Identification of genes required for cell fusion during mating in the yeast Saccharomyces cerevisiae
Elia, Lisa Patricia
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Cell fusion during mating in the yeast, Saccharomyces cerevisiae, provides a good system to study signal-induced changes at the cell surface. Many of the components involved in yeast cell fusion remain uncharacterized. To characterize the molecular details of cell fusion, a multi-tiered genetic screen was performed to isolate mutants defective in the fusion stage of yeast mating. The screen required that mutants complete early stages of mating and retain the ability to elicit responses in a chosen mating partner, but not complete cell fusion.;Three of the tightest mutations isolated in the screen were identified as partial-function alleles of the ABC transporter gene STE6 required for transport of a-factor. The ste6(cef1-1) allele was recovered, sequenced, and found to contain a stop codon predicted to truncate Ste6p at amino acid residue 862 (of 1290). The ste6(cef) mutations reduced expression of a-factor. Light and electron microscopy revealed that unlike ste6 null mutations, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material termed the fusion wall. Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes. A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating indicating a tight threshold of a-factor signaling required for mating might exist. However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain. Overexpression of the structural gene for a-factor also did not suppress the fusion defect. It is possible that a-factor or STE6 plays multiple roles in cell fusion.;The AXL1 gene, encoding a metalloendoprotease, was identified as required for cell fusion in both mating types. AXL1 is a pheromone-inducible gene required for axial bud site selection in haploid yeast and for proteolytic maturation of a-factor. Two other bud site selection genes, RSR1, encoding a small GTPase, and BUD3, were also required for efficient cell fusion. Based on double mutant analysis, AXL1 acted genetically in the same pathway with FUS2, a fusion-dedicated gene. Electron microscopy of axl1, rsr1, and fus2 prezygotes revealed similar defects in nuclear migration, vesicle accumulation, cell wall degradation, and membrane fusion during cell fusion. AXL1 protease function was required for fusion during mating, but was dispensible for bud site selection during vegetative growth. The ability of the Rsr1p GTPase to cycle was required for efficient cell fusion, as it is for bud site selection. Since Rsr1p is known to physically associate with components of the pheromone response pathway, I suggest that Rsr1p and Axl1p may act in a branch of the pheromone response pathway controlling Fus2p-based fusion events.