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）之间融合的分子机制的理解 在有性生殖过程中是非常有限的。在对单细胞绿色果蝇的研究中， 衣原体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HAP 2（也称为GCS 1）是必需的， 在配子融合过程中的双层合并。HAP 2在配子浆紧密粘附后起作用 膜，并且仅存在于两个配子中的一个上（例如，交配型减去配子 衣原体），因此功能单方面的，因为这样做病毒融合蛋白在其进入 进入宿主细胞值得注意的是，最近的合作结构/功能研究表明， 衣原体HAP 2在结构上与许多衣原体的II类融合蛋白同源， 包膜病毒，包括登革热和寨卡病毒。虽然重组形式的胞外域的 衣原体HAP 2在体外形成三聚体，疏水融合环对于 虽然在体内发挥作用，但我们对这类真核生物的分子机制仍知之甚少 在任何HAP 2生物体中配子融合期间的II融合蛋白。内源性三聚体， 据推测，在配子融合过程中形成的，尚未被发现，其机制是 未知的限制触发三聚体形成，直到HAP 2轴承配子经历 与其配偶配子的膜的特异性膜相互作用。初步 在实验中，我发现了一种稳定的寡聚体，它只在配子融合后出现， 可能是内源性HAP 2三聚体。我还确定，与病毒类不同， II蛋白，低pH不能触发HAP 2的寡聚体形成。相反，含有HAP 2的 负交配结构必须与正交配结构上的粘附蛋白FUS 1结合， 确保触发仅在正确的时间和地点发生。本研究旨在定义 一个广泛保守的真核生物功能的分子机制 跨界生物体中配子融合所必需的膜融合素。的 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策略， 性繁殖和传播危害人类的生物体，包括 毁灭性的疟原虫在这里，我将测试一个新的HAP 2寡聚体模型， 在融合后的配子中检测到的是三聚体。我将研究所需的HAP 2的结构特征， 用于低聚。并且，我将研究FUS 1依赖的机制， 引发HAP 2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.