Studying Arabidopsis Envelope Protein Localization and Topology Using Thermolysin and Trypsin Proteases
Studying Arabidopsis Envelope Protein Localization and Topology Using Thermolysin and Trypsin Proteases
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DOI：
10.1007/978-1-61779-234-2_21
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Froehlich, John
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作者：
Froehlich, John
叶绿体是新陈代谢中重要的细胞器，在植物细胞中执行许多基本功能。叶绿体可以细分为六个不同的亚室，蛋白质可能最终以这些亚室为靶标。这些亚室被定义为外被膜(OEM)、内被膜(EM)、类囊体膜，以及膜间间隙(IMS)、基质和类囊体腔三个水中亚室。在细胞生物学中，蛋白质靶向于叶绿体包膜的过程仍然是一个具有挑战性的问题。我们对蛋白质靶向OEM的了解非常有限，而将膜蛋白靶向IEM似乎至少利用了两种靶向途径，即停止转移途径和保守的分选(或进口后)途径。此外，一旦膜蛋白到达包膜，我们对其如何实现最终拓扑的了解仍然有限。一种可以用来确定包膜蛋白的拓扑结构的方法是应用“双酶”策略。这一方法包括几个步骤：首先，进行体外进口分析；其次，使用热裂解酶和胰蛋白酶进行“双酶”保护分析；最后，分离和分析叶绿体亚细胞级分(即总膜和可溶部分)。通过使用这种多步骤方法，人们可以获得关于OEM或IEM蛋白质的最终拓扑的关键信息。同样，“双酶”方法可能有助于阐明膜蛋白在插入被膜之前可能利用的靶向途径。
Chloroplasts are metabolically important organelles that perform many essential functions within plant cells. The chloroplasts can be subdivided into six distinct sub-compartments to which a protein may be ultimately targeted. These sub-compartments are defined as the outer envelope membrane (OEM), the inner envelope membrane (EM), the thylakoid membrane, and three aqueous sub-compartments the intermembrane space (IMS), the stroma, and the thylakoid lumen. The process by which proteins are targeted to the chloroplastic envelope membrane remains a challenging question in cell biology. Our understanding of protein targeting to the OEM is very limited, whereas targeting of membrane proteins to the IEM appears to utilize at least two targeting pathways called the stop-transfer and the conservative sorting (or post-import) pathways. Furthermore, once a membrane protein arrives at the envelope membrane, our understanding of how it achieves its final topology remains limited. One method that can be used to determine the topology of an envelope membrane protein is to apply the "dual protease" strategy. This approach involves several steps: first, performing an in vitro import assay; second, applying a "dual protease" protection assay using thermolysin and trypsin; and finally, isolating and analyzing chloroplastic subcellular fractionations (i.e., total membrane and soluble fractions). By using this multistep approach, one can gain critical information regarding the final topology of an OEM or IEM protein. Likewise, the "dual protease" approach may help in elucidating the possible targeting pathway that a membrane protein utilizes prior to its insertion into the envelope membrane.