A cell fusion interaction network during the mating of Saccharomyces cerevisiae
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Date
2023
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Abstract
Cell-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.
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Keywords
Cell fusion, Loss-of-function screening, V-ATPase, Mediator complex