+ultra-high+performance+LC-MS+method+for+lipid+profiling.">A versatile ultra-high performance LC-MS method for lipid profiling.</a> Journal of Chromatography B. 951-952, 119-128 (2014).</li><li>Ruiz, J. I., Ochoa, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+in+the+subnanomolar+range+of+phospholipids+and+neutral+lipids+by+monodimensional+thin-layer+chromatography+and+image+analysis.">Quantification in the subnanomolar range of phospholipids and neutral lipids by monodimensional thin-layer chromatography and image analysis.</a> Journal of Lipid Research. 38 (7), 1482-1489 (1997).</li><li>Bui, Q., Sherma, J., Hines, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high+performance+thin+layer+chromatography-densitometry+to+study+the+influence+of+the+prion+[RNQ+]+and+its+determinant+prion+protein+Rnq1+on+yeast+lipid+profiles.">Using high performance thin layer chromatography-densitometry to study the influence of the prion [RNQ+] and its determinant prion protein Rnq1 on yeast lipid profiles.</a> Separations. 5 (1), 5010006 (2018).</li><li>Pronk, J. T., de Steensma, H., Van Dijken, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyruvate+metabolism+in+Saccharomyces+cerevisiae.">Pyruvate metabolism in Saccharomyces cerevisiae.</a> Yeast. 12 (16), 1607-1633 (1996).</li><li>Buttke, T. M., Pyle, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unsaturated+fatty+acid+deprivation+on+neutral+lipid+synthesis+in+Saccharomyces+cerevisiae.">Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae.</a> Journal of Bacteriology. 152 (2), 747-756 (1982).</li><li>Touchstone, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatographic+procedures+for+lipid+separation.">Thin-layer chromatographic procedures for lipid separation.</a> Journal of Chromatography B: Biomedical Sciences and Applications. 671 (1-2), 169-195 (1995).</li><li>Folch, J., Lees, M., Sloane Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> The Journal of Biological Chemistry. 226 (1), 497-509 (1957).</li><li>Breil, C., Abert Vian, M., Zemb, T., Kunz, W., Chemat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Bligh+and+Dyer"+and+Folch+methods+for+solid-liquid-liquid+extraction+of+lipids+from+microorganisms.+Comprehension+of+solvatation+mechanisms+and+towards+substitution+with+alternative+solvents.">"Bligh and Dyer" and Folch methods for solid-liquid-liquid extraction of lipids from microorganisms. Comprehension of solvatation mechanisms and towards substitution with alternative solvents.</a> International Journal of Molecular Sciences. 18 (4), 708 (2017).</li><li>Fuchs, B., S&#252;&#223;, R., Teuber, K., Eibisch, M., Schiller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+analysis+by+thin-layer+chromatography-A+review+of+the+current+state.">Lipid analysis by thin-layer chromatography-A review of the current state.</a> Journal of Chromatography A. 1218 (19), 2754-2774 (2011).</li></ol>

Here, a versatile radiolabeling protocol to quantitatively monitor the synthesis of NL species in yeast is presented. This protocol is very modular, which allows for the procedure to be finished within 3-6 days. Additionally, a wealth of literature exists on the use of TLC to separate lipid species and metabolites, which should permit the user to detect several lipid species of interest with a simple change of TLC solvent systems16,19.This protocol is conducive t...

Acetyl-CoA is the fundamental building block of diverse biomolecules including neutral lipids (NLs), which serve as a versatile biomolecular currency for building membranes, generating ATP, and regulating cell signaling1,2. The availability of NLs to be shunted into any of these respective pathways is, in part, regulated by their storage. Lipid droplets (LDs), cytoplasmic organelles composed of hydrophobic cores of triglycerides (TGs) and sterol-esters (SEs), are the main storage compartments of most cellular NLs. As such, LDs sequester and regulate NLs, which can be degraded and subsequently utilized for biochemical and metabolic processes3,4. It is known that the mis-regulation of NL and LD-associated proteins is correlated with the onset of pathologies including lipodystrophy and metabolic syndromes5,6. Because of this, current LD research is intensely focused on how NL synthesis is regulated spatially, temporally, and across distinct tissues of multi-cellular organisms. Due to the ubiquitous cellular roles for NLs, many enzymes responsible for the synthesis and regulation of NLs are conserved throughout eukaryotes7. Indeed, even some prokaryotes store NLs in LDs8. Therefore, genetically tractable model organisms such as Saccharomyces cerevisiae (budding yeast) have been useful for the study of NL synthesis and regulation.
The separation and quantification of NLs from cell extracts can be accomplished in a myriad of ways, including gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)9,10,11. Perhaps the simplest method for separating NLs is via thin layer chromatography (TLC), which allows for subsequent densitometric quantification from a standard curve12,13. Although TLC provides only a course-grained separation of NLs, it remains a powerful technique because it is inexpensive, and it allows for the rapid separation of NLs from several samples simultaneously. Two of the most considerable challenges facing the study of NLs via TLC are: 1) the broad range of cellular abundances of NL species and their intermediates, and 2) the range of hydrophilicity/hydrophobicity of lipid intermediates within NL synthesis pathways. Consequently, the quantification of NL species via TLC is typically restricted to the most abundant species; however, introduction of a 14C-acetic acid radiolabel can significantly enhance the detection of low abundance intermediates within NL pathways. Acetic acid is rapidly converted into acetyl-CoA by the acetyl-CoA synthetase ACS214, which makes 14C-acetic acid a suitable radiolabeling substrate in yeast15. Additionally, separation of both hydrophobic NLs and hydrophilic intermediates of NLs can be achieved by TLC through the use of multiple solvent systems16. Here, a method is presented for the separation of NLs using 14C-acetic acid metabolic labeling in yeast. Lipids labeled during the pulse period are subsequently isolated by a well-established total lipid isolation protocol17, followed by the separation of NL species by TLC. Developing of TLC plates by both autoradiography to visualize labeled lipids, and a chemical spray to visualize total lipids, permits for multiple methods of quantification. Individual lipid bands can also be easily extracted from the TLC plate using a razor blade, and scintillation counting can be used to quantify amount of radiolabeled material within the band.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[1-C14] Acetic acid sodium salt specific activity: 45-60mCi</td>
			<td>PerkinElmer</td>
			<td>NEC084H001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>18:1 1,2 dioleoyl-sn-glycerol</td>
			<td>Avanti</td>
			<td>800811O</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof absolute ethanol</td>
			<td>Sigma</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid washed glass beads 425-600um</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber bulbs for Pastuer pipettes</td>
			<td>Fisher</td>
			<td>03-448-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulfate &#62;99%</td>
			<td>Sigma</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman LS6500 scintillation counter</td>
			<td>PerkinElmer</td>
			<td>A481000</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (HPLC grade)</td>
			<td>Fisher</td>
			<td>C607SK</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol &#62;99%</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesteryl-linoleate &#62;98%</td>
			<td>Sigma</td>
			<td>C0289</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated sulfuric acid</td>
			<td>Sigma</td>
			<td>339741</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50mL conical tubes, polypropylene with centristar cap</td>
			<td>Sigma</td>
			<td>CLS430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose, anhydrous grade</td>
			<td>Sigma</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether anhydrous grade</td>
			<td>Sigma</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Fisher</td>
			<td>11-475-155</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoLume scintillation liquid</td>
			<td>VWR</td>
			<td>IC88247001</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R centrifuge</td>
			<td>Fisher</td>
			<td>05-401-205</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Storage phosphor screen</td>
			<td>Sigma</td>
			<td>GE28-9564-75</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Typhoon FLA9500 imager</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid, ACS grade</td>
			<td>Sigma</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass 6mL scintillation vials</td>
			<td>Sigma</td>
			<td>M1901</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass centrifuge tube caps</td>
			<td>Fisher</td>
			<td>14-595-36A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass centrifuge tubes</td>
			<td>Fisher</td>
			<td>14-595-35A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, anhydrous grade</td>
			<td>Sigma</td>
			<td>296090</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Adenine &#62;99%</td>
			<td>Sigma</td>
			<td>A8626</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine &#62;98%</td>
			<td>Sigma</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine &#62;99%</td>
			<td>Sigma</td>
			<td>A1270000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine &#62;98%</td>
			<td>Sigma</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartate &#62;98%</td>
			<td>Sigma</td>
			<td>A9256</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine &#62;97%</td>
			<td>Sigma</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt monohydrate &#62;98%</td>
			<td>Sigma</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine &#62;99%</td>
			<td>Sigma</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glycine &#62;99%</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine &#62;99%</td>
			<td>Sigma</td>
			<td>H8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine &#62;98%</td>
			<td>Sigma</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine &#62;98%</td>
			<td>Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine &#62;98%</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine, HPLC grade</td>
			<td>Sigma</td>
			<td>M9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine, reagent grade</td>
			<td>Sigma</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline &#62;99%</td>
			<td>Sigma</td>
			<td>P0380</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine &#62;99%</td>
			<td>Sigma</td>
			<td>S4500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Theronine, reagent grade</td>
			<td>Sigma</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophan &#62;98%</td>
			<td>Sigma</td>
			<td>T0254</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine &#62;98%</td>
			<td>Sigma</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Uracil &#62;99%</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine &#62;98%</td>
			<td>Sigma</td>
			<td>V0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, ACS grade</td>
			<td>Fisher</td>
			<td>A412</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid &#62;99%</td>
			<td>Sigma</td>
			<td>O1008</td>
			<td></td>
		</tr>
		<tr>
			<td>p-anisaldehyde</td>
			<td>Sigma</td>
			<td>A88107</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum ether, ACS grade</td>
			<td>Sigma</td>
			<td>184519</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylcholine, dipalmitoyl &#62;99%</td>
			<td>Sigma</td>
			<td>P1652</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td>2231000713</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, ACS grade</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide pellets, certified ACS</td>
			<td>Fisher</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene &#62;98%</td>
			<td>Sigma</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic Acid crystalline/certified</td>
			<td>Fisher</td>
			<td>110-15-6</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC saturation pad</td>
			<td>Sigma</td>
			<td>Z265225</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC silica gel 60G glass channeled plate</td>
			<td>Fisher</td>
			<td>NC9825743</td>
			<td>No fluorescent indicators</td>
		</tr>
		<tr>
			<td>Transparency plastic film</td>
			<td>Apollo</td>
			<td>829903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>Sigma</td>
			<td>T0377</td>
			<td></td>
		</tr>
		<tr>
			<td>Triolein &#62;99%</td>
			<td>Sigma</td>
			<td>T7140</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman exposure cassette</td>
			<td>Sigma</td>
			<td>WHA29175523</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without ammonium sulfate and amino acids</td>
			<td>Sigma</td>
			<td>Y1251</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of neutral lipid synthesis in saccharomyces cerevisiae by metabolic labeling and thin layer chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

In this protocol, we have demonstrated that the labeling, detection, and quantification of NL species can be accomplished by 14C-acetic acid metabolic labeling. Major NL species can be separated in a solvent system of 50:40:10:1 (v/v/v/v%) Hexane:Petroleum ether:Diethyl ether:Acetic acid (Figure 1A,B). Phosphor imaging allows for visualization of labeled free fatty acid (FFA), triacylglycerol (TG), diacylglycerol (DG), cholesterol (Chol), and squalene (SQ) (<stron...

Watch this Scientific Journal Video about Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography at JoVE.com

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

This technique uses instruments commonly found in most laboratory environments and allows for the analysis of several samples simultaneously. This method can provide key insights into how lipid metabolism enzymes are regulated in a cellular context and in different genetic backgrounds. Inoculate a colony from a yeast culture plate into 20 milliliters of SC media containing 2%dextrose and incubate at 30 degrees Celsius for overnight with shaking at 200 rpm.On the next day, dilute the culture in 50 milliliters of fresh media to a final OD of 0.2 and grow it for 24 hours until the stationary phase is reached. Prepare two 20 milliliter aliquots of quenching buffer for each sample and store them at minus 80 degrees Celsius. Simultaneously, prepare radiolabeling media by adding acetic acid sodium salt to dextrose free SC media at a final concentration of 10 microcurie per milliliter.Collect the cells by centrifuging at 4, 100 x g for 10 minutes. Remove the supernatant and wash the cell pellet once with 20 milliliters of dextrose free SC media. Collect the cells again by centrifuging at 4, 100 x g for five minutes and transfer them to a labeled two milliliter microcentrifuge tube using dextrous free media.Centrifuge the cells for another two minutes. Resuspend the cells in 500 microliters of dextrose free SC media and begin the radiolabeling period by quickly adding 500 microliters of radiolabeling media to each cell suspension. Incubate the tubes in a rotating incubator at 30 degrees Celsius for 20 minutes.Meanwhile, pre cool the centrifuge equipped for 50 milliliter conical tubes to minus 10 degrees Celsius and thaw one 20 milliliter aliquots of quenching buffer. Once the radiolabeling period has ended use a pipette to plunge the entire one milliliter sample into thawed quenching buffer on ice. Collect the cell pellet by spinning in a pre cooled centrifuge at 5, 000 x g for three minutes and add 20 milliliters of fresh cold quenching buffer.Vortex and shake the samples to fully resuspend them in quenching buffer and centrifuge the samples again to collect the cells. Once the cells are pelleted, thoroughly remove all quenching buffer from the samples by pouring off the supernatant and removing the excess with a pipette. Store the tubes at minus 80 degrees Celsius for further processing.Weigh 0.3 grams of acid washed glass beads for each sample and store them in two milliliter microcentrifuge tubes on ice. Remove the cell pellets from the minus 80 degree freezer and place them on ice. Then, add 350 microliters of methanol and 700 microliters of chloroform to each sample and resuspend the cells.Transfer them to micro centrifuge tubes containing pre-weighed glass beads. Lyse the cells by vortexing three times for one minute, with 30 second incubation on ice between agitations. Pour the cells into a 15 milliliter glass centrifuge tube and label the tube as tube A.Wash the microcentrifuge tube with one milliliter of methanol, vortex it, and pour the methanol into tube A.Add two milliliters of chloroform followed by 400 microliters of water to tube A for a final sample volume of 4.45 milliliters.Vortex the samples for one minute followed by a five minutes centrifugation at 1000 x g to clearly separate the aqueous and organic phases with cell debris lying at the interface. Using a glass pasture pipette, collect the organic phase into a new glass centrifuge tube labeled B and add one milliliter of one molar potassium chloride. Add one milliliter of methanol and two milliliters of chloroform to tube A and repeat the vortexing and centrifugation steps.Vortex and centrifuge tube B to separate the aqueous and organic phase. Remove the upper aqueous layer and collect the entire bottom organic layer into a four milliliter glass vial. Completely evaporate the solvent from the lipid extracts by drying under inert gas and preheat an oven to 145 degrees Celsius for heating the TLC plate.Determine relative amounts of radiolabel taken up by the cells before loading them onto the TLC plate. Reconstitute the sample lipids in 40 to 50 microliters of chloroform and methanol mixed at a one-to-one ratio by vortexing for five minutes. Prepare 101 milliliters of the mobile phase solvent in a glass graduated cylinder.Then, pour the solvent into a glass TLC chamber containing a 20 by 20 TLC saturation pad and a tight fitting lid. Prepare a channeled 20 by 20 silica gel 60G plate and mark a line 1.5 centimeters above the bottom of the plate using a pencil. Once the plate has been sufficiently heated and the TLC saturation pad is saturated with solvent, remove the TLC plate from the oven and immediately proceed to loading it.Using a pipette, spot five microliters of sample onto the origin of each lane located 1.5 centimeters above the bottom of the TLC plate and repeat loading until 20 to 40 microliters of sample has been loaded into each lane. Once the standard and the samples have been loaded, place the plate in the developing chamber and wait until the solvent has reached the top of the plate. When the plate is fully developed, remove it from the chamber and allow it to dry in the fume hood for 20 minutes.After the plate is dried, cover it with plastic film and place it in a developing cassette with an autoradiography screen. Remove the screen from the developing cassette and place it inside of a phosphor imager. Spray the TLC plate with p-Anisaldehyde reagent until the silica is saturated, then bake it in a 145 degree oven for five minutes or until bands have appeared.To quantify lipid species, scrape the silica gel containing individual lipid species and transfer them to glass scintillation vials. Add six milliliters of scintillation fluid and vortex vigorously until the silica band has been reduced to small pieces. Place the rack with the scintillation vials into a scintillation counter and count by adjusting the counting time to two minutes per vial.Phosphor imaging allows for visualization of autoradiogram of label-free fatty acid, triacylglycerol, diacylglycerol, cholesterol, and squalane. It was demonstrated that purified lipid species can be separated in this method and subsequently visualized by spraying of the TLC plate with p-Anisaldehyde reagent. Visualized species include all previously mentioned lipids in addition to sterile esters.By applying a 10 minute chase period in radiolabel-free media following the pulse, it was observed at the major pool of squalane disappeared and total cholesterol was elevated. Similarly, the appearance of diacylglycerol in the chase period correlated with a decrease in the free fatty acids signal. Prior to performing the full protocol, it is important to determine whether the cells will uptake the acetic acid radiolabel in the growth condition and genetic background of interest.

analysis-neutral-lipid-synthesis-saccharomyces-cerevisiae-metabolic

Research

JoVE Journal

Biochemistry

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker&#039;s Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Locus-Specific Chromatin Isolation from Yeast Cells

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

Author Spotlight: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...