Department of Molecular Cell Biology of Plants, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany.
Department of Molecular Cell Biology of Plants, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany; Frankfurt Institute for Advanced Studies, Ruth-Moufang-Straße 1, D-60438 Frankfurt, Germany.
Biochim Biophys Acta Proteins Proteom. 2019 Jun;1867(6):627-636. doi: 10.1016/j.bbapap.2019.01.002. Epub 2019 Jan 3.
The GTPases Toc159 and Toc34 of the translocon of the outer envelope of chloroplasts (TOC) are involved in recognition and transfer of precursor proteins at the cytosolic face of the organelle. Both proteins engage multiple interactions within the translocon during the translocation process, including dimeric states of their G-domains. The units of the Toc34 homodimer are involved in the recognition of the transit peptide representing the translocation signal of precursor proteins. This substrate recognition is part of the regulation of the GTPase cycle of Toc34. The Toc159 monomer and the Toc34 homodimer recognize the transit peptide of the small subunit of Rubisco at the N- and at the C-terminal region, respectively. Analysis of the transit peptide interaction by crosslinking shows that the heterodimer between both G-domains binds pSSU most efficiently. While substrate recognition by Toc34 homodimer was shown to regulate nucleotide exchange, we provide evidence that the high activation energy of the GTPase Toc159 is lowered by substrate recognition. The nucleotide affinity of Toc34 homodimer and Toc159 monomer are distinct, Toc34 homodimer recognizes GDP and Toc159 GTP with highest affinity. Moreover, the analysis of the nucleotide association rates of the monomeric and dimeric receptor units suggests that the heterodimer has an arrangement distinct from the homodimer of Toc34. Based on the biochemical parameters determined we propose a model for the order of events at the cytosolic side of TOC. The molecular processes described by this hypothesis range from transit peptide recognition to perception of the substrate by the translocation channel.
叶绿体外壳体转位通道（TOC）的 GTPasesToc159 和 Toc34 参与细胞器胞质侧前体蛋白的识别和转运。这两种蛋白质在转运过程中都在转位通道内进行多次相互作用，包括其 G 结构域的二聚体状态。Toc34 同源二聚体的单位参与识别代表前体蛋白转运信号的转运肽。这种底物识别是 Toc34 GTP 酶循环调节的一部分。Toc159 单体和 Toc34 同源二聚体分别在 N 端和 C 端区域识别 Rubisco 小亚基的转运肽。通过交联分析对转运肽相互作用的研究表明，两个 G 结构域之间的异二聚体最有效地结合 pSSU。虽然 Toc34 同源二聚体的底物识别被证明可以调节核苷酸交换，但我们提供的证据表明，底物识别降低了 GTPaseToc159 的高活化能。Toc34 同源二聚体和 Toc159 单体的核苷酸亲和力不同，Toc34 同源二聚体以最高亲和力识别 GDP 和 Toc159 GTP。此外，对单体和二聚体受体单元的核苷酸结合速率的分析表明，异二聚体的排列与 Toc34 同源二聚体不同。基于确定的生化参数，我们提出了 TOC 胞质侧事件顺序的模型。该假设描述的分子过程范围从转运肽识别到转运通道对底物的感知。
