Analysis of the structure function and regulation of the Rho1-specific GTP-exchange proteins of the yeast cell wall integrity pathway

The cells that make up every organism are delicate and intricate machines that must carry out many complex tasks to stay alive. The single celled fungus, the budding yeast, although modest in size, shares with our cells many of these intricate mechanisms. Yeast has the huge advantage over humans in scientific research: it is relatively easy and cheap to study. Many of the insights gained into how yeast cells work apply, in one form or another, to other organisms, including ourselves. Among the key tasks shared between yeast and human cells is the ability to grow bigger without bursting. Another is to survive changes in the immediate environment that threaten lysis (bursting), such as changes in temperature or nasty chemicals. Yeast possesses one main system that senses a variety of threats to the cell's integrity and responds so as to maintain that integrity (and thereby keep the cell alive) - the cell wall integrity (CWI) pathway. Many of the components of this system are shared with humans but some are not - these latter may be a fungus' Achilles' heel, to which drugs could be developed that cause fungal cells (many pathogenic) to blow up (die) leaving human cells undisturbed. The CWI pathway is worth understanding. In addition, the CWI pathway presents scientific puzzles that challenge our understanding of how living systems work. Multiple signals feed into this pathway, and the pathway can activate a variety of distinct responses: how can one pathway integrate many inputs and 'decide' to make a sensible response? Key regulators of the CWI pathway are proteins called GEFs. CWI-GEFs appear to come in two distinct flavours that appear to perform distinct roles in activating the pathway. In this proposal, we seek to better understand how these GEFs are regulated, how they differ from each other both structurally and functionally and how information is processed by these GEFs to affect CWI outputs in the appropriate way. We hope to better understand how the complex and important CWI pathway is regulated.

The Cell Wall Integrity pathway acts to maintain fungal cell integrity and is a target for antifungal agents such as cercosporamide. The pathway does not only affect the fungal cell wall, but also affects a variety of surface-related processes that are shared with other eukaryotes, such as actin organization, vesicle trafficking and phospholipids synthesis. The CWI pathway is heavily branched, with multiple inputs feeding into it and multiple effectors/processes affected by it. Information channels through the pathway's small GTPase Rho1 which can activate, when in its GTP-bound form, 5 distinct effectors. How signals are integrated by Rho1 and if, or how, it preferentially affects particular targets are not known. Rho1-GTP exchange factors (Rho1-GEFs) likely integrate input signals to stimulate formation of active Rho1-GTP. The Rom2 GEF is best understood and may directly link the sensors of surface state to Rho1. Another GEF, Tus1, was recently discovered: its role in the CWI pathway is not well understood. The Rho1-specific GEFs are the only yeast proteins to possess a citron-homology (CNH) domain. Our data indicate that Tus1 and Rom2 play distinct roles in the CWI pathway either by responding to distinct signals or directing Rho1 to distinct effectors or both. Rom2 and Tus1 are also structurally distinguishable: Rom2 possesses a DEP domain and a degenerate PH domain whereas Tus1 possesses a non-degenerate PH domain. We have recently identified a novel activator of CWI signalling, Ack1, that somehow acts at the level of, and binds to, Rom2 and Tus1. Here we seek to understand 1) the role of Ack1 in the CWI pathway and in Tus1 and Rom2 activity 2) the role of Tus1 in the CWI pathway 3) how Tus1 and Rom2 play distinct roles in the pathway 4) the functions for the Rho1-GEF domains and to 5) identify novel regulators of Tus1 and Rom2 activity.