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J Biol Chem

Title:		Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1
Author(s):		Maruoka T; Nagasoe Y; Inoue S; Mori Y; Goto J; Ikeda M; Iida H;
Address:		"Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan"
Journal Title:		J Biol Chem
Year:		2002
Volume:		20020116
Issue:		14
Page Number:		11645 - 11652
DOI:		10.1074/jbc.M111603200
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
Abstract:		"The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-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 Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). 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 Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) 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"
Keywords:		"Alanine/chemistry Alleles Asparagine/chemistry Binding Sites Calcium/*metabolism Calcium Channels/metabolism Codon Codon, Nonsense Cysteine/*chemistry Fungal Proteins/*chemistry/metabolism Hydroxylamine/pharmacology Immunoblotting Membrane Glycoproteins/*;"
Notes:		"MedlineMaruoka, Takashi Nagasoe, Yurika Inoue, Shinobu Mori, Yasunori Goto, June Ikeda, Mitsunobu Iida, Hidetoshi eng Research Support, Non-U.S. Gov't 2002/01/18 J Biol Chem. 2002 Apr 5; 277(14):11645-52. doi: 10.1074/jbc.M111603200. Epub 2002 Jan 16"