Journal
MICROBIOLOGY-SGM
Volume 159, Issue -, Pages 970-979Publisher
MICROBIOLOGY SOC
DOI: 10.1099/mic.0.064030-0
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Funding
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [21026009, 23120509]
- Japan Society for the Promotion of Science [21370017]
- Grants-in-Aid for Scientific Research [21026009, 21370017] Funding Source: KAKEN
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The yeast Saccharomyces cerevisiae CCH1 gene encodes a homologue of the pore-forming alpha(1) subunit of mammalian voltage-gated calcium channels. Cch1 cooperates with Midi, a candidate for a putative, functional homologue of the mammalian regulatory subunit alpha(2)/delta, and is essential for Ca2+ influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2). The product of CCH1* displayed a marked increase in Ca2+ uptake activity in the presence and absence of alpha-factor, and its increased activity was still dependent on Mid1. Mutations in CCH1* did not affect its susceptibility.to regulation by calcineurin. In addition, not only was the N1066D mutation in the cytoplasmic loop between domains II and III responsible for the increased activity of Cch1*, but also substitution of another negatively charged amino acid Glu for Asn(1066) resulted in a significant increase in the Ca2+ uptake activity of Cch1. This is the first report of a hyperactive mutation in Cch1. On the other hand, the cch1-2 allele possesses the P1228L mutation located in the extracellular S1-S2 linker of domain III. The Pro(1228) residue is highly conserved from fungi to humans, and the P1228L mutation led to a partial loss in Cch1 function, but did not affect the localization and expression of Cch1. The results extend our understanding of the structure-function relationship and functional regulation of Cch1.
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