Aksnes Henriette, Goris Marianne, Strømland Øyvind, Drazic Adrian, Waheed Qaiser, Reuter Nathalie, Arnesen Thomas
From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.
the Computational Biology Unit, Department of Informatics, University of Bergen, N-5020 Bergen, and.
J Biol Chem. 2017 Apr 21;292(16):6821-6837. doi: 10.1074/jbc.M116.770362. Epub 2017 Feb 14.
α-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, assays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C terminus was unstructured in solution and only folded into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 toward membranes containing the phosphatidylinositol PI(4)P, thus possibly explaining the primary residency of Naa60 at the PI(4)P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.
α-乙酰转移酶60（Naa60或NatF）最近被鉴定为一种非常规的N端乙酰转移酶（NAT），因为它定位于细胞器，特别是高尔基体，并且优先乙酰化跨膜蛋白的N端。这一发现挑战了N端乙酰化是一种与共翻译核糖体相关过程的主流观点，并为负责这种日益被认可的蛋白质修饰的酶提出了一种新的作用机制。对Naa60的晶体学研究未能解析负责细胞器定位的Naa60 C末端尾巴。在这里，我们结合建模、实验和细胞定位研究来探究Naa60的二级结构和膜相互作用能力。结果表明，Naa60是一种外周膜蛋白。Naa60 C末端的两个两亲性螺旋通过疏水和静电相互作用相对于脂质双层以平行位置直接结合膜。对应于C末端的肽在溶液中无结构，仅在脂质体存在下折叠成α螺旋构象。计算建模和细胞突变分析表明，两个α螺旋的疏水面对膜定位至关重要。此外，我们发现Naa60对含有磷脂酰肌醇PI(4)P的膜具有强烈且特异性的结合偏好，这可能解释了Naa60在富含PI(4)P的高尔基体中的主要驻留位置。总之，我们已经确定了胞质Naa60锚定到高尔基体的模式，最有可能发生在翻译后，并特别促进许多跨膜蛋白的翻译后N端乙酰化。
