Linster Eric, Layer Dominik, Bienvenut Willy V, Dinh Trinh V, Weyer Felix A, Leemhuis Wiebke, Brünje Annika, Hoffrichter Marion, Miklankova Pavlina, Kopp Jürgen, Lapouge Karine, Sindlinger Julia, Schwarzer Dirk, Meinnel Thierry, Finkemeier Iris, Giglione Carmela, Hell Ruediger, Sinning Irmgard, Wirtz Markus
Centre for Organismal Studies Heidelberg, Heidelberg University, Heidelberg, 69120, Germany.
Heidelberg University Biochemistry Center, Heidelberg, 69120, Germany.
New Phytol. 2020 Oct;228(2):554-569. doi: 10.1111/nph.16747. Epub 2020 Jul 16.
In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.
在人类和植物中，N 端乙酰化在蛋白质稳态中起着核心作用，影响细胞质中 80%的蛋白质，由五种核糖体相关的 N - 乙酰转移酶（NatA - E）催化。人类还拥有一种与高尔基体相关的 NatF（HsNAA60），它对高尔基体的完整性至关重要。值得注意的是，NAA60 在真菌中不存在，在植物中也未被鉴定出来。在这里，我们通过反向遗传学、全局蛋白质组学、活细胞成像、微量热泳动、圆二色光谱、纳米差示扫描荧光法、内源色氨酸荧光和 X 射线晶体学等方法相结合，在模式植物拟南芥中鉴定并表征了首个质膜锚定的翻译后作用的 N - 乙酰转移酶 AtNAA60。我们证明，AtNAA60 与 HsNAA60 一样，在体内通过其 C 端的α - 螺旋膜锚定定位在膜上，但与 HsNAA60 不同的是，AtNAA60 定位于质膜。AtNAA60 的晶体结构为底物结合、广泛的底物特异性以及基于结构的诱变所探究的催化机制提供了见解。对 NAA60 功能缺失突变体（naa60 - 1 和 naa60 - 2）的表征揭示了 AtNAA60 的一个质膜定位底物以及 NAA60 在高盐胁迫期间的重要性。我们的研究结果为质膜锚定的 N - 乙酰转移酶的植物特异性进化提供了证据，该酶对于适应胁迫至关重要。
