Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Summary

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.

Abstract

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.

Introduction

Mitochondria are essential organelles of eukaryotic cells that play crucial roles in bioenergetics, cellular metabolism, and signaling pathways¹. To properly execute these tasks, mitochondria rely on a unique set of proteins and lipids responsible for their structure and function. The budding yeast Saccharomyces cerevisiae has been widely used as a model system for investigations on mitochondrial processes, as well as for other organelles². The mitochondrial genome codes for only eight proteins in yeast; the vast majority of mitochondrial proteins (~99%) are encoded by nuclear genes, which are translated on cyto....

Protocol

1. Growth of yeast cells

1. Isolate single colonies of the strain of interest by streaking a small portion of the cells from a -80 °C glycerol stock onto a YPD (1% yeast extract, 2% peptone, 2% glucose) agar plate. Incubate the plate at 30 °C for 2-3 days.
   NOTE: The S. cerevisiae strain used in this protocol is derived from BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0). With the exception of the auxotrophic marker genes, this strain does not contain any deleted gene and does not carry any plasmid. Thus, it can be successfully cu....

Results

The success of submitochondrial fractionation protocol depends on obtaining highly purified intact mitochondria. For this, it is essential that during the yeast cell lysis, the intactness of the organelles remains almost totally preserved. This is achieved by using a cell lysis protocol that combines the enzymatic digestion of the cell wall followed by physical disruption of the plasma membrane by using a Dounce homogenizer. The mitochondrial contents are then collected by differential centrifugation. This subcellular fr.......

Discussion

The protocol presented here has been successfully used and continuously optimized for a long-time to determine the protein localization in the submitochondrial compartments^{13,14,18,21,22,23}. The reliability and reproducibility of this protocol are strongly dependent on the purity and integrity of mitochondrial preparations

Materials

Name	Company	Catalog Number	Comments
Bacto Peptone	BD	211677
Bacto Yeast extract	BD	212750
Beckman Ultra-Clear Centrifuge Tubes, 14 x 89 mm	Beckman Coulter	344059
Bovine serum albumin (BSA fatty acid free)	Sigma-Aldrich	A7030	Component of Homogenization buffer
DL-Dithiothreitol	Sigma-Aldrich	43815	Component of DDT buffer
D-Sorbitol	Sigma-Aldrich	S1876
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E9884
Galactose	Sigma-Aldrich	G0625
Glucose	Sigma-Aldrich	G7021
MOPS	Sigma-Aldrich	M1254
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	P7626	Used to inactivate proteinase K
Potassium phosphate dibasic	Sigma-Aldrich	P3786
Potassium phosphate monobasic	Sigma-Aldrich	P0662
Proteinase K	Sigma-Aldrich
Sucrose	Sigma-Aldrich	S8501
Trichloroacetic acid (TCA)	Sigma-Aldrich	T6399
Trizma Base	Sigma-Aldrich	T1503
Zymolyase-20T from Arthrobacter luteus	MP Biomedicals, Irvine, CA	320921	Used to lyse living yeast cell walls to produce spheroplast