Evidence for amino acid snorkeling from a high-resolution, in vivo analysis of Fis1 tail anchor insertion at the mitochondrial outer membrane

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.

由羧基末端尾锚定（carboxyl-terminal tail anchor, TA）序列定位于线粒体的蛋白质，参与细胞凋亡、线粒体动态调控及线粒体蛋白导入过程。为揭示可能在线粒体靶向过程中发挥关键作用的尾锚定序列特征，本研究聚焦酿酒酵母（Saccharomyces cerevisiae）Fis1蛋白的尾锚定序列。具体而言，我们构建了与Gal4转录因子（Gal4 transcription factor）融合的Fis1p尾锚定序列变异体文库，随后借助下一代测序技术，筛选出可抑制膜插入并使Gal4p在细胞核内发挥活性的Fis1p尾锚定序列突变体。基于全局分析结果的提示，我们后续又针对单个Fis1p尾锚定序列突变体的线粒体靶向能力展开了分析。研究结果表明，Fis1p尾锚定序列的膜结合结构域可能具有二元结构特征；同时我们获得的实验证据显示，Fis1蛋白羧基末端的正电荷聚集区，对于膜插入过程与细胞器靶向特异性均不可或缺。此外，将Fis1p尾锚定序列延长或缩短至多3个氨基酸残基，并未影响其线粒体靶向能力，这与“尾锚定序列长度决定其靶向不同细胞器的插入过程”的模型相悖。尤为关键的是，在Fis1p尾锚定序列的膜结合结构域内的多个位点，带正电的氨基酸残基相比带负电的氨基酸残基更易被耐受。本研究通过深度突变扫描（deep mutational scanning）首次完成了细胞器靶向序列的高分辨率分析，所得发现为“赖氨酸与精氨酸可通过将侧链末端电荷定位于膜界面，从而稳定整合至脂质双分子层中的所谓"snorkel"现象”提供了强有力的体内实验证据。