Department Biology I, Plant Sciences, LMU München Martinsried, Germany ; Munich Center for Integrated Protein Science CiPS München, Germany ; Lysando GmbH Regensburg, Germany.
Department Biology I, Plant Sciences, LMU München Martinsried, Germany ; Munich Center for Integrated Protein Science CiPS München, Germany.
Front Plant Sci. 2014 May 28;5:239. doi: 10.3389/fpls.2014.00239. eCollection 2014.
Protein import into chloroplasts relies on specific targeting of preproteins from the cytosol to the organelles and coordinated translocation processes across the double envelope membrane. Here, two complex machineries constitute the so called general import pathway, which consists of the TOC and TIC complexes (translocon at the outer envelope of chloroplasts and translocon at the inner envelope of chloroplasts, respectively). The majority of canonical preproteins feature an N-terminal cleavable transit peptide, which is necessary for targeting and recognition at the chloroplast surface by receptors of TOC, where Toc159 acts as the primary contact site. We identified a non-canonical preprotein without the classical transit peptide, the superoxide dismutase (FSD1), which was then used in chemical crosslinking approaches to find new interaction partners at the outer envelope from pea chloroplasts. In this way we could link FSD1 to members of the Toc159 family in pea, namely psToc132 and psToc120. Using deletion mutants as well as a peptide scanning approach we defined regions of the preprotein, which are involved in receptor binding. These are distributed across the entire sequence; however the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the non-canonical targeting information and recognition by a specific receptor this preprotein follows a similar way across the envelope as the majority of plastid preproteins.
蛋白质向叶绿体的输入依赖于前体蛋白从细胞质到细胞器的特异性靶向和协调的跨双层膜转运过程。在这里，两个复杂的机制构成了所谓的一般导入途径，它由 TOC 和 TIC 复合物（叶绿体的外膜转位体和叶绿体的内膜转位体）组成。大多数规范的前体蛋白都具有可切割的 N 端转运肽，这对于在叶绿体表面被 TOC 受体识别和靶向是必要的，其中 Toc159 作为主要接触位点。我们鉴定了一种没有经典转运肽的非规范前体蛋白，即超氧化物歧化酶（FSD1），然后用化学交联方法从豌豆叶绿体的外膜中寻找新的相互作用伙伴。通过这种方式，我们可以将 FSD1 与豌豆中的 Toc159 家族成员 psToc132 和 psToc120 联系起来。使用缺失突变体和肽扫描方法，我们定义了参与受体结合的前体蛋白区域。这些区域分布在整个序列中；然而，极端的 N 端以及 C 端近端结构域对于靶向和输入是必需的。在进入质体的过程中，FSD1 与一般导入途径的成分结合，这意味着尽管前体蛋白具有非规范的靶向信息和由特定受体识别，但它沿着与大多数质体前体蛋白相似的途径穿过包膜。
