Henderson Matthew P A, Hwang Yeen Ting, Dyer John M, Mullen Robert T, Andrews David W
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8N 3Z5.
Biochem J. 2007 Feb 1;401(3):701-9. doi: 10.1042/BJ20060990.
The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002-3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.
由尾锚序列靶向定位于膜的蛋白质的正确亚细胞定位的分子机制目前还不清楚。此前，我们发现油桐（Vernicia (Aleurites) fordii）的两种细胞色素b5（Cb5）尾锚亚型在体内和体外都能特异性靶向内质网（ER）膜【Hwang, Pelitire, Henderson, Andrews, Dyer和Mullen（2004年），《植物细胞》16卷，3002 - 3019页】。在本研究中，我们检测了各种油桐Cb5融合蛋白和截短突变体在体外对纯化的细胞内膜的靶向作用，以评估带电荷的C末端序列（CTS）在靶向特定膜中的重要性。从油桐Cb5蛋白中去除CTS后，其能有效结合内质网和线粒体。细胞器竞争、脂质体结合和膜蛋白水解实验结果表明，去除CTS会导致油桐Cb5蛋白自发插入脂质双层。我们的结果表明，植物Cb5蛋白的CTS通过防止自发插入错误的亚细胞膜来提供内质网特异性。
