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 supernatant.</li>
	<li>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 break the disulfide bonds within the cell wall, thereby improving hydrolysis.</li>
	<li>Incubate the cells for 10 min at 30 &#176;C with gentle shaking. Meanwhile, weigh 6 mg zymolyase per 1 g of cells and suspend it in Zymolyase Buffer.</li>
	<li>Centrifuge cells at 3,000 x g for 5 min at room temperature and discard the supernatant.</li>
	<li>Resuspend the pellet in 1 ml Zymolyase Buffer per 0.15 g of cells. Do not vortex.</li>
	<li>Measure OD600 of 10 &#956;l aliquot of the cells in 990 &#956;l of water.</li>
	<li>Add Zymolyase (step 1.5) to the cells to hydrolyze glucose polymers at the &#946;-1,3-glucan linkages and generate spheroplasts. From this step, spheroplasts should be kept in an isotonic solution in order to avoid lysis.</li>
	<li>Incubate cells for 15 min at 30 &#176;C (for optimal activity of zymolyase) with gentle shaking. To verify hydrolysis of the cell wall and spheroplasts generation, mix 10 &#956;l of the cells with 990 &#956;l of water. Spheroplasts are expected to lyse due to osmotic difference, and the OD600 should be at least 10-fold less than the value determined in step 1.9. If not, continue incubation with zymolyase for another 15 min.</li>
	<li>Centrifuge cells at 3,000 x g for 5 min at room temperature. Carefully discard the supernatant, as the pellet might be unstable.</li>
	<li>Wash spheroplasts with Zymolyase Buffer and discard the supernatant.</li>
	<li>Resuspend spheroplasts with 100 ml Recovery medium. Transfer spheroplasts to an Erlenmeyer flask and incubate for 1 hr at 30 &#176;C with shaking. This recovery step is necessary since during the Zymolyase treatment translation is arrested and mRNA localization is disrupted (Figure 1B).</li>
	<li>Add 0.1 mg/ml CHX and transfer spheroplasts to a precooled 50 ml conical tube. CHX addition is important to freeze ribosomes&#8217; association with mRNAs. Thus, ribosomes-dependent mRNA association with mitochondria is maintained.</li>
	<li>Centrifuge spheroplasts at 3,000 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Wash twice with cold Mannitol Buffer.</li>
	<li>Resuspend spheroplasts with 4 ml cold Mannitol Buffer and transfer them to a Dounce homogenizer of 15 ml capacity equipped with tight fitting pestle. Gently break spheroplasts with 15 strokes and transfer the lysate to a 13 ml tube.</li>
	<li>Centrifuge the lysate at 1,500 x g for 6 min at 4 &#176;C to pellet nuclei and unbroken cells.</li>
	<li>Carefully transfer the supernatant to a new tube. Set aside 1 ml (25%) of the sample as an unfractionated sample (&#8220;Total&#8221; sample). Transfer 50 ml of this sample to a new tube and add 15 ml of 4X LSB for western blot analysis. &#160;Precipitate RNA from the rest of the &#8220;Total&#8221; sample (See Protocol 3, RNA precipitation).</li>
	<li>Centrifuge the supernatant at 10,000 x g for 10 min at 4 &#176;C to pellet the mitochondria.</li>
	<li>Transfer the supernatant (~3 ml) to a new tube and keep it on ice. This is the &#8220;Cytosolic&#8221; fraction. Transfer 50 ml of this sample to a new tube and add 15 ml of 4x&#160;LSB for western blot analysis. Precipitate RNA from the rest of the &#8220;Cytosolic&#8221; sample (See Protocol 3, RNA precipitation).</li>
	<li>Wash the pellet with 3 ml Mannitol Buffer and centrifuge again at 10,000 x g for 10 min at 4 &#176;C.</li>
	<li>Resuspend the pellet with 3 ml Mannitol Buffer. This sample contains the mitochondria, and referred to as &#8220;Mitochondria&#8221; fraction. Transfer 50 ml of this sample to a new tube and add 15 ml of 4x&#160;LSB for western blot analysis.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Add to each sample one volume of 8 M guanidinium-HCl and two volumes of 100% ethanol. Vortex and incubate for at least 2 hr at -20 &#176;C.</li>
	<li>Centrifuge samples at 10,000 x g for 20 min at 4 &#176;C. Discard supernatant. Be careful as the pellet might be unstable.</li>
	<li>Wash the pellet with 70% EtOH.</li>
	<li>Resuspend the pellet with 400 ml of RNase-free water and transfer the sample to a new Eppendorf tube.</li>
	<li>Precipitate the RNA again by adding 0.1 volume of 3 M sodium acetate pH 5.2 and two volumes of 100% EtOH. Vortex and incubate for at least 2 hr at -20 &#176;C.</li>
	<li>Centrifuge samples at 20,000 x g for 20 min at 4 &#176;C. Discard the supernatant and wash with 70% EtOH.</li>
	<li>Air dry the pellet and resuspend with RNase-free water. Store RNA samples at -80 &#176;C. Samples can be used for northern analysis19 or microarray analysis18 (See Protocol 3).</li>
</ol>
3. Preparing the RNA for Microarray Analysis
<ol>
	<li>Resuspend the mRNA from step 2.2 with 650 ml of RNase-free water.</li>
	<li>To remove any residual DNA or proteins, add equal volume (650 ml) of acidic phenol:chloroform (5:1, pH 4.7) and vortex vigorously. Centrifuge at maximum speed for 2 min.</li>
	<li>Transfer 500 ml of the upper phase (the aqueous phase) to a new tube.&#160;Avoid taking the interphase, as it contains DNA. Also be careful of taking phenol, which can inhibit the reverse transcription reaction.</li>
	<li>The RNA sample contains a significant amount of heparin, which is a potent inhibitor of reverse transcriptase. Thus, for RT-PCR or microarray labeling it needs to be removed. Remove heparin by LiCl precipitation: add LiCl to a final concentration of 2 M and incubate samples over night at -20 &#176;C.</li>
	<li>Thaw the samples at 4 &#176;C and centrifuge at 20,000 x g for 20 min at 4 &#176;C.</li>
	<li>Carefully discard the supernatant and wash the pellet with 80% ethanol. Centrifuge again as described in step 3.5.</li>
	<li>Discard the supernatant, air dry and resuspend the pellet in 150 ml of RNase-free water.</li>
	<li>To remove any residual LiCl, precipitate again with 0.1 volume of 3 M sodium acetate pH 5.3 and 3 volumes of 100% ethanol. Incubate at -20 &#176;C for at least 2 hr.</li>
	<li>Centrifuge at top speed for 20 min at 4 &#176;C. Wash carefully the transparent pellet with 80% ethanol and air dry.</li>
	<li>Resuspend the pellet with 25 ml RNase-free water and keep the samples at -80 &#176;C.</li>
	<li>Follow the steps for RNA labeling and hybridization described in18,20.</li>
</ol>

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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.

<table><tbody><tr><td>Yeast Extract</td><td></td><td></td><td></td></tr><tr><td>BactoPeptone</td><td></td><td></td><td></td></tr><tr><td>Galactose</td><td></td><td></td><td>Do not autoclave Galactose</td></tr><tr><td>Growth medium</td><td></td><td></td><td>For mitochondrial enrichment, you should use any nonfermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose</td></tr><tr><td>0.1 M Tris-HCl, pH 9.4</td><td></td><td></td><td></td></tr><tr><td>30 mM Tris-HCl, pH 7.6</td><td></td><td></td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td></td><td></td><td></td></tr><tr><td>10 mM DTT Buffer</td><td></td><td></td><td>0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time</td></tr><tr><td>1.2 M Sorbitol</td><td></td><td></td><td></td></tr><tr><td>4 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>16 mM K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;#956;m filter</td><td></td><td></td><td></td></tr><tr><td>Zymolyase Buffer</td><td></td><td></td><td>1.2 M Sorbitol, 4 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; , 16 mM K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;. Filter this buffer (0.2 &amp;#956;m) and keep at room temperature for future use</td></tr><tr><td>Zymolyase 20T</td><td></td><td></td><td>20,000 U/g</td></tr><tr><td>Recovery medium</td><td></td><td></td><td>Galactose-based growth medium supplemented with 1 M Sorbitol</td></tr><tr><td>0.1 mg/ml Cycloheximide (CHX)</td><td></td><td></td><td></td></tr><tr><td>0.6 M Mannitol</td><td></td><td></td><td></td></tr><tr><td>5 mM MgAc</td><td></td><td></td><td></td></tr><tr><td>100 mM KCl</td><td></td><td></td><td></td></tr><tr><td>0.5 mg/ml Heparin</td><td></td><td></td><td></td></tr><tr><td>1 mM Phenylmethanesulfonylfluoride (PMSF)</td><td></td><td></td><td></td></tr><tr><td>Mannitol Buffer</td><td></td><td></td><td>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</td></tr><tr><td>8 M Guanidinium-HCl</td><td></td><td></td><td></td></tr><tr><td>100% and 70% Ethanol (EtOH)</td><td></td><td></td><td></td></tr><tr><td>3 M Sodium Acetate, pH 5.2</td><td></td><td></td><td></td></tr><tr><td>10 M LiCl stock solution</td><td></td><td></td><td></td></tr><tr><td>250 mM Tris HCl pH 6.8</td><td></td><td></td><td></td></tr><tr><td>SDS</td><td></td><td></td><td></td></tr><tr><td>Glycerol</td><td></td><td></td><td></td></tr><tr><td>&amp;#946;-Mercaptoethanol</td><td></td><td></td><td></td></tr><tr><td>Bromophenol blue</td><td></td><td></td><td></td></tr><tr><td>4x LSB</td><td></td><td></td><td>250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% &amp;#946;-mercaptoethanol, and 0.02% Bromophenol blue</td></tr><tr><td>Dounce homogenizer of 15 ml capacity equipped with tight fitting pestle</td><td></td><td></td><td></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...

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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 ...

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Universidad de Cartagena UDEC

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Biology

12.8K Views.  Technion - Israel Institute of Technology. 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.

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

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Genetics

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Biolistic Transformation of Yeast Mitochondria for Mutagenetic Analysis

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Author Spotlight: Advancing Techniques and Discoveries in Protein Synthesis and Assembly Through Innovative Mitochondrial Research

This work explains how to transform yeast mitochondria using a biolistic method. We also show how to select and purify the transformants and how to introduce the desired mutation in the target position within the mitochondrial genome.

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

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.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

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.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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

Summary

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Chapters in this video

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Quantitative Immunofluorescence to Measure Global Localized Translation

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Eukaryotic Polyribosome Profile Analysis

An Improved Method to Isolate Mitochondrial Contact Sites

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA