Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Room E5132, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Traffic. 2011 Sep;12(9):1124-38. doi: 10.1111/j.1600-0854.2011.01232.x. Epub 2011 Jul 7.
Trafficking of soluble proteins to the apicoplast in Plasmodium falciparum is determined by an N-terminal transit peptide (TP) which is necessary and sufficient for apicoplast import. Apicoplast precursor proteins are synthesized at the rough endoplasmic reticulum, but are then specifically sorted from other proteins in the secretory pathway. The mechanism of TP recognition is presently unknown. Apicoplast TPs do not contain a conserved sequence motif; therefore, we asked whether they contain an essential structural motif. Using nuclear magnetic resonance to study a model TP from acyl carrier protein, we found a short, low-occupancy helix, but the TP was otherwise disordered. Using an in vivo localization assay, we blocked TP secondary structure by proline mutagenesis, but found robust apicoplast localization. Alternatively, we increased the helical content of the TP through mutation while maintaining established TP characteristics. Apicoplast import was disrupted in a helical mutant TP, but import was then restored by the further addition of a single proline. We conclude that structure in the TP interferes with apicoplast import, and therefore TPs are functionally disordered. These results provide an explanation for the amino acid bias observed in apicoplast TPs.
疟原虫中可溶性蛋白向顶质体的转运由一个 N 端转运肽（TP）决定，该转运肽对于顶质体的输入是必要且充分的。顶质体前体蛋白在粗面内质网上合成，但随后在分泌途径中与其他蛋白特异性分拣。目前尚不清楚 TP 识别的机制。顶质体 TPs 不包含保守的序列基序；因此，我们询问它们是否包含一个必需的结构基序。我们使用核磁共振研究酰基辅酶 A 蛋白的模型 TP，发现了一个短的、低占据的螺旋，但 TP 其他部分是无序的。使用体内定位测定，我们通过脯氨酸突变阻断 TP 的二级结构，但发现顶质体的定位仍然很强。或者，我们通过突变增加了 TP 的螺旋含量，同时保持了已建立的 TP 特征。在螺旋突变 TP 中，顶质体的导入被破坏，但通过进一步添加一个脯氨酸，导入又得到恢复。我们得出结论，TP 中的结构干扰了顶质体的导入，因此 TPs 在功能上是无序的。这些结果为顶质体 TPs 中观察到的氨基酸偏性提供了一个解释。
