Polarized Morphogenesis Regulator Spa2 Is Required for the Function of Putative Stretch-Activated Ca 2+ -Permeable Channel Component Mid1 in Saccharomyces cerevisiae

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Abstract
Mid1 is a putative stretch-activated Ca 2+ channel component and is required for the maintenance of viability in the mating process. In response to mating pheromone, the mid1 mutant normally forms a pointed mating projection but eventually dies. This phenotype is called the mid phenotype. To identify a protein regulating Mid1 or regulated by Mid1, we isolated a multicopy suppressor that rescues the mid1 - 1 mutant from mating pheromone-induced death and found that it encodes a truncated Spa2 protein lacking an amino-terminal region responsible for interaction with components of the mitogen-activated protein kinase cascades. One of these SPA2 alleles was SPA2 Δ N , whose product lacked the region from Ser 5 to Leu 230 . SPA2 Δ N on a multicopy plasmid (YEpSPA2ΔN) complemented the mid phenotype but not another phenotype, low Ca 2+ accumulation, of the mid1 - 1 mutant. Neither SPA2 Δ N on a low-copy plasmid nor wild-type SPA2 on a multicopy plasmid had suppressive activity. The SPA2 gene is involved in the formation of a pointed mating projection, and cells of the spa2 Δ mutant lacking Spa2 are viable and develop a peanut shell-like structure when exposed to mating pheromone. Like the spa2 Δ mutant, the mid1 - 1 spa2 Δ double mutant and the mid1 - 1/ YEpSPA2ΔN strain developed the peanut shell-like structure. The mid1 - 1 spa2 Δ double mutant did not have the mid phenotype, indicating that SPA2 is epistatic to MID1 . Overexpression of Spa2ΔN abolished the localization of Spa2-green fluorescent protein to the tip of the mating projection. These results suggest that the Spa2ΔN protein interferes with the localization of the normal Spa2 protein and thereby prevents cells from entering the mating process. Therefore, we suggest that Mid1 function is influenced by Spa2 function through polarized morphogenesis.