Polarized Morphogenesis Regulator Spa2 Is Required for the Function of Putative Stretch-Activated Ca2+-Permeable Channel Component Mid1 in Saccharomyces cerevisiae

doi:10.1128/EC.4.8.1353-1363.2005

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Polarized Morphogenesis Regulator Spa2 Is Required for the Function of Putative Stretch-Activated Ca²⁺-Permeable Channel Component Mid1 in Saccharomyces cerevisiae

Shigeko Noma,¹^,2^, Kazuko Iida,³ and Hidetoshi Iida¹^,2^*

Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501,¹ CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012,² Medical R & D Center, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan³

Received 5 May 2005/ Accepted 6 June 2005

Mid1 is a putative stretch-activated Ca²⁺ channel componentand is required for the maintenance of viability in the matingprocess. In response to mating pheromone, the mid1 mutant normallyforms a pointed mating projection but eventually dies. Thisphenotype is called the mid phenotype. To identify a proteinregulating Mid1 or regulated by Mid1, we isolated a multicopysuppressor that rescues the mid1-1 mutant from mating pheromone-induceddeath and found that it encodes a truncated Spa2 protein lackingan amino-terminal region responsible for interaction with componentsof the mitogen-activated protein kinase cascades. One of theseSPA2 alleles was SPA2 ${Delta}$ N, whose product lacked the region fromSer⁵ to Leu²³⁰. SPA2 ${Delta}$ N on a multicopy plasmid (YEpSPA2 ${Delta}$ N) complementedthe mid phenotype but not another phenotype, low Ca²⁺ accumulation,of the mid1-1 mutant. Neither SPA2 ${Delta}$ N on a low-copy plasmid norwild-type SPA2 on a multicopy plasmid had suppressive activity.The SPA2 gene is involved in the formation of a pointed matingprojection, and cells of the spa2 ${Delta}$ mutant lacking Spa2 are viableand develop a peanut shell-like structure when exposed to matingpheromone. Like the spa2 ${Delta}$ mutant, the mid1-1 spa2 ${Delta}$ double mutantand the mid1-1/YEpSPA2 ${Delta}$ N strain developed the peanut shell-likestructure. The mid1-1 spa2 ${Delta}$ double mutant did not have the midphenotype, indicating that SPA2 is epistatic to MID1. Overexpressionof Spa2 ${Delta}$ N abolished the localization of Spa2-green fluorescentprotein to the tip of the mating projection. These results suggestthat the Spa2 ${Delta}$ N protein interferes with the localization of thenormal Spa2 protein and thereby prevents cells from enteringthe mating process. Therefore, we suggest that Mid1 functionis influenced by Spa2 function through polarized morphogenesis.

* Corresponding author. Mailing address: Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan. Phone and fax: 81-42-329-7517. E-mail: iida{at}u-gakugei.ac.jp.

Present address: Department of Medical Genome Sciences, GraduateSchool of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha,Kashiwa, Chiba 277-8562, Japan.

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