A minimal peptide sequence that targets fluorescent and functional proteins into the mitochondrial intermembrane space
A minimal peptide sequence that targets fluorescent and functional proteins into the mitochondrial intermembrane space
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DOI：
10.1021/cb600492a
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发表时间：
2007-03-01
影响因子：
4
通讯作者：
Umezawa, Yoshio
中科院分区：
文献类型：
--
作者：
Ozawa, Takeaki;Natori, Yutaka;Umezawa, Yoshio
基于蛋白质的荧光和功能探针被广泛用于各种生物分子的实时可视化、纯化和调控。基于蛋白质的探针通常可以通过特定的短肽序列靶向真核细胞的亚细胞区室。然而，很少有人知道的序列，目标探针进入线粒体膜间隙（IMS）。为了识别IMS靶向序列，我们开发了一种简单的遗传筛选方法来区分位于IMS中的蛋白质与线粒体基质中的蛋白质，从而揭示IMS靶向的最小必需序列。随机突变IMS定位的蛋白质Smac/DIABLO，并分析每个突变体的线粒体定位。我们发现Ala-Val-Pro-Ile的四个残基是IMS定位所需的，并且与基质靶向信号融合的这四个残基的序列足以将Smac/DIABLO靶向IMS。该序列被证明可以很容易地将三种不同的感兴趣的蛋白质定向到IMS，这将为阐明IMS在活细胞中的功能开辟途径。
Protein-based fluorescent and functional probes are widely used for real-time visualization, purification, and regulation of a variety of biological molecules. The protein-based probes can generally be targeted into subcellular compartments of eukaryotic cells by a particular short peptide sequence. Little is known, however, about the sequence that targets probes into the mitochondrial intermembrane space (IMS). To identify the IMS-targeting sequence, we developed a simple genetic screening method to discriminate the proteins localized in the IMS from those in the mitochondrial matrix, thereby revealing the minimum requisite sequence for the IMS targeting. An IMS-localized protein, Smac/DIABLO, was randomly mutated, and the mitochondrial localization of each mutant was analyzed. We found that the four residues of Ala-Val-Pro-Ile are required for IMS localization, and a sequence of these four residues fused with matrix-targeting signals is sufficient for targeting the Smac/DIABLO into the IMS. The sequence was shown to readily direct three dissimilar proteins of interest to the IMS, which will open avenues to elucidating the functions of the IMS in live cells.