Systematische funktionelle Analyse der Wsc- und Mid-Sensorfamilie in der Hefe Saccharomyces cerevisiae

As all living organisms, the microorganism Saccharomyces cerevisiae has to adapt to a constantly changing environment. For this purpose, the fungus needs sensing systems, which help to recognize the outer impacts and to properly react to them. Cell wall disturbing environmental conditions are recognized and processed by the yeast sensors and proteins of the conserved Cell-Wall-Integrity pathway (CWI). These conditions are for example mechanical or chemical stresses, which are sensed by the CWI-proteins of the Wsc- and Mid-family. The Wsc protein group consists of the sensors Wsc1, Wsc2 and Wsc3, which are characterized by the essential head group, called Wsc- or cysteine-rich domain (CRD). In the second group, the Mid-proteins with Mid2 and Mtl1, this CRD is not found. One aim of this work was to clarify, if the five CWI-sensors had specific functions in cells of the yeast strain ∑1278b. In favour of that, mutant strains with only one or no active sensor were constructed. It was shown, that cells without any sensor were able to grow without osmotic support. In addition to that, ∑1278b Wsc1 was identified to be unnecessary for providing resistance to Caspofungin and caffeine, but important for sensing the cell wall stressor Congo Red. In contrast, the important CWI-kinase Slt2 was not needed for signal transduction in presence of this chemical stress compound. On top of that, a Wsc1 function in biofilm formation on semi-solid surfaces was confirmed, thought this function seemed to be nutrient dependent. Wsc2 showed a supporting role in survival rate of the stress exposed yeast cells. None of the other sensors revealed any specific function, however a mutual influence of all sensors was indicated and a sensor function in adhesion, pseudohyphal growth, DNA-, reductive or amino acid-stress was excluded. Moreover, this work uncovered differences in sensing and signal transduction of the CWI in different strain backgrounds of S. cerevisiae. Furthermore, it should be analysed, if clustered aromatic residues, which are exposed on the surface of the Wsc1 CRD-structure, were needed for sensor function. It was shown, that the investigated amino acid clusters were essential for the resistance of yeast cells exposed to the chemical cell wall stresses Congo Red or Caspofungin. On top of that, the exchanged amino acids led to a localization shift of the sensors into the cytoplasm, which was previously shown for Wsc1-sensors with mutations in the conserved cysteine residues in a similar way.