Cellular and molecular mechanisms regulating function of a broadly conserved gamete membrane fusogen

项目摘要 我们对生殖细胞(配子)融合的分子机制的理解 在有性繁殖过程中是令人惊讶的有限的。在对单细胞绿藻的研究中， 衣藻和疟疾病原体疟原虫，斯内尔实验室已经表明 广泛保守的配子特异性膜蛋白HAP2(也称为GCS1)是 配子融合过程中的双层合并。HAP2在配子血浆紧密粘连后起作用 膜，只存在于两个配子中的一个配子上(例如，交配型减去配子 衣藻)，因此起单边作用，正如病毒融合蛋白在进入体内时一样 进入宿主细胞。值得注意的是，最近的协作结构/功能研究表明 衣藻HAP2在结构上与许多 包膜病毒，包括登革热和寨卡病毒。通过重组形式的胞外结构域 衣藻HAP2在体外形成三聚体，疏水融合环是 在体内发挥作用，但我们对这类真核生物的分子机制仍然知之甚少。 II任何HAP2生物体配子融合过程中的融合蛋白。内源性三聚体 可能是在配子融合过程中形成的，目前还没有发现，其机制是 在携带HAP2的配子完成之前，限制三聚体形成的未知因素 特异膜与配子配子的膜相互作用。在预赛中 实验中，我发现了一种稳定的低聚物，它只有在配子融合后才出现，并且是 可能是内源性HAP2三聚体。我还确定，与病毒课不同的是 II蛋白，低pH不能引发HAP2寡聚体的形成。相反，含有HAP2的 负交配结构必须与正交配结构上的黏附蛋白FUS1结合， 确保触发只在正确的时间和地点发生。这项研究旨在定义 广泛保守的真核生物功能的分子机制 跨王国生物体中配子融合所必需的膜FusoGen。这个 从这项研究中获得的见解将提高我们对基本的、保守的 配子融合的机制，并有可能产生改进的干扰策略 有性繁殖和危害人类的有机体的传播，包括 毁灭性的疟疾生物疟原虫。在这里，我将测试一种新的HAP2齐聚物的模型 融合后的配子中检测到一种三聚体。我将调查所需的HAP2的结构特征 用于寡聚反应。并且，我将研究依赖于FUS1的机制 触发HAP2结构重排。

Project Summary Our understanding of the molecular mechanisms of fusion between reproductive cells (gametes) during sexual reproduction is surprisingly limited. In studies with the unicellular green alga, Chlamydomonas and the malaria pathogen Plasmodium, the Snell laboratory has shown that the broadly conserved, gamete-specific membrane protein HAP2 (also called GCS1) is essential for bilayer merger during gamete fusion. HAP2 acts after tight adhesion of gamete plasma membranes, and is present on only one of the two gametes (e.g., mating type minus gametes in Chlamydomonas) and therefore functions unilaterally, as do viral fusion proteins during their entry into host cells. Remarkably, recent collaborative structure/function studies demonstrated that Chlamydomonas HAP2 is structurally homologous to the class II fusion proteins of many enveloped viruses, including dengue and Zika. Though recombinant forms of the ectodomain of Chlamydomonas HAP2 form trimers in vitro and the hydrophobic fusion loop is essential for function in vivo, we still know very little about the molecular mechanism of this eukaryotic class II fusion protein during gamete fusion in any HAP2 organism. Endogenous trimers that presumably form during gamete fusion have yet to be detected, and the mechanisms are unknown that restrict triggering of trimer formation until the HAP2-bearing gamete undergoes specific membrane interaction with the membrane of its partner gamete. In preliminary experiments, I have discovered a stable oligomer, which appears only after gamete fusion, and is likely to be the endogenous HAP2 trimer. I have also determined that, unlike with the viral class II proteins, low pH fails to trigger oligomer formation of HAP2. Rather, the HAP2-containing minus mating structure must bind to the adhesion protein FUS1 on the plus mating structure, ensuring that triggering takes place only at the right time and place. This research aims to define the molecular mechanisms that underlie the function of a broadly conserved eukaryotic membrane fusogen that is essential for fusion of gametes in organisms across kingdoms. The insights gained from this study will improve our understanding of fundamental, conserved mechanisms of gamete fusion, and have the potential to yield improved strategies for interfering with sexual reproduction and transmission of organism that harm humans, including the devastating malaria organism Plasmodium. Here, I will test the model that a new HAP2 oligomer detected in gametes after fusion is a trimer. I will investigate structural features of HAP2 required for oligomerization. And, I will investigate the mechanisms that underlie FUS1-dependent triggering of HAP2 structural rearrangements.