Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.

植物具有两种类型的内共生细胞器，叶绿体和线粒体。过境肽和前序列分别作为信号序列，分别特异性导入叶绿体和线粒体。但是，这些高度相似的信号序列如何赋予蛋白质导入特异性仍然难以捉摸。在这里，我们显示线粒体或叶绿体特异性导入涉及两个不同的步骤，即特异性确定和跨包膜转运，这分别由转运肽和序列的N端区域和功能上可互换的C端区域介导。在该序列的N-末端区域中具有多个精氨酸和疏水序列基序的结构域被鉴定为线粒体特异性因子。该结构域的存在和其他常见靶向信号的N末端区域中精氨酸残基的缺失分别赋予蛋白质导入线粒体和叶绿体的特异性。叶绿体输入受体AtToc159也有助于确定叶绿体输入特异性。我们提出，通过在N末端区域分别存在和不存在多个精氨酸和疏水序列基序，共同祖先序列被功能化为线粒体和叶绿体特异性信号序列。