Membrane Targeting and Retargeting of Polarity Proteins

极性蛋白质的膜靶向和重定向 摘要 建立和维持顶端-基底端的极性对上皮组织的完整性和功能至关重要 无论是在正常情况下还是在压力下。一组所谓的极性蛋白质发挥着至关重要的作用 在无脊椎动物和脊椎动物中都具有调节细胞极性的保守功能。其中的一个关键特征 极性蛋白质是指与质膜(PM)或细胞皮质的结合对于它们在体内的作用是至关重要的 功能。除了少数例外，大多数极性蛋白被认为是通过 蛋白质之间的相互作用。潜在的直接相互作用背后可调节的分子机制 PM和大多数极性蛋白质之间的关系长期以来一直难以捉摸。多元基域，富含 带正电荷的Arg和Lyn残基在PM特异性靶向中具有公认的作用，基于其 带负电荷的聚磷脂酰肌醇PI4P和PIP2的静电相互作用 在粉末冶金内表面有浓缩物。然而，几十年来，PM中多碱结构域的功能研究 靶向仅限于少数具有不同功能的蛋白质，并且没有多碱结构域 在任何一种极性蛋白质中都有特征。 我们最近确认LGL是第一个含有进化上保守的和 其与质膜上的PI4P和PIP2的静电结合直接介导的可磷酸化多碱结构域 LGL的PM本地化。在这个提议中，我们已经确定了多个额外的极性蛋白质，以及 作为果蝇和人类基因组中成百上千种蛋白质，也含有潜在的多元 域名。我们将使用果蝇和培养的哺乳动物细胞来研究这一假设 多碱结构域与质膜之间的静电结合是一个关键的可调节分子 控制多个多碱性极性蛋白亚细胞定位的机制。我们将首先 证实多个多碱基蛋白质与PM的静电结合。更重要的是，我们会 研究磷酸化、变构调节和重合蛋白等分子机制 可能控制PM和多碱性极性蛋白之间的直接结合以实现其 极化的亚细胞定位和激活。最后，我们的研究首次强调了低氧 和耗竭ATP通过耗竭而急性和可逆地抑制PM靶向的多碱性结构域蛋白 PI4P和PIP2在PM上，揭示了细胞在以前没有意识到但在生物学上具有重大意义的挑战 在缺氧/缺血后，将多碱性极性蛋白重定向到原始的PM结构域。我们将专注于识别 主动将多碱极性蛋白质重定向至其原始PM结构域的机制。 我们的研究旨在建立一种新的范式，即多元碱基之间如何可调节的结合 结构域和PM是整合蛋白质-蛋白质相互作用的基本机制 极化PM在正常和应激条件下对极性蛋白质的靶向。

Membrane Targeting and Retargeting of Polarity Proteins ABSTRACT Establishing and maintaining apical-basal polarity is essential for epithelial tissue integrity and function under both normal and stressed conditions. A group of so-called polarity proteins play essential and conserved functions in regulating cell polarity in both invertebrates and vertebrates. One key feature of all polarity proteins is that association with plasma membrane (PM) or cell cortex is critical for their in vivo functions. With few exceptions, most polarity proteins are assumed to be localized to PM/cell cortex through protein-protein interactions. Regulatable molecular mechanisms underlying the potential direct interaction between PM and majority of polarity proteins have long been elusive. Polybasic domains that are rich in positively charged Arg and Lyn residues have a well-established role in PM-specific targeting, based on its electrostatic interactions with negatively charged polyphosphoinositides PI4P and PIP2 that are uniquely enriched on the PM inner surface. However, for decades functional studies on polybasic domain in PM targeting have been limited to a small number of proteins of diverse functions and no polybasic domains had been characterized in any of the polarity proteins. We recently identified Lgl as the first polarity protein that contains an evolutionarily conserved and phosphorylatable polybasic domain whose electrostatic binding to PI4P and PIP2 on the PM directly mediate the PM localization of Lgl. In this proposal we have identified that multiple additional polarity proteins, as well as hundreds to thousands of proteins in Drosophila and human genomes, also contain potential polybasic domains. We will use both Drosophila and cultured mammalian cells to investigate the hypothesis that electrostatic binding between polybasic domain and plasma membrane serves a key regulatable molecular mechanism for controlling the subcellular localizations of multiple polybasic polarity proteins. We will first confirm the electrostatic binding of PM by multiple polybasic polarity proteins. More importantly, we will investigate the molecular mechanisms such as phosphorylation, allosteric regulation and coincident protein interactions that may control the direct binding between PM and polybasic polarity proteins to achieve their polarized subcellular localization and activations. Finally, our research highlighted for the first time that hypoxia and depletion of ATP acutely and reversibly inhibit polybasic domain proteins PM targeting through depleting PI4P and PIP2 on the PM, revealing a previously unappreciated but biologically significant challenge for cells to retarget polybasic polarity proteins to original PM domains after hypoxia/ischemia. We will focus on identifying mechanisms that actively direct the retargeting of polybasic polarity proteins to their original PM domains. Our research aims to establish a new paradigm regarding how regulatable binding between polybasic domains and PM serves as a fundamental mechanism to integrate the protein-protein interactions in achieving polarized PM targeting of polarity protein under both normal and stress conditions.