Phosphoinositide signalling and fungal cell wall integrity: investigating the essential role of the conserved protein Ypp1

Living cells can tolerate extensive changes to their environment. Several signalling mechanisms exist which detect environmental changes, leading to activation of an appropriate response. We have identified Ypp1, a protein in baker's yeast, that is a key modulator of a particular signalling pathway contributing to maintenance of the fungal cell wall. Human cells don't have a cell wall, so this structure is regarded as a principal target for the development of antifungal agents which are non-toxic to humans. Signalling pathways in pathogenic fungi and baker's yeast are similar, so we will use baker's yeast as a model organism for investigating the role of Ypp1 in maintaining fungal cell wall integrity. The precise nature by which Ypp1 affects signalling will be determined, by measuring key biochemical intermediates of the signalling pathway involved. This will be followed by assessing how loss of Ypp1 affects the responses activated by the signalling pathway, with emphasis on how the chemical structure of the cell wall is re-modelled. Also, because the genome of baker's yeast can be manipulated with ease, we will identify pathways and proteins that involve the action of Ypp1. In this way, we will identify additional targets for antifungal drug design.

Ypp1 is an essential protein found in all fungi. We have made conditional mutants of the corresponding gene in the baker's yeast Saccharomyces cerevisiae. The lytic phenotype of these mutants at non-permissive conditions is rescued by osmotic stabilization of media, and at semi-permissive conditions the mutants are sensitive to agents that de-stabilise the cell wall. Accordingly, our preliminary data indicates that Ypp1 is required for maintenance of the fungal cell wall. The phosphatidylinositol 4-kinase Stt4 is a multicopy-suppressor of the phenotype displayed by our ypp1 mutants, implying that Ypp1 plays a crucial role in phosphoinositide metabolism. Phosphatidylinositol phosphates are widely recognized as key regulators of a broad range of signal transduction processes. We propose a multidisciplinary approach to determine how Ypp1 controls metabolism of phosphoinositide second messengers. We will measure levels of phosphoinositides in our conditional ypp1 mutants followed by assay of lipid kinase and lipid phosphatase activity from cell extracts. The physiological effects of losing Ypp1 function will be followed, especially in terms of signalling through the MAPK cascade that controls cell wall re-modelling. Screens for genetic and physical interactions with Ypp1 could reveal proteins acting downstream and upstream of this protein, with the latter being of special interest given our poor understanding of how phosphoinositide signalling is controlled at the fungal plasma membrane. Many aspects of cell wall construction and signalling are conserved between S. cerevisiae and fungal pathogens. Investigating Ypp1 function in S. cerevisiae should aid understanding of the role played by phosphoinositides in maintaining fungal cell wall integrity, and identify additional genes affecting target structures in cell walls. Given the lack of new, effective small molecule anti-fungals the work proposed should provide insight into potential novel anti-fungal drug targets.