Molecular dissection of the hydrophobic segments H3 and H4 of the yeast Ca2+ channel component Mid1.
The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca(2+)-permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the alpha1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca(2+) accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca(2+) accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.
Tada
Tomoko
T
Department of Biology, Tokyo Gakugei University, Tokyo 184-8501, Japan.
Ohmori
Masayuki
M
Iida
Hidetoshi
H
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D008164:Luminescent Proteins; C090463:MID1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D002118:Calcium