Name: isolation of mrnas associated with yeast mitochondria to study mechanisms of localized translation
Uploaded: 2014-03-14T17:50:00
Description: Watch this Scientific Journal Video about Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation at JoVE.com

我们感谢博士。埃雷兹埃利亚胡，丹尼尔·梅拉梅德，Ophry松树和多伦拉帕波特建立这个协议的过程中帮助和意见。这项工作是由ISF（批准号1193至1109年）的资助。

1。线粒体提纯权衡细胞沉淀。大约0.6克是用100ml的细胞获得。 <ol><li>增长100-150毫升酵母细胞的OD 600 = 1-1.5 30 °C于一个非发酵生长介质中，例如半乳糖基生长培养基中，以富集线粒体。 </li><li>离心细胞在3000×g离心5分钟，在室温下，弃上清。 </li><li>用双蒸水，离心再次洗涤沉淀。弃上清。 </li><li>每0.5 G细胞的悬浮颗粒1毫升缓冲液的DTT。它治疗的细胞与一种...

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U., Fisher, P. R. <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+nuclear-encoded+mitochondrial+proteins:+a+cotranslational+perspective.">Import of nuclear-encoded mitochondrial proteins: a cotranslational perspective.</a> Int. Rev. Cell Biol. 273, 49-68 (2009).</li><li>Eliyahu, E., Melamed, D., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+RNA+extracted+from+isolated+mitochondria.">Genome-wide analysis of RNA extracted from isolated mitochondria.</a> Methods Mol. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tracking+of+single+mRNA+particles+in+Saccharomyces+cerevisiae+using+a+double-helix+point+spread+function.">Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 17864-17871 (2010).</li><li>Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+RNA+distribution+and+dynamics+in+living+cells.">Imaging intracellular RNA distribution and dynamics in living cells.</a> Nat. Methods. 6, 331-338 (2009).</li><li>Garcia, M., Darzacq, X., Devaux, F., Singer, R. H., Jacq, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+transcriptomics.">Yeast mitochondrial transcriptomics.</a> Methods Mol. Biol. 372, 505-528 (2007).</li><li>Reinders, J., Sickmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+yeast+mitochondria.">Proteomics of yeast mitochondria.</a> Methods Mol. Biol. 372, 543-557 (2007).</li><li>Meisinger, C., Sommer, T., Pfanner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Saccharomcyes+cerevisiae+mitochondria+devoid+of+microsomal+and+cytosolic+contaminations.">Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations.</a> Anal. Biochem. 287, 339-342 (2000).</li><li>Glick, B. S., Pon, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+highly+purified+mitochondria+from+Saccharomyces+cerevisiae.">Isolation of highly purified mitochondria from Saccharomyces cerevisiae.</a> Methods Enzymol. 260, 213-223 (1995).</li><li>Boldogh, I. R., Pon, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+subfractionation+of+mitochondria+from+the+yeast+Saccharomyces+cerevisiae.">Purification and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae.</a> Methods Cell Biol. 80, 45-64 (2007).</li><li>Rieder, S. E., Emr, S. D., Bonifacino, J. 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在成像技术的进步已经产生了高解析度的工具来研究mRNA的定位。今天，人们可以在毫秒时间尺度23-26测量甚至单个mRNA分子的移动。然而，传统的生化方法，如上面所描述的，也都是有用的资讯和是优选的情况。生化隔离允许的mRNA和蛋白质的大型剧目的净化，因此是最好的全基因组的研究。在单个隔离，可以得到足够的mRNA水平，以允许数以千计的基因的表征，无论是由DNA微阵列或RNA-SEQ <su...

真核细胞被组织在具有特定功能的不同的隔间。为了实现它的功能，每个隔间中包含了一套独特的蛋白质，是其活性所必需的。通过这些蛋白质接近他们的隔间一种可能的机制是通过本地化翻译1,2。在这个过程中，蛋白质是由核糖体和mRNA的是位于那里合成了其目的地。其中本地化翻译的可能优点是增加蛋白质定位的效率，减少需要的蛋白质分子伴侣，并使特定地点的调节机制。另外，局部的mRNA和核糖体可以是机器翻译的一个僻静的水库在细胞应激，当一般翻译被抑制的案件。 线粒体在最近几年成为研究的本地化翻译中心模型。大多数线粒体蛋白质编码在细胞核中，翻译在胞质溶胶中，并导入到细胞器。各行的证据表明，许多这些蛋白质是通过一个本地翻译过程中产生的。最初，电子显微镜和生物化学分馏研究发现线粒体3-5相关核糖体。这些研究的地方，然后通过特定的mRNA，被发现要导入只在共翻译方式6,7的工作证实在体内 。与线粒体的mRNA关联的全基因组的研究显示，mRNAs的一个显著馏分被定位于线粒体附近8-10。一些这些mRNA的进一步特征在于在体内荧光的方法，如鱼或MTAG 9,11。该协会的一个直接的解释是，这些mRNA作为本地化翻译的模板。 由这些mRNA接近线粒体的机制是未知的。非编码区域（最significantly 3的&#39;非编码区）被证明是参与mRNA联想到线粒体12。这些领域有可能成为一个结合位点的RNA结合蛋白介导的运输。研究表明酵母，蛋白质的PUM系列（Puf3）的成员支持与线粒体8,13 mRNA的关联。对于Puf3，这是基于对其他家庭成员函数的一个似是而非的作用，是抑制翻译而表达的是途中 14。因此，可能的mRNA在非翻译状态被输送，由与非编码区相互作用的RNA结合蛋白。此外，庞大的身躯的工作表明，当蛋白质被合成的交通工具发生。特别是，翻译抑制剂被证明影响mRNA的结社8,13。此外，翻译等功能的AUG，线粒体定位序列（MTS），或ORF地区被证明，以协助定位8,11,15。热休克蛋白70家族的蛋白质通道aperone和蛋白质受体在线粒体外膜也显示，支持mRNA的关联，进一步暗示编码蛋 ​​白的功能是mRNA定位16重要。这是一个模型，其中核糖体蛋白新兴用作靶向的mRNA-核糖体-蛋白质复合物的线粒体17识别元件是一致的。 附近线粒体本地化翻译，研究了各种方法，包括电子显微镜（可视化核糖体）3，鱼9，绿色的RNA（检测特定mRNA的）11，和生化分馏（检测RNA和核糖体）10,18。而前者的方法检测定位在体内 ，并且可以允许传输动力学的可视化，后者允许检测在单次实验中多个不同的mRNA。此外，用于生化分离的编码或非编码的域不需要是人tered，因此其具体的作用进行评估。生化分馏已成功使用多年，对许多不同的细胞区室的隔离。它的校长和局限性已经非常成熟，并且可以很容易地修改为不同目的的现有协议。必要的仪器在许多实验室的标准，因此它通常是选择的第一个方法用于研究细胞内的定位。我们描述，这对于mRNA的分离优化，而核糖体与线粒体相关的协议。因此，该协议是最佳的学习参与线粒体附近本地化翻译的因素。

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			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
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			<td height="21" style="height:21px;">BactoPeptone</td>
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			<td height="21" style="height:21px;">Galactose</td>
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			<td style="width:563px;">Do not autoclave Galactose</td>
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			<td height="60" style="height:60px;">Growth medium</td>
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			<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>
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			<td height="21" style="height:21px;">0.1 M TrisHCl pH9.4</td>
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			<td height="21" style="height:21px;">30 mM TrisHCl pH7.6</td>
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			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
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			<td height="21" style="height:21px;">&#160;10 mM DTT Buffer</td>
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			<td style="width:563px;">0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time</td>
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			<td height="21" style="height:21px;width:216px;">1.2 M Sorbitol</td>
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			<td height="25" style="height:25px;width:216px;">4 mM KH2PO4</td>
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			<td height="25" style="height:25px;width:216px;">16 mM K2HPO4</td>
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			<td height="21" style="height:21px;width:216px;">0.2&#956;m filter</td>
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			<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>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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

大多数线粒体蛋白质编码的细胞核和需要被导入到细胞器。而蛋白质在接近线粒体合成的，可能会出现导入。支持这种可能性是从最近的研究，在其中有许多mRNA的编码线粒体蛋白质被证明是定位于线粒体附近而得。连同早前与外膜核糖体协会的示威活动，这些结果表明本地化的翻译过程。这种本地化的翻译可能会提高进口效率，提供了独特的调控位点，并尽量减少异位表达的情况下。多种方法已被用于表征介导的本地化翻译的因素和因素。在这些标准是亚细胞分级差速离心。这个协议在单个程序的mRNA，核糖​​体和蛋白质的分离的优点。然后这些可以表征通过各种分子和双辉化工方法。此外，转录组学和蛋白质组学的方法可以应用到所得到的材料，从而使基因组范围的见解。酵母的利用率作为模式生物对这些研究具有速度快，成本和简单性的优点。此外，先进的基因工具和可用的缺失株便于核实候选人的因素。

该协议允许从胞浆成分含有线粒体组分的分离。以测试其成功的最好方法是从不同的隔离措施进行分析，北部和西部的分析（ 图1）的样品。三个参数的隔离质量均来自这些分析。首先，无论在样品中的RNA或蛋白是完整的 - 这些将被检测为不同的频带中进行分析。降级事件将导致额外的，更短的波段或污点的外观。其次，从每一步从输入（ 即总）的信号进行比较的信号提供了?...

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

编码线粒体蛋白质多的mRNA与线粒体外膜有关。我们描述了亚细胞分离过程，旨在酵母线粒体与它相关的基因和核糖体的隔离。这个协议可以被应用到不同的条件下生长的细胞，以揭示mRNA定位的机制，并靠近线粒体本地化的翻译。

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 ...

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Purification of Mitochondria from Yeast Cells

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

使用膜片钳技术研究线粒体的产热能力

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

从田间收集的蚊子中分离出蚊相关病毒

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two ...

相关的mRNA分离与酵母线粒体研究本地化翻译的机制