Identifying the molecular mechanism by which the conserved Hook/Fts/Fhip complex controls kinesin-3 and dynein attachment to early endosomes

细胞的组织和功能需要沿着细胞骨架的蛋白质纤维进行沿着的细胞内运输。就像“高速公路”一样，这些纤维为蛋白质机器提供了“轨道”，所谓的分子马达，利用化学能在整个细胞中移动它们的“货物”。一种这样的“货物”是早期内体（EE）。这些膜状容器沿着细胞骨架纤维的一个子类微管移动。它们的运输发生在相反的方向（双向），并通过抵消马达驱动蛋白-3和动力蛋白介导。这个过程对我们体内的细胞非常重要，EE运动性、驱动蛋白-3或动力蛋白功能的缺陷被认为是严重人类疾病的基础。病原真菌入侵我们的作物的能力，这同样需要电机和EE，再次强调了这一过程中的细胞的普遍重要性。丝状真菌有重大的技术优势，我们最近已经利用，以深入了解控制EE的运动和分布在病原体玉米黑粉菌的因素。利用这个模式系统，这也是一个重要的作物病原体，我们最近发现了一个蛋白质复合物，使电机驱动蛋白-3和动力蛋白结合EE。这种复合物（命名为Hok 1/Fts 1/Fhp 1复合物）也存在于人类中，我们提供了将马达连接到其“货物”的类似功能的证据。在这个拟议的项目中，我们将阐明Hok 1/Fts 1/Fhp 1靶向“货物”的分子环境和详细机制，以及复合物如何协调相对的马达驱动蛋白3和动力蛋白的结合。我们发现的初步证据表明，这两种机制（锚定“货物”和绑定电机）涉及额外的，但身份不明的蛋白质因素。在准备这个应用程序，我们已经进行了扩展的生化实验，导致了一系列假定的相互作用蛋白质。我们将测试这些蛋白质是否与EE结合，以及当从细胞中取出时，EE运动性，运动结合或Hok 1/Fts 1/Fhp 1锚定是否受损。我们还将以完全无偏见的方式解决双向EE运动的过程，通过使用随机产生的突变体，EE分布和运输缺陷的遗传筛选。该筛选在之前进行，并导致在U. maydis（见上文）。我们将遵循相同的实验策略，并将在其他突变株中检测更多推定因子在EE运动和分布中的作用。我们已经证实了3个蛋白质从这个屏幕上的附件EE，并将阐明其在电机结合和电机调节或Hok 1/Fts 1/Fhp 1招聘的“货物”膜推定的作用。此外，这种方法有可能揭示完全意想不到的方式，使细胞能够双向运输和均匀分布的EEs.In总结，这一建议解决了一个基本的过程中发现的动物，人类和丝状真菌在一个公正和全面的方式。我们广泛的初步结果是指导我们在一个非常结构化的方式来阐明电机移动EE在细胞中的分子机制。了解这一过程将为医学研究提供信息，同时也为抗真菌药物的开发提供了新的途径。

The organisation and function of cells requires intracellular transport along protein fibres of the cytoskeleton. Like "highways", these fibres provide the "tracks" for protein machines, the so-called molecular motors, which use chemical energy to move their "cargo" throughout the cell. One such "cargo" is the early endosomes (EE). These membranous containers move along a sub-class of cytoskeletal fibres, the microtubules. Their transport occurs in opposite direction (bi-directional) and is mediated by the counteracting motors kinesin-3 and dynein. This process is high importance for the cells in our body, and defects in EE motility, kinesin-3 or dynein function is thought to underlie severe human diseases. The same motors and EEs are required for the ability of pathogenic fungi to invade our crops, which, again, highlights the general importance of this process in cells.Filamentous fungi have major technical advantages that we have exploited recently to gain insight into the factors that control EE motility and distribution in the pathogen Ustilago maydis. Making use of this model system, which is also an important crop pathogen, we found recently a protein complex that enables the motors kinesin-3 and dynein to bind EEs. This complex (named Hok1/Fts1/Fhp1 complex) is also found in humans and we provided evidence for similar functions in linking motors to their "cargo". In this proposed project, we will elucidate the molecular environment and detailed mechanism by which Hok1/Fts1/Fhp1 is targeted to the "cargo", and how the complex coordinates the binding of the opposing motors kinesin-3 and dynein. We found preliminary evidence that both mechanisms (anchorage to "cargo" and binding motors) involves additional, yet unidentified protein factors. In preparation for this application, we have performed extended biochemical experiments that led to a list of putative interacting proteins. We will test if these proteins bind to EEs and if, when removed from the cell, EE motility, motor binding or Hok1/Fts1/Fhp1 anchorage is impaired. We will also address the process of bi-directional EE motility in an entirely unbiased way, by using genetic screening for randomly-generated mutants, defective in EE distribution and transport. This screen was undertaken previously and led to the identification of the Hok1/Fts1/Fhp1 complex in U. maydis (see above). We will follow the same experimental strategy and will test more putative factors in additional mutant strains for a role in EE motility and distribution. We have already confirmed the attachment of 3 proteins from this screen to EEs, and will elucidate their putative role in motor binding and motor regulation or Hok1/Fts1/Fhp1 recruitment to "cargo" membranes. In addition, this approach has the potential to reveal entirely unexpected ways by which the cell enables bi-directional transport and even distribution of EEs.In summary, this proposal addresses a fundamental process found in animals, humans and filamentous fungi in an unbiased and comprehensive way. Our extensive preliminary results are guiding us in a very structured way to elucidate the molecular mechanism by which motors move EEs in the cell. Understanding this process will inform medical research, but also provides new avenues towards the development of anti-fungal drugs.