The yeast polarisome as nucleus for organizing the cytosol at the cell tip

在酵母中检测到的大量蛋白质-蛋白质相互作用表明其细胞组织的隐藏复杂性。然而，大多数相互作用既不是结构上的，也不是功能上的，大多数蛋白质相互作用网络的拓扑结构也没有足够的分辨来将这些信息转化为细胞理解。拨款提案提出了这样的想法，即某些蛋白质相互作用网络反映并负责胞浆的特定时间和位置的架构。我们遵循这一概念，通过询问Epo1p-极化体相互作用网络对酵母芽尖胞浆组织的影响。我们已经证明，这个网络的两个成员Scs2p和Epo1p之间的一个复合体将皮质内质网连接到细胞顶端。然而，这个网络中其他配体的身份已经暗示了在组织极化分泌的其他元素方面具有更一般的作用，尽管人们还不太清楚。使用我们已建立的方法，我们将首先构建网络结构，然后专注于非传统肌球蛋白Myo2p和极化体成分Pea2p之间的中心相互作用。这种方法和体外和体内技术的结合将确定网络中的哪些蛋白质通过与Pea2p的相互作用连接到Myo2p。我们将在酵母基因组中插入突变，这些突变专门中断这些配体与Pea2p之间的相互作用，或Pea2p与Myo2p之间的相互作用。对这些表达GFP融合的突变菌株进行成像，标记细胞器或其他基于蛋白质的组装，将解决Myo2p-Pea2p相互作用对这些细胞结构的定位和动力学的意义。这将识别需要主动运动才能到达和/或停留在细胞顶端的蛋白质复合体或细胞器。类似的分析将讨论Epo1p-极化体网络成员的功能意义，这些成员由RhoGTP酶CDC42p的活性激活或调节，以及参与极化分泌和货物运输的网络成员。为了对相互作用网络破坏突变的表型分析，我们将跟踪两个细胞器或蛋白质复合体的相对位置和运动。对Epo1p-Scs2p相互作用的仔细研究将揭示Epo1p与Scs2p的两个结合位点是否会在内质网的膜上诱导Scs2p的高阶结构。在缺乏Epo1p-Scs2p结合界面的菌株中进行GFP-Scs2p的光动力学实验将证明这些Scs2p-簇是否存在，以及它们是否对芽尖附近ER膜中的扩散障碍负责。我们将通过极化体的结构特征以及我们不断努力重建在此定义的货物在肌动蛋白轨道上的移动来补充我们的网络分析。

The great number of protein-protein interactions detected in yeast suggests a hidden complexity of its cellular organization. However, the majority of interactions are neither structurally nor functionally understood nor are the topologies of most of the protein interaction networks sufficiently resolved to transform this information into cellular understanding. The grant proposal puts forward the idea that certain protein interaction networks reflect and are responsible for a time and location-specific architecture of the cytosol. We follow this concept by interrogating the influence of the Epo1p-polarisome interaction network on the organization of the cytosol at the bud tip of the yeast. We could already show that a complex between Scs2p and Epo1p, two members of this network, attaches the cortical ER to the cell tip. However, the identities of the other ligands within this network already hint to a more general although less well understood role in organizing other elements of polarized secretion. Using our established approaches we will first structure the network to then focus on a central interaction between the unconventional Myosin Myo2p and the polarisome component Pea2p. This approach and a combination of in vitro and in vivo techniques will determine which proteins of the network are connected to Myo2p through their interaction with Pea2p. We will insert mutations into the yeast genome that specifically interrupt the interactions between these ligands and Pea2p, or the interaction between Pea2p and Myo2p. Imaging of these mutant strains expressing GFP fusions that mark organelles or other protein-based assemblies will address the significance of the Myo2p-Pea2p interaction on the localization and dynamics of these cellular structures. This will identify the protein complexes or organelles that require active movement to reach and/or to stay at the tip of the cell. A similar analysis will address the functional significance of the members of the Epo1p-polarisome network that are activated by or regulate the activity of the RhoGTPase Cdc42p, and the members of the network that are involved in polarized secretion and cargo delivery.For the phenotypic analysis of our interaction network-disrupting mutations we will track the relative location and movements of two organelles or protein complexes at a time. A closer inspection of the Epo1p-Scs2p interaction will reveal whether the two identified binding sites of Epo1p for Scs2p might induce a higher-order structure of Scs2p in the membrane of the ER. Photokinetic experiments of GFP-Scs2p in strains lacking the Epo1p-Scs2p binding interface will prove whether these Scs2p-clusters exist and whether they are responsible for a diffusion barrier in the ER membrane close to the bud tip. We will complement our network analysis by a structural characterization of the polarisome as well as by our ongoing efforts to reconstitute the movement of the herein defined cargo on actin tracks.