Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Biozentrum, Halle (Saale), Germany.
PLoS One. 2019 Mar 13;14(3):e0213364. doi: 10.1371/journal.pone.0213364. eCollection 2019.
We report a systematic analysis of chloroplast high-molecular mass protein complexes using a combination of native gel electrophoresis and absolute protein quantification by MSE. With this experimental setup, we characterized the effect of the tic56-3 mutation in the 1-MDa inner envelope translocase (TIC) on the assembly of the chloroplast proteome. We show that the tic56-3 mutation results in a reduction of the 1-MDa TIC complex to approximately 10% of wildtype levels. Hierarchical clustering confirmed the association of malate dehydrogenase (MDH) with an envelope-associated FtsH/FtsHi complex and suggested the association of a glycine-rich protein with the 1-MDa TIC complex. Depletion of this complex leads to a reduction of chloroplast ATPase to approx. 75% of wildtype levels, while the abundance of the FtsH/FtsHi complex is increased to approx. 140% of wildtype. The accumulation of the major photosynthetic complexes is not affected by the mutation, suggesting that tic56-3 plants can sustain a functional photosynthetic machinery despite a significant reduction of the 1-MDa TIC complex. Together our analysis expands recent efforts to catalogue the native molecular masses of chloroplast proteins and provides information on the consequences of impaired accumulation of the 1-MDa TIC translocase for chloroplast proteome assembly.
我们使用天然凝胶电泳和 MSE 绝对蛋白定量相结合的方法对叶绿体高分子质量蛋白复合物进行了系统分析。通过这种实验设置，我们研究了 1-MDa 内膜转运蛋白（TIC）中的 tic56-3 突变对叶绿体蛋白质组组装的影响。结果表明，tic56-3 突变导致 1-MDa TIC 复合物减少到野生型水平的约 10%。层次聚类证实了苹果酸脱氢酶（MDH）与包膜相关的 FtsH/FtsHi 复合物的关联，并提示甘氨酸丰富蛋白与 1-MDa TIC 复合物的关联。该复合物的耗尽导致叶绿体 ATP 酶减少到野生型水平的约 75%，而 FtsH/FtsHi 复合物的丰度增加到野生型的约 140%。该突变不影响主要光合复合物的积累，表明 tic56-3 植物可以维持功能齐全的光合作用机制，尽管 1-MDa TIC 复合物的积累显著减少。我们的分析扩展了最近对叶绿体蛋白天然分子量进行编目的努力，并提供了关于 1-MDa TIC 转运蛋白积累受损对叶绿体蛋白质组组装的影响的信息。
