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

Summary

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.

Abstract

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

Introduction

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 signaling^1,2. 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 bioc....

Protocol

1. Growth and labeling of yeast cells with ¹⁴C-acetic acid

1. 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 ˚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 .......

Discussion

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 systems^16,19.This protocol is conducive t.......

Materials

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