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，大多数的相互作用既没有结构上也没有功能上的理解，也不是拓扑结构的大多数蛋白质相互作用网络充分解决这些信息转化为细胞的理解。该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细胞质的时间和位置特异性结构。我们按照这个概念，通过询问的Epo 1 p-极化体相互作用网络的组织在芽尖的酵母细胞质的影响。我们已经可以证明，这个网络的两个成员Scs 2 p和Epo 1 p之间的复合物将皮质ER连接到细胞尖端。然而，在这个网络中的其他配体的身份已经暗示了一个更普遍的，但不太清楚的组织极化分泌的其他元素的作用。 使用我们建立的方法，我们将首先构建网络，然后专注于非常规肌球蛋白Myo 2 p和极化体组件Pea 2 p之间的中心相互作用。这种方法以及体外和体内技术的组合将确定网络的哪些蛋白质通过与Pea 2 p的相互作用连接到Myo 2 p。我们将在酵母基因组中插入突变，特异性地中断这些配体和Pea 2 p之间的相互作用，或Pea 2 p和Myo 2 p之间的相互作用。这些表达标记细胞器或其他基于蛋白质的组件的GFP融合的突变株的成像将解决Myo 2 p-Pea 2 p相互作用对这些细胞结构的定位和动力学的重要性。这将识别需要主动运动以到达和/或停留在细胞尖端的蛋白质复合物或细胞器。 类似的分析将解决Epo 1 p-极化体网络的成员的功能意义，激活或调节的RhoGT 3 Cdc 42 p的活性，并参与极化分泌和货物交付网络的成员。对于我们的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物的相对位置和运动的时间。Epo 1 p-Scs 2 p相互作用的仔细检查将揭示Epo 1 p的两个识别的Scs 2 p结合位点是否可能诱导ER膜中Scs 2 p的高阶结构。缺乏Epo 1 p-Scs 2 p结合界面的菌株中GFP-Scs 2 p的光动力学实验将证明这些Scs 2 p簇是否存在以及它们是否负责靠近芽尖的ER膜中的扩散屏障。我们将补充我们的网络分析的极性体的结构特征，以及我们正在进行的努力，以重建本文定义的货物肌动蛋白轨道上的运动。

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.