Protein Import into Mitochondria

小行星9418257 该提议涉及蛋白质共翻译导入线粒体的主题。 这是一个至关重要的问题，因为“教科书”观点是蛋白质输入线粒体是通过翻译后输入（从未附着到靶向膜的多核糖体释放肽）发生的。 这一观点源于高知名度的研究，该研究可能对体外输入反应与体内情况的依赖程度不合理。 这项备受瞩目的研究表明，翻译后输入可以在体外发生，无论它是否在体内发生，排除共翻译机制，是这个建议所解决的问题。 提出的实验研究线粒体前体蛋白的性质，可能会影响他们的合成和进口的耦合，并探讨胞质伴侣蛋白的功能，以及其他胞质组分的进口反应。 在与前体蛋白质结构相关的实验中，将采用基因融合技术来产生各种人工蛋白质，这些人工蛋白质将在体外和体内输入反应中进行研究。 线粒体前序列的影响和整体前体结构的相对稳定性将被检查，以确定前体蛋白的特性，影响其能力被导入的共翻译或翻译后机制。 此外，将进行体外输入反应，以评价线粒体生物发生中其他胞质组分的参与。 将使用标准生物化学程序鉴定和分离这些组分。 从拟议的研究中获得的结果预计将大大增加我们对蛋白质输入线粒体和其他细胞器的理解。 所提出的大部分实验旨在探索线粒体生物合成与其他细胞现象的整合，如胞质蛋白质合成，翻译速率，以及前体蛋白和胞质组分之间的相互作用。 这是一个重要的步骤，在查看进口过程中的整体复杂性的活胞质溶胶的背景下，并将补充其他工作领域中的过程往往被研究得非常详细，但往往在隔离的其他细胞反应。 ***

9418257 Verner This proposal addresses the topic of cotranslational import of proteins into mitochondria. This is a question of fundamental importance, since the "textbook" view is that protein import into mitochondria occurs via post-translation import (release of peptide from polysome unattached to the targeted membrane). This view has resulted from high profile research which places perhaps an unwarranted degree of reliance on in vitro import reactions vs the in vivo situation. This high profile research has shown that post translational import can occur in vitro, whether it does occur in vivo, to the exclusion of the co-translational mechanisms, is the question addressed by this proposal. The experiments proposed examine the properties of mitochondrial precursor proteins that may influence the coupling of their synthesis and import and explore the function of cytosolic chaperone proteins, as well as other cytosolic components in the import reaction. In experiments related to the structure of precursor proteins, gene fusion technology will be employed to generate various artificial proteins that will be studied in both in vitro and in vivo import reacitons. The effect of mitochondrial presequences and the relative stability of overall precursor structure will be examined in order to determine the characteristics of precursor proteins that influence their ability to be imported by either a cotranslational or posttranslational mechanism. In addition, in vitro import reactions will be performed to evaluate the involvement of other cytosolic components in mitochondrial biogenesis. Such components will be identified and isolated using standard biochemical procedures. %%% Results obtained from the proposed studies are anticipated to add significantly to our understanding of protein import into mitochondria and other organelles. The bulk of the proposed experiments are designed to explore the integration of mitochondrial biogenesis with other cellular phenomena such as cytosolic protein synthesis, translation rates, and interactions between precursor proieins and cytosolic components. This is an important step in viewing the import process in the context of the overall complexity of the living cytosol and will be complementary to other work in the field in which the process tends to be studied in great detail but often in isolation of other cellular reactions. ***