yam8 + ,a Schizosaccharomyces pombe Gene, Is a Potential Homologue of the Saccharomyces cerevisiae MID1 Gene Encoding a Stretch- Activated Ca 2+ -Permeable Channel Yasushi Tasaka,* Yuko Nakagawa,† Chikara Sato,‡ Masanobu Mino,* Nobuyuki Uozumi,§ Norio Murata, ¶ Shoshi Muto,§ and Hidetoshi Iida† ,1 *Research Institute for Biological Sciences, 7549-1 Yoshikawa, Kayo, Joubo, Okayama 716-1241, Japan; †Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei, Tokyo 184-8501, Japan; ‡Supermolecular Science Division, Electrotechnical Laboratory, 1-1-4 Umezono, Tsukuba, Ibaraki 305-8568, Japan; §Nagoya University Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan; and ¶ Department of Regulation Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki 444-8585, Japan Received January 19, 2000 The Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca 2 -permeable channel. In a pro- tein database, we found a Schizosaccharomyces pombe gene whose predicted protein shows 26% identical and 62% similar to the Mid1 channel in amino acid se- quence. cDNA derived from this gene, designated yam8  , was isolated by reverse transcription-poly- merase chain reaction (RT-PCR). Further analysis showed that the Yam8 protein consists of 486 amino acids and has 6 hydrophobic segments. The yam8  cDNA, placed under the S. cerevisiae TDH3 promoter, partially complemented the mating pheromone-in- duced death (mid) phenotype of the S. cerevisiae mid1 mutant. The expression of the yam8  cDNA in the mid1 mutant cells partially remediated the mid phenotype and resulted in a slight increase in Ca 2 uptake activ- ity. These findings suggest that Yam8 is a potential homologue of Mid1. © 2000 Academic Press The MID1 gene product (Mid1) is an N-glycosylated, integral membrane protein required for Ca 2+ influx stimulated by mating pheromone (1). Cells lacking MID1 die because of limited Ca 2+ influx when incu- bated with mating pheromone. Electrophysiological and cell biological studies on Chinese hamster ovary (CHO) cells expressing Mid1 have revealed that it is a Ca 2+ -permeable, stretch-activated nonselective cation channel (SA Cat channel) (2). SA Cat channels are suggested to transduce mechan- ical strain into a cellular response, and thus play a crucial role in touch sensation, hearing, detecting grav- ity, and sensing osmotic changes (3, 4). Bacterial SA Cat channels are considerably studied because their genes and homologues are isolated, enabling ones to characterize them biochemically, electrophysiologi- cally and structurally in conjunction with in vitro mu- tagenesis (5, 6). By contrast, such studies on eukaryotic SA Cat channels have just started after the finding of Mid1 as an SA Cat channel. Besides going into deeper analyses on Mid1, we need to find and analyze Mid1 homologues in other organisms for better understand- ing of eukaryotic SA Cat channels. Here we report the isolation and basic characterization of a potential MID1 homologue, the yam8 + cDNA, of the fission yeast S. pombe. MATERIALS AND METHODS Microbial strains and culture conditions. S. cerevisiae strains H207 (MATa his3-1 leu2-3,112 trp1-289 ura3-52 sst1-2) and H301 (MATa his3-1 leu2-3,112 trp1-289 ura3-52 sst1-2 mid1-1) was grown in rich medium (YPD) or synthetic media (SD and SD.Ca100). The compositions of these media were described previously (1). The S. pombe strain JY282 (h + ura4), provided by Dr. M. Yamamoto (University of Tokyo, Japan), was grown in YPD medium for the isolation of a cDNA clone of yam8 + . Escherichia coli strain DH5 was used as a host of plasmid DNAs. Isolation of the S. pombe yam8 + cDNA and construction of a plasmid expressing the yam8 + cDNA. A S. pombe genomic clone (yam8 + , SPAC1F5.08C in Chromosome I, SWISS-PROT accession No. Q10063, GeneBank Accession No. Z68136) was identified as a potential MID1 homologue through a BLAST search (7) on the S. cerevisiae MID1 gene. To isolate the cDNA fragment encoding the yam8 + ORF of S. pombe, RT-PCR was performed. Total RNA was extracted from S. pombe cells using YeaStar RNA extraction kit (Zymo Research, Orange, CA), and a poly (A) + RNA was isolated using Oligotex-dT30 Super (Takarashuzo, Kyoto, Japan). Total first- 1 To whom correspondence should be addressed. Fax: 81-42-329- 7517. E-mail: iida@u-gakugei.ac.jp. Biochemical and Biophysical Research Communications 269, 265–269 (2000) doi:10.1006/bbrc.2000.2278, available online at http://www.idealibrary.com on 265 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.