Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Summary

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Abstract

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes’ association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Introduction

Eukaryotic cells are organized in distinct compartments having specific functions. To accomplish its function, each compartment contains a unique set of proteins that are essential for its activity. A possible mechanism through which these proteins approach their compartment is by localized translation^1,2. In this process, the protein is synthesized at its destination by ribosomes and mRNAs that are located there. Among the probable advantages of localized translation are increased efficiency of protein targeting, decreased need for protein chaperones, and enabling site-specific regulation mechanisms. Also, localized mRNAs and ribosomes can be a secluded re....

Protocol

1. Mitochondria Purification

Weigh the pellet of cells. Roughly 0.6 g are obtained from 100 ml cells.

1. Grow 100-150 ml of yeast cells to OD₆₀₀ = 1-1.5 at 30 °C on a nonfermentable growth medium, such as galactose-based growth medium, in order to enrich for mitochondria.
2. Centrifuge cells at 3,000 x g for 5 min at room temperature and discard the supernatant.
3. Wash the pellet with double distilled water and centrifuge again. Discard the supernatant.
4. Resuspend the pellet in 1 ml DTT Buffer per 0.5 g of cells. It is important to treat the cells with a reducing agent in order to ....

Results

This protocol allows separation of a mitochondria-containing fraction from cytosolic components. The best way to test its success is to perform northern analysis and western analysis (Figure 1) to samples from the different isolation steps. Three parameters for the isolation quality are derived from these analyses. First, whether the RNA or proteins in the samples are intact – these will be detected as distinct bands in the analyses. Degradation events will induce the appearance of additional, shorter ba.......

Discussion

Technological advancements in imaging had yielded high resolution tools to study mRNA localization. Today, one can measure the movement of even a single mRNA molecule at the msec time scale^23-26. Yet, traditional biochemical approaches, as the one described above, are also informative and are preferable in some cases. Biochemical isolation allows purification of a large repertoire of mRNAs and proteins, and is therefore preferable for genome-wide studies. In a single isolation, one can obtain sufficient mRNA l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yeast Extract
BactoPeptone
Galactose			Do not autoclave Galactose
Growth medium			For mitochondrial enrichment, you should use any nonfermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose
0.1 M Tris-HCl, pH 9.4
30 mM Tris-HCl, pH 7.6
Dithiothreitol (DTT)
10 mM DTT Buffer			0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time
1.2 M Sorbitol
4 mM KH2PO4
16 mM K2HPO4
0.2 μm filter
Zymolyase Buffer			1.2 M Sorbitol, 4 mM KH2PO4 , 16 mM K2HPO4. Filter this buffer (0.2 μm) and keep at room temperature for future use
Zymolyase 20T			20,000 U/g
Recovery medium			Galactose-based growth medium supplemented with 1 M Sorbitol
0.1 mg/ml Cycloheximide (CHX)
0.6 M Mannitol
5 mM MgAc
100 mM KCl
0.5 mg/ml Heparin
1 mM Phenylmethanesulfonylfluoride (PMSF)
Mannitol Buffer			0.6 M Mannitol, 30 mM Tris-HCl pH7.6, 5 mM MgAc, 100 mM KCl. Add freshly: 0.5 mg/ml Heparin, 0.1 mg/ml CHX and 1mM (PMSF). Filter this buffer and use it ice-cold
8 M Guanidinium-HCl
100% and 70% Ethanol (EtOH)
3 M Sodium Acetate, pH 5.2
10 M LiCl stock solution
250 mM Tris HCl pH 6.8
SDS
Glycerol
β-Mercaptoethanol
Bromophenol blue
4x LSB			250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% β-mercaptoethanol, and 0.02% Bromophenol blue
Dounce homogenizer of 15 ml capacity equipped with tight fitting pestle