Mitochondrial Respiration Quantification in Yeast Whole Cells

Summary

Mitochondrial respiration in yeast whole cells is a valuable indicator of cell bioenergetics. Here, we present a protocol to quantify this phenotype applicable to different yeast species.

Abstract

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.

Introduction

Mitochondria plays a fundamental role in cellular bioenergetics since it is the main source of ATP for most cells, and several pathways converge and depend on the activity of mitochondrial pathways¹. Oxidative phosphorylation is needed for ATP synthesis that combines electron transport through the electron transport chain to reduce oxygen and F₁F₀- ATPase activity, synthesizing ATP using the mitochondrial membrane potential produced due to electron flux². Thus, mitochondrial respiration is part of the oxidative phosphorylation³.

The mitochondrial r....

Protocol

1. Culture media and inoculum preparation

1. Prepare 100 mL of yeast extract-peptone-dextrose medium (YPD) by adding 1 g of yeast extract, 2 g of casein peptone, and 2 g of glucose in 95 mL of distilled water. Distribute 3 mL into 10 mL glass test tubes, sterilize at 121 °C, and 1.5 psi for 15 min.
   NOTE: The medium can be stored at 4 - 8 °C for up to 1 month.
2. Inoculate a 10 mL glass test tube containing 3 mL of YPD medium with 250 µL of -20 °C glycerol-preser.......

Discussion

Mitochondrial respiration phosphorylation plays a fundamental role in several pathways that depend on mitochondrial membrane potential and maintain ATP levels through oxidative phosphorylation. Understanding how environmental and nutritional conditions impact yeasts' mitochondrial respiration serves as a tool to elucidate molecular mechanisms.

It is essential to consider the following critical steps to obtain reliable results from this method. Agitation >200 rpm is critical to obtainin.......

Materials

Name	Company	Catalog Number	Comments
2- thenoyltrifluoroacetone (TTFA)	Merck	T27006	Inhibitor complex II
3-chlorophenylhydrazone carbonyl cyanide (CCCP)	Merck	C2759	Mitochondrial respiration uncoupler
Absolut ethanol	Merck	107017	For dissolving quercetin
Agar	Merck	A1296	YPD agar preparation
Ammonium sulfate granular (NH4)2SO4	J.T. Baker	0792-05	SC broth preparation
Antimycin A (AA)	Merck	A8674	Inhibitor complex III
aYSI5300A	----	----	Monitor
Centrifuge	Hermle	Z 206 A	For cells centrifugation
Clark-type oxygen	----	----	Electrode
Computer	----	----	For data acquisition
Dipotassium phosphate K2HPO4	J.T. Baker	3252-05	SC broth preparation
Glucose	Merck	G7021	YPD broth preparation
Glycerol	Merck	G5516	Substrate medium supplementation
Lactate	Merck	L1250	Substrate medium supplementation
Oligomycin from Streptomyces diastatochromogenes	Merck	O4876	Inhibition of mitochondrial ATP synthase
Orbital Shaker	Thermo Fisher	SHKE6000	Inoculum incubation glass tubes and flask
Peptone from casein, enzymatic digest	Merck	82303	YPD broth preparation
Quercetin	Merck	337951	For decreasing mitochondrial respiration
Uracil	Merck	U0750	SC broth preparation
Yeast extract	Merck	Y1625	YPD broth preparation
Yeast nitrogen base without amino acids and ammonium sulfate	Merck	Y1251	SC broth preparation
Yeast Synthetic Drop-out medium supplements without uracil	Merck	Y1501	SC broth preparation