We thank Drs. Erez Eliyahu, Daniel Melamed, Ophry Pines and Doron Rapaport for help and comments during the establishment of this protocol. This work is funded by the ISF (grant number 1193/09).

1. Mitochondria Purification
Weigh the pellet of cells. Roughly 0.6 g are obtained from 100 ml cells.
<ol>
	<li>Grow 100-150 ml of yeast cells to OD600 = 1-1.5 at 30 &#176;C on a nonfermentable growth medium, such as galactose-based growth medium, in order to enrich for mitochondria.</li>
	<li>Centrifuge cells at 3,000 x g for 5 min at room temperature and discard the supernatant.</li>
	<li>Wash the pellet with double distilled water and centrifuge again. Discard the su...

<ol>
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Changes in the amount of bound ribosomes in response to changes in metabolic state.</a> J. Biol. Chem. 249, 3304-3310 (1974).</li><li>Suissa, M., Schatz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Import+of+proteins+into+mitochondria.+Translatable+mRNAs+for+imported+mitochondrial+proteins+are+present+in+free+as+well+as+mitochondria-bound+cytoplasmic+polysomes.">Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes.</a> J. Biol. 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The authors declare no competing financial interests.

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 scale23-26. 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...

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 translation1,2. 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 reservoir of translation machinery in cases of cellular stress, when general translation is inhibited.
Mitochondria became in recent years a central model to study localized translation. &#160;Most mitochondria proteins are encoded in the nucleus, translated in the cytosol, and imported into the organelle. Various lines of evidence indicate that many of these proteins are produced through a local translation process. Initially, electron microscopy and biochemical fractionation studies detected ribosomes associated with mitochondria3-5. These studies where then corroborated in vivo by work on specific mRNAs, that were found to be imported only in a cotranslational manner6,7.&#160;Genome-wide studies of mRNAs association with mitochondria revealed that a significant fraction of mRNAs are localized to the mitochondria vicinity8-10. Some of these mRNAs were further characterized by in vivo fluorescence methods, such as FISH or mTAG9,11. &#160;A straight-forward interpretation of this association is that these mRNAs serve as templates for localized translation.
The mechanisms by which these mRNAs approach the mitochondria are unknown. Noncoding domains (most significantly 3&#8217; UTRs) were shown to be involved in mRNA association to the mitochondria12. These domains are likely to serve as a binding site to RNA-binding proteins which mediate their transport. Studies in yeast revealed that a member of the PUM family of proteins (Puf3) supports mRNA association with mitochondria8,13. A plausible role for Puf3, which is based on functions of other family members, is to inhibit translation while the mRNA is en route14. Thus, mRNAs may be transported in a nontranslated status, by RNA binding proteins that interact with noncoding regions. Alternatively, a large body of work suggests that transport occurs while the protein is being synthesized. In particular, translation inhibitors were shown to affect mRNAs association8,13. Furthermore, translated features such as the AUG, mitochondrial targeting sequence (MTS) or ORF regions were shown to assist in localization8,11,15. Hsp70-family protein chaperone and protein receptor on the mitochondria outer membrane were also shown to support mRNA association, further implying that encoded-protein features are important for mRNA localization16. This is consistent with a model in which the ribosome-emerging protein serves as recognition element for targeting mRNA-ribosome-protein complex to the mitochondria17.
Localized translation near the mitochondria was studied by various methods, including electron microscopy (to visualize ribosomes)3,&#160; FISH9,&#160; green RNA (to detect specific mRNAs)11, and biochemical fractionation (to detect both RNA and ribosomes)10,18. While the former methods detect localization in vivo and may allow visualization of transport dynamics, the latter allows detection of many different mRNAs in a single experiment. Furthermore, for biochemical fractionation coding or noncoding domains do not need to be altered, therefore their specific roles can be evaluated. Biochemical fractionation has been successfully used for many years, for isolation of many different cellular compartments. Its principals and limitations are well established, and one can easily modify existing protocols for different purpose. The necessary instrumentation is standard in many labs, therefore it is usually the first method of choice for studying intracellular localization. We describe a protocol that was optimized for isolation of mRNAs while ribosomes are associated with mitochondria. This protocol is therefore optimal for studying factors involved in localized translation near mitochondria.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BactoPeptone</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Galactose</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Do not autoclave Galactose</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Growth medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">For mitochondrial enrichment, you should use any non- fermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 M TrisHCl pH9.4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30 mM TrisHCl pH7.6</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 mM DTT Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.2 M Sorbitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">4 mM KH2PO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">16 mM K2HPO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2&#956;m filter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Zymolyase Buffer&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">1.2 M Sorbitol, 4 mM KH2PO4 , 16 mM K2HPO4. Filter this&#160; buffer (0.2&#956;m) and keep at room temperature for future use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Zymolyase 20T&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">20,000 U/gr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recovery medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Galactose-based growth medium supplemented with 1 M Sorbitol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 mg/mL Cycloheximide (CHX)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.6 M Mannitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
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			<td height="21" style="height:21px;">5 mM MgAc</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mM KCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5 mg/mL Heparin</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 mM phenylmethanesulfonylfluoride (PMSF)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mannitol Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.6 M Mannitol, 30 mM TrisHCl 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</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8M Guanidinium-HCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;100% and 70% Ethanol (EtOH)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;3 M Sodium Acetate pH5.2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 M LiCl stock solution</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mM Tris HCl pH 6.8</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Glycerol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenolblue</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;4xLSB</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% &#946;-mercaptoethanol and 0.02% Bromophenol blue</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer of 15-ml capacity equipped with tight- fitting pestle</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
	</tbody>
</table>

isolation of mrnas associated with yeast mitochondria to study mechanisms of localized translation

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&#8217; 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.

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 &#8211; these will be detected as distinct bands in the analyses. Degradation events will induce the appearance of additional, shor...

Watch this Scientific Journal Video about Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation at JoVE.com

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

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&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

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 ...

isolation-mrnas-associated-with-yeast-mitochondria-to-study

Research

JoVE Journal

Biology

12.8K ビュー.  Technion - Israel Institute of Technology. ミトコンドリアタンパク質をコードするmRNAの多くはミトコンドリア外膜に関連している。私たちは、それに関連するmRNAおよびリボソームと酵母ミトコンドリアの分離を目的とした細胞下分画の手順が記載されている。このプロトコルは、mRNAの局在化およびミトコンドリア付近の局所的な翻訳の機構を明らかにするために、多様な条件下で増殖させた細?...