|Authors:||Mainye, Sheila Kemunto|
|Title:||Discovery and characterization of Pun1p, a putative component of the yeast fertilization synapse|
|Abstract:||Cell-cell fusion is a fundamental process in sexual reproduction and development but the mechanisms mediating and regulating this process are only now beginning to be uncovered. Despite recent progress in identifying key players in the plant and protist kingdoms, large gaps remain in other Eukaryotic lineages, including fungi. The cell fusion in the mating of the yeast Saccharomyces cerevisiae therefore presents itself as an ideal system to understand the molecular mechanisms underlying cell fusion in fungi. In an effort to identify novel components of the yeast fusion machinery and associated regulators, this thesis reports on the discovery and characterization of a membrane protein that negatively regulates the fusion of the plasma membrane (PM) which resembles tight junction claudins found in mammals. A proteomics analysis of pheromone-regulated membrane proteins revealed that components of the MCC/eisosomes, a specialized furrow-like, membrane domain important for PM organization, were preferentially down-regulated during the pheromone response. An exception was Pun1p, a four-pass membrane-spanning protein with a conserved claudin motif, which was up-regulated in response to pheromone and was targeted to the transient junction established between two mating cells. Whereas deletion of PUN1 and its paralogs presented mild fusion defects, its function was distinctly revealed in a sensitized prm1Δ null background, where high-copy expression of PUN1 resulted in a partial inhibition of cell fusion. Further examination of the Pun1p inhibition activity in prm1Δ mutants revealed that PUN1 expression had no effect on lysis or formation of cytoplasmic bubbles, two prm1Δ phenotypes that occur after the cell wall (CW) has been remodeled. Instead, PUN1 expression inhibited fusion by enhancing the formation of a flat PM interface between two cells. However, contrary to previous reports, further analysis revealed that in a proportion of these mating pairs, the flat PM interface was structurally stabilized without any underlying CW material, suggesting the arrest occurred at a stage after CW remodeling via an unknown mechanism. Notably, the Pun1p inhibition activity was dosage and concentration-dependent and was enhanced with increasing concentration of Pun1p molecules across the mating junction, indicating additive functional trans-interactions. Analysis of the mechanistic basis of Pun1p activity revealed a claudin-like activity as a Pun1p mutant version of the conserved claudin motif lost its inhibitory activity. Additionally, the mutant protein exhibited localization defects and was modified at the monomeric level. Together, these results suggest that Pun1p is expressed in mating conditions and localized at the mating junction where it interacts with other unknown proteins to promote the formation of a PM junction-like structure similar to mammalian claudin-mediated tight junctions. The PM junction-like structure would function as a fusion fidelity checkpoint that negatively regulates PM fusion in the event of a compromised fusion machinery. Finally, the identification of Pun1p as a novel component of the fusion machinery prompted further characterization of the mating junction by employing a Horseradish Peroxidase (HRP)-based proximity labeling approach. Using a HRP-Fus1p recombinant protein as a proof of principle, specific HRP-mediated labeling was observed at the shmoo tip of polarized cells, consistent with the pheromone-dependent localization of Fus1p. Although the ultimate objective of characterizing the labelled proteins was not plausible during the course of this thesis, these initial findings demonstrate the feasibility of this approach in yeast studies and its applicability in elucidating the molecular architecture of other similar junctions, a structure which we here refer to as a fertilization synapse.|
|Subject Headings (RSWK):||Proteomics|
|Appears in Collections:||Chemische Biologie|
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