Komparative Funktionsanalyse der GPI-spezifischen Metallophosphoesterasen Cdc1, Ted1 und Dcr2 aus Saccharomyces cerevisiae

AbstractAbstract In eukaryotes, biosynthesis of glycosylphosphatidylinositol (GPI) and transport of GPI-anchored proteins from the ER to the plasma membrane or the cell wall are mediated by a complex process- ing and sorting network involving more than 20 known enzymes. In the budding yeast Saccha- romyces cerevisiae, the metallophosphoesterases (MPEs) Cdc1 together with Ted1 and Dcr2 ca- talyze two essential steps of GPI-anchor processing. In one step, the ethanolaminephosphate moiety of mannose-2 (EtNP-2) of the GPI-core is removed by Ted1 in the ER, a reaction that is required for efficient ER export. In addition, EtNP-2 can be removed by Dcr2 in the Golgi apparatus. In another step, Golgi-localized Cdc1 removes the EtNP moiety from mannose-1 (EtNP-1) of the GPI-core, a reaction that is thought to be essential for sorting of GPI-anchored proteins from the plasma mem- brane to the cell wall. While a substantial body of genetic and cell biological evidence has unco- vered the essential function of these three enzymes in protein sorting, potential specific roles of these MPEs during cell wall stress or biofilm formation has not be investigated in detail. In addi- tion, the structural basis for specific substrate recognition and processing by these enzymes is largely unknown. In the first part of this work, the specific functions of CDC1, TED1 and DCR2 genes during cell wall stress and biofilm formation were investigated by gene dosage variation and exposure of re- spective yeast strains to different cell wall perturbing agents or biofilm-inducing conditions. These investigations indicate that Cdc1 is specifically sensitive against the cell wall perturbing agents Congo red and Calcofluor white, while Ted1 and Dcr2 appear to be highly sensitive against the β1- 3-glucan synthase inhibitor Caspofungin. These findings can be explained by different substrate specificities of the three enzymes. In contrast, no specific function of the three MPEs was observed during biofilm formation. In a second part, a structure-guided analysis of Cdc1, Ted1 and Dcr2 was performed, in order to identify residues that are essential for function of the enzymes. In summary, a total of 30 previously unknown essential residues were identified that could be involved in co-factor binding, substrate binding or structural integrity of the enzymes. In addition, a total of 15 further residues were found, which are required for full functionality of the proteins under cell wall stress. In the last part, it was attempted to obtain a variant of Cdc1 with a Ted1-like function, in order to identify residues that might confer substrate specificity. By employing an in vitro evolution-based approach, a Cdc1 variant carrying 33 amino acids mutations (Cdc1MORF) could be isolated, which I was able to complement the Zn2+-dependent growth defect of a yeast strain lacking Ted1, but not the lethal growth phenotype caused by the absence of both Ted1 and Dcr2. This finding suggests that Cdc1MORF does not exhibit an altered specificity towards binding and processing of EtNP-2, but might instead have acquired a so far unknown function conferring increased Zn2+-resistance. Impor- tantly, however, Cdc1MORF was also not able to complement the lethal growth phenotype of a strain lacking Cdc1. By performing a fine analysis of the 33 mutations, this phenotype enabled the identi- fication of further residues essential for the functionality of Cdc1. Taken together, this work identifies structural hotspots on the surface of GPI-specific MPEs that ap- pear to be involved in co-factor binding, substrate binding or structural integrity of the enzymes as well as residues that confer robust function under cell wall stress.