MOLECULAR MECHANISMS OF V-ATPASES: ASSEMBLY, BIOGENESIS, REGULATION, AND FUNCTION

摘要 囊泡型或液泡型三磷酸腺苷酶(V-ATPase)是由多组分的ATP驱动的质子 泵由具有ATPase活性的V1复合体和用于质子转移的Vo复合体组成 膜。V-ATPase在细胞内小泡、细胞器和细胞内的酸化过程中起着重要作用 细胞外环境，对维持内小体、溶酶体和细胞内的pH平衡至关重要。 高尔基体存在于所有真核细胞中。哺乳动物的V-ATPase缺陷是胚胎致死的，而V-ATPase功能障碍 V-ATPase与多种疾病有关，包括微生物感染、肾小管酸中毒、 骨质疏松症、感觉神经性耳聋、神经退行性疾病和癌症。尽管具有以下关键功能 V-ATPase，我们对哺乳动物的生物发生、组装、调节和信号转导的了解有限 V-ATPase。研究哺乳动物V-ATPase的一个主要挑战是，纯的复合体很难 获取用于生化和生物物理实验的材料。我们开发了一种创新的方法来提纯大量的 将人的V-ATPase直接从细胞转化为同质。我们的初步低温电子显微镜(CRYO-EM) 人类V-ATPase的结构在高达3.1°的分辨率下显示出三种功能状态，并具有所有已知的亚基， 它们共同代表了迄今为止最完整的V-ATPase机制模型。我们的研究显示， 哺乳动物的V-ATPase由蛋白质、多糖、糖脂和脂类组成。因此，我们定义了V- ATPase是一种糖蛋白脂复合体。我们的研究为全面了解 V-ATPase的生物发生、组装、调节和信号转导。在我们以前工作的基础上，我们将补充 用生化和功能分析、酵母遗传学和质谱学确定冷冻-EM结构 分析以解决该领域的基本问题，包括糖脂在V-ATPase中的作用 组装和功能，可逆组装对V-ATPase的调节，质子的详细机制 转移，以及V-ATPase介导的细胞信号转导机制。这一项目的完成不仅将 提供关于V-ATPase组装、调节和信号传递的概念创新，但也激励 V-ATPase相关疾病的新治疗策略。

Summary Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps consisting of a V1 complex that possesses ATPase activity and a Vo complex for proton transfer across the membrane. V-ATPases play important roles in the acidification of intracellular vesicles, organelles, and the extracellular milieu, and are essential for maintaining the pH homeostasis of endosomes, lysosomes, and the Golgi apparatus in all eukaryotic cells. V-ATPase deficiency in mammals is embryonic lethal, and malfunction of V-ATPases is associated with numerous diseases, including microbial infection, renal tubular acidosis, osteoporosis, sensorineural deafness, neurodegenerative diseases, and cancer. Despite the critical functions of V-ATPases, we have limited understanding on the biogenesis, assembly, regulation, and signaling of mammalian V-ATPases. A major challenge in studying mammalian V-ATPases is that the pure complexes are difficult to obtain for biochemical and biophysical experiments. We developed an innovative method to purify large amounts of human V-ATPase to homogeneity directly from cells. Our preliminary cryo-electron microscopy (cryo-EM) structures of human V-ATPases show three functional states at up to 3.1 Å resolution and with all known subunits, which together represent the most complete mechanistic model of V-ATPase to date. Our study revealed that mammalian V-ATPases are composed of proteins, glycans, glycolipids, and lipids. Therefore, we defined the V- ATPase as a glycoproteolipid complex. Our study opened up the field for comprehensively understanding the biogenesis, assembly, regulation, and signaling of V-ATPases. Based on our prior work, we will complement cryo-EM structure determination with biochemical and functional assays, yeast genetics, and mass spectrometry analysis to address fundamental questions in the field, including the roles of glycolipids in the V-ATPase assembly and function, the regulation of V-ATPases by reversible assembly, the detailed mechanism of proton transfer, and the mechanisms of V-ATPase mediated cell signaling. The completion of this project will not only provide conceptual innovations regarding the V-ATPases assembly, regulation, and signaling, but also inspire new therapeutic strategies for treating V-ATPase-related diseases.