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

• Applicant: Professor Dr. Nils Johnsson
• Subject Area: Cell Biology
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 279190516

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.

• DFG Programme: Research Grants