Lesnik Chen, Arava Yoav
Department of Biology, Technion - Israel Institute of Technology.
Department of Biology, Technion - Israel Institute of Technology;
J Vis Exp. 2014 Mar 14(85):51265. doi: 10.3791/51265.
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.
大多数线粒体蛋白是由细胞核编码的，需要导入到该细胞器中。导入可能在蛋白质在线粒体附近合成时发生。近期研究支持了这种可能性，其中许多编码线粒体蛋白的mRNA被证明定位于线粒体附近。连同早期关于核糖体与外膜结合的证明，这些结果表明存在局部翻译过程。这种局部翻译可能提高导入效率，提供独特的调控位点，并将异位表达的情况降至最低。已使用多种方法来表征介导局部翻译的因子和元件。其中标准的方法是通过差速离心进行亚细胞分级分离。该方案的优点是可以在单个步骤中分离mRNA、核糖体和蛋白质。然后可以通过各种分子和生化方法对它们进行表征。此外，转录组学和蛋白质组学方法可以应用于所得材料，从而实现全基因组层面的洞察。将酵母用作此类研究的模式生物具有速度快、成本低和操作简单的优点。此外，先进的遗传工具和可用的缺失菌株有助于验证候选因子。
