Essential Hydrophilic Carboxyl-terminal Regions Including Cysteine Residues of the Yeast Stretch-activated Calcium-permeable Channel Mid1

• Published in: The Journal of biological chemistry Vol. 277; no. 14; pp. 11645 - 11652
• Main Authors: Maruoka, Takashi, Nagasoe, Yurika, Inoue, Shinobu, Mori, Yasunori, Goto, June, Ikeda, Mitsunobu, Iida, Hidetoshi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.04.2002; American Society for Biochemistry and Molecular Biology
• Subjects: Alanine - chemistry; Alleles; Asparagine - chemistry; Binding Sites; Calcium - metabolism; Calcium Channels - metabolism; Codon; Codon, Nonsense; Cysteine - chemistry; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Hydroxylamine - pharmacology; Immunoblotting; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Mid1 gene; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Phosphorylation; Plasmids - metabolism; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Serine - chemistry; Threonine - chemistry; Time Factors; Tryptophan - chemistry; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111603200

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca2+-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp441, generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn389 to Thr548. In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe522 (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca2+ uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn389 to Asn521 is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.