The Microalgal Biotechnology Laboratory, The French Associates Institute for Agriculture and Biotechnology, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus, Be'er Sheva, 84990, Israel.
J Phycol. 2020 Dec;56(6):1642-1663. doi: 10.1111/jpy.13063. Epub 2020 Oct 30.
Lipid droplets (LDs) are an organelle conserved amongst all eukaryotes, consisting of a neutral lipid core surrounded by a polar lipid monolayer. Many species of microalgae accumulate LDs in response to stress conditions, such as nitrogen starvation. Here, we report the isolation and proteomic profiling of LD proteins from the model oleaginous pennate diatom Phaeodactylum tricornutum, strain Pt4 (UTEX 646). We also provide a quantitative description of LD morphological ontogeny, and fatty acid content. Novel cell disruption and LD isolation methods, combined with suspension-trapping and nanoflow liquid chromatography coupled to high resolution mass spectrometry, yielded an unprecedented number of LD proteins. Predictive annotation of the LD proteome suggests a broad assemblage of proteins with diverse functions, including lipid metabolism and vesicle trafficking, as well as ribosomal and proteasomal machinery. These proteins provide mechanistic insights into LD processes, and evidence for interactions between LDs and other organelles. We identify for the first time several key steps in diatom LD-associated triacylglycerol biosynthesis. Bioinformatic analyses of the LD proteome suggests multiple protein targeting mechanisms, including amphipathic helices, post-translational modifications, and translocation machinery. This work corroborates recent findings from other strains of P. tricornutum, other diatoms, and other eukaryotic organisms, suggesting that the fundamental proteins orchestrating LDs are conserved, and represent an ancient component of the eukaryotic endomembrane system. We postulate a comprehensive model of nitrogen starvation-induced diatom LDs on a molecular scale, and provide a wealth of candidates for metabolic engineering, with the potential to eventually customize LD contents.
脂滴（LDs）是一种在所有真核生物中都保守的细胞器，由中性脂质核心组成，周围环绕着极性脂质单层。许多微藻物种在氮饥饿等应激条件下积累 LDs。在这里，我们报告了从模式产油的不等鞭毛硅藻菱形藻菌株 Pt4（UTEX 646）中分离和蛋白质组学分析 LD 蛋白。我们还提供了 LD 形态发生和脂肪酸含量的定量描述。新的细胞破碎和 LD 分离方法，结合悬浮捕获和纳米流液相色谱与高分辨率质谱联用，产生了前所未有的 LD 蛋白数量。LD 蛋白质组的预测注释表明存在具有多种功能的广泛组装蛋白，包括脂质代谢和囊泡运输，以及核糖体和蛋白酶体机制。这些蛋白质为 LD 过程提供了机制见解，并为 LD 与其他细胞器之间的相互作用提供了证据。我们首次确定了硅藻 LD 相关三酰甘油生物合成的几个关键步骤。LD 蛋白质组的生物信息学分析表明存在多种蛋白质靶向机制，包括两亲性螺旋、翻译后修饰和易位机制。这项工作与其他菱形藻菌株、其他硅藻和其他真核生物的最新发现相吻合，表明协调 LD 的基本蛋白质是保守的，是真核内体膜系统的古老组成部分。我们在分子水平上提出了氮饥饿诱导的硅藻 LD 的综合模型，并提供了丰富的代谢工程候选物，具有最终定制 LD 含量的潜力。
