Evidence for Amino Acid Snorkeling from a High-Resolution, In Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane.
Evidence for Amino Acid Snorkeling from a High-Resolution, In Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane.
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DOI：
10.1534/genetics.116.196428
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发表时间：
2017-02
期刊：
影响因子：
3.3
通讯作者：
Dunn CD
中科院分区：
文献类型：
--
作者：
Keskin A;Akdoğan E;Dunn CD
通过羧基末端尾锚（TA）定位于线粒体的蛋白质在细胞凋亡、线粒体动力学和线粒体蛋白质输入中发挥作用。为了揭示可能对线粒体靶向重要的TA的特征，我们将注意力集中在酿酒酵母Fis1蛋白的TA上。具体来说，我们产生了一个库的Fis1p TA变体融合的Gal4转录因子，然后，使用下一代测序，揭示了Fis1p TA突变抑制膜插入，并允许Gal4p在细胞核中的活性。通过我们的全局分析，我们随后分析了单个Fis1p TA突变体定位于线粒体的能力。我们的研究结果表明，膜相关域的Fis1p TA可能是二分的性质，我们遇到的证据表明，带正电荷的补丁在羧基末端的Fis1p是所需的膜插入和细胞器特异性。此外，延长或缩短Fis1p TA最多三个氨基酸并没有抑制线粒体靶向，反对TA长度指导TA插入不同细胞器的模型。最重要的是，带正电荷的残基是更可接受的在膜相关领域的Fis1p TA内的几个位置比带负电荷的残基。这些发现，出现在第一个高分辨率分析的细胞器靶向序列的深度突变扫描，提供了强有力的，在体内的证据表明，赖氨酸和精氨酸可以“浮潜”，或成为稳定地纳入脂质双层内放置在膜界面的侧链的终端电荷。
Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. To reveal characteristics of TAs that may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. Specifically, we generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then, using next-generation sequencing, revealed which Fis1p TA mutations inhibited membrane insertion and allowed Gal4p activity in the nucleus. Prompted by our global analysis, we subsequently analyzed the ability of individual Fis1p TA mutants to localize to mitochondria. Our findings suggest that the membrane-associated domain of the Fis1p TA may be bipartite in nature, and we encountered evidence that the positively charged patch at the carboxyl terminus of Fis1p is required for both membrane insertion and organelle specificity. Furthermore, lengthening or shortening of the Fis1p TA by up to three amino acids did not inhibit mitochondrial targeting, arguing against a model in which TA length directs insertion of TAs to distinct organelles. Most importantly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings, emerging from the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning, provide strong, in vivo evidence that lysine and arginine can “snorkel,” or become stably incorporated within a lipid bilayer by placing terminal charges of their side chains at the membrane interface.