Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France.
Université de Bordeaux, CNRS, IBGC, UMR5095, Bordeaux, France.
PLoS Genet. 2023 Aug 16;19(8):e1010848. doi: 10.1371/journal.pgen.1010848. eCollection 2023 Aug.
N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.
多肽的 N 端对于 N 端修饰酶的选择性共翻译募集至关重要。然而，目前尚不清楚特定的 N 端特征是否根据其细胞功能差异调节蛋白质命运。在这项工作中，我们开发了一种在细胞 N 端组中检测功能偏好的计算方法，并在 S. cerevisiae 中鉴定了 200 多个具有特定 N 端特征的基因本体术语。特别是，我们发现线粒体靶向序列（MTS）在位置 2 上强烈且特异性地过表达，已知该位置 2 定义了 N 端乙酰转移酶 NatC 的潜在底物的疏水性残基。我们通过选择性纯化翻译核糖体来验证线粒体前体是 NatC 的共翻译靶标，并发现它们的 N 端特征在 Saccharomycotina 酵母中是保守的。最后，对酵母线粒体蛋白原型中位置 2 的系统诱变证实了其在线粒体蛋白输入中的关键作用。我们的工作强调了 MTS N 端残基的疏水性及其被 NatC 靶向作为定义线粒体蛋白质组的重要特征，为在酵母或人类 NatC 耗尽细胞中观察到的线粒体缺陷提供了分子解释。因此，N 端残基的功能映射有可能支持对蛋白质调节或靶向的新机制的发现。
