Phosphatidylinositol (4,5)-bisphosphate turnover by INP51 regulates the cell wall integrity pathway in "Saccharomyces cerevisiae"

Signal transduction pathways are important for the cell to transduce external or internal stimuli where second messengers play an important role as mediators of the stimuli. One important group of second messengers are the phosphoinositide family present in organisms ranging from yeast to mammals. The dephosphorylation and phosphorylation cycle of the phosphatidylinositol species are thought to be important in signaling for recruitment or activation of proteins involved in vesicular transport and/or to control the organization of the actin cytoskeleton. In mammals, phosphatidylinositol (4,5)bisphosphate (PI(4,5)P2) signaling is essential and regulated by various kinases and phosphatases. In the model organism Saccharomyces cerevisiae PI(4,5)P2 signaling is also essential but the regulation remains unclear. My dissertation focuses on the regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae. The organization of the actin cytoskeleton in Saccharomyces cerevisiae is regulated by different proteins such as calmodulin, CMD1, and here I present data that CMD1 plays a role in the regulation of the only phosphatidylinositol 4-phosphate 5-kinase, MSS4, in Saccharomyces cerevisiae. CMD1 regulates MSS4 activity through an unknown mechanism and thereby controls the organization of the actin cytoskeleton. MSS4 and CMD1 do not physically interact but MSS4 seems to be part of a large molecular weight complex as shown by gel filtration chromatography. This complex could contain regulators of the MSS4 activity. The complex is not caused by dimerization of MSS4 since MSS4 does not interact with itself. Two pathways, the cell wall integrity pathway and TORC2 (target of rapamycin complex 2) signaling cascade are important for the organization of the actin cytoskeleton. Loss of TOR2 function results in a growth defect that can be suppressed by MSS4 overexpression. To further characterize the link between MSS4 and the TORC2 signaling pathway and the cell wall integrity pathway we looked for targets of PI(4,5)P2. The TORC2 pathway and the cell wall integrity pathway signal to the GEF ROM2, an activator of the small GTPase RHO1. In our study we identified ROM2 as a target of PI(4,5)P2 signaling. We observed that the ROM2 localization changes in an mss4 conditional mutant. This suggests that the proper localization needs PI(4,5)P2. This could be mediated by the putative PI(4,5)P2 binding pleckstrin homology (PH) domain of ROM2. To better understand the regulation of PI(4,5)P2 levels in Saccharomyces cerevisiae we
focused on one of the PI(4,5)P2 5-phosphatases, INP51. Here we present evidence that
INP51 is a new negative regulator of the cell wall integrity pathway as well as the TORC2
pathway. INP51 probably regulates these two pathways by the turnover of PI(4,5)P2
thereby inactivating the effector/s. The deletion of INP51 does not result in any phenotype,
but when combined with mutations of the cell wall integrity pathway we observe synthetic
interaction.
INP51 together with the GTPase activating protein (GAP) SAC7, responsible for the
negative regulation of RHO1, negatively regulates the cell wall integrity pathway during
vegetative growth. One of the targets of cell wall integrity pathway, the cell wall
component chitin, which is normally deposited at the bud end, bud neck and forms bud
scars, is delocalized in the mother cell in the sac7 inp51 double deletion mutant. In
addition, another downstream component of the cell wall integrity pathway, the MAP
kinase MPK1, has increased phosphorylation and protein level in the sac7 inp51 double
deletion mutant. This suggests that INP51 is important for the negative regulation of the
cell wall integrity pathway.
Furthermore, we show evidence that INP51 forms a complex with TAX4 or IRS4, with two
EH-domain containing proteins, that positively regulates the activity of INP51 and in this
manner negatively regulate the cell wall integrity pathway. The EH-domain is known to
bind the NPF-motif. This motif is present in INP51 and is important for INP51 interaction
with TAX4 or IRS4. The EH-NPF interaction is a conserved mechanism to build up
protein networks. The interaction between an EH-domain containing protein and a
PI(4,5)P2 5-phosphatase is conserved. This is demonstrated by the epidermal growth factor
substrate EPS15 (EH) interaction with the PI(4,5)P2 5-phosphatase synaptojanin the
mammalian orthologue of the Saccharomyces cerevisiae INP proteins.
In summary, INP51 together with TAX4 and IRS4, forms complexes important for
regulation of PI(4,5)P2 levels. The complexes are linked to the TORC2 signaling pathway
and the cell wall integrity pathway, specifically regulating MPK1 activation and chitin
biosynthesis. The work presented in this dissertation facilitates the development of a model
of the complex regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae.