Department of Botany, University of Wisconsin-Madison, Madison, WI 53706, USA.
J Exp Bot. 2017 Nov 2;68(18):5029-5043. doi: 10.1093/jxb/erx318.
Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms.
为了建立和维持这些细胞器特有的高度分化的膜，必须将输入叶绿体的膜蛋白准确地靶向。关于参与的靶向信息或途径知之甚少，特别是对于具有多个跨膜结构域的蛋白质。我们研究了叶绿体中两个 SEC 转运体的 SCY 成分的靶向。SCY1 和 SCY2 具有相似的、高度保守的结构，有 10 个跨膜结构域，但靶向不同的膜：类囊体和内囊体。我们使用原生质体转染和共焦显微镜成像测定法结合结构域交换方法来研究分选途径，并鉴定这些蛋白质中的重要靶向元件。我们表明，SCY1 的 N 端区域包含靶向决定因素，允许 SCY1 被招募到信号识别颗粒途径。此外，用 SCY2 的 N 端区域替代 SCY1 的 N 端区域会导致 SCY2 从内囊体中移位。包含跨膜结构域 3 和 4 的 SCY2 区域是定位于内囊体所必需的，并且可以作为膜锚，通过停止转移或进口后机制增强其他跨膜结构域的整合。
