Raunser, StefanHagemeier, Angela2023-11-132023-11-132023http://hdl.handle.net/2003/42197http://dx.doi.org/10.17877/DE290R-24031Cell-cell fusion is essential for sexual reproduction and occurs when the lipid membranes of two distinct cells merge into one continuous bilayer. While in recent years some general aspects have been uncovered, the underlying molecular mechanism remains poorly understood. Mating of haploid Saccharomyces cerevisiae cells of the opposite sex provides an ideal model system to study plasma membrane (PM) fusion in eukaryotic organisms. In this work, a multicolor flow cytometry assay based on fluorescent complementation (BiFC) of split-GFP was adapted to screen a customized yeast knockout library (YKO) for fusion defects. In total, 28 mutants were identified that exhibited fusion levels at least as defective as .prm1, a known regulator of this step. Like .prm1, the majority displayed a bilateral fusion defect. The remaining part of the work focused on an in-depth analysis of select gene of interest (GOI) mutants. Investigations of synergistic relationships in trans revealed an interaction network operating during PM fusion involving at least four independent yet partially overlapping fusion pathways. Previously two pathways with ERG6 and PRM1 have been reported. VMA2, a gene encoding a subunit of the vacuolar membrane ATPase (V-ATPase), was revealed in this work to operate on a third pathway. The findings show that the V-ATPase i) promotes cell fusion indirectly by acidifying endomembrane organelles, ii) facilitates both cell wall (CW) remodeling and PM fusion stages approximately equally, and iii) synergistically interacts with 12 other genes identified in this study. CAX4 was identified to operate on the fourth pathway and the only novel gene found to synergize with PRM1. Further investigations revealed that Prm1p was less abundant in a .cax4 sensitized background, while its localization is not affected. Finally, this work discovered that several subunits of the RNA polymerase II mediator complex are involved in promoting early and late stages of yeast mating. The deletion of subunits leads to varying degrees of defects in cell pairing and pheromone secretion as well as CW remodeling and PM fusion. Together, these findings suggest that the mediator complex acts as a master regulator of cell fusion perhaps by synchronizing the expression of mating genes needed at crucial time points starting from the digestion of the CW up to the merging of the PMs.enCell fusionLoss-of-function screeningV-ATPaseMediator complex570540TextZellfusionScreeningV-Typ-ATPasenPlasmamembran