There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immunecompromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and morphogenesis. These studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The principal mechanism by which yeast cells detect and respond to wall stress is a signaling pathway mediated by two families of cell surface sensors, a small GTPase (Rho1), protein kinase C (Pkc1), and a MAP kinase cascade, although additional pathways also contribute to the structural integrity of the wall.
The specific aims of this project are 1) To determine if Pkc1 contributes to the G2/M transition by regulating the Mps1 protein kinase during spindle formation. Considerable evidence has accumulated to support a role for Pkc1 in mitosis. Our data implicates Pkc1 in the regulation of the Mps1 mitotic checkpoint kinase. We will test the hypothesis that Mps1 is a Pkc1 target and explore the mitotic effects of this phosphorylation. 2) To determine if Mpk1 acts as a transcription factor under conditions of cell wall stress. We have exciting evidence revealing that the Mpk1 MAP kinase can regulate the SBF transcription factor in a manner that is independent of its protein kinase activity. We propose to test the unusual notion that Mpk1 forms a ternary complex with SBF on the DMA and to explore the mechanism by which Mpk1 drives transcription. 3) To determine if the Wsc1 sensor is recruited to the site of polarized growth through a beta-1,3- glucan-binding domain. The extracellular region of Wsc1 possesses a cys-rich domain that we propose binds to glucan chains and is responsible for localization of Wsc1 to the glucan synthase. We will test this model by a combination of biochemical and molecular genetic approaches. 4) To understand how the PH domain proteins, Ask10 and Ypr115w, contribute to the maintenance of cell wall integrity. We have identified two redundant proteins whose loss of function results in cell lysis. We propose a multifaceted approach to establishing their role in the maintenance of cell wall integrity. ? ? ?
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