Abstract
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.
Highlights
The MID1 gene product, Mid1, is an N-glycosylated, integral membrane protein required for viability of differentiated yeast cells and Ca2ϩ influx induced by the mating pheromone ␣-factor [1]
The channel activity of Cch1 has not yet been revealed experimentally and Mid1 has no homology to the auxiliary subunits, Mid1 has been shown to cooperate with Cch1 in mating pheromone-induced Ca2ϩ uptake [8, 9, 17], store-operated or capacitative Ca2ϩ entry [10], endoplasmic reticulum stress-induced Ca2ϩ uptake [18], and a hyperosmotic stress-induced increase in cytosolic Ca2ϩ [19]
H3 Is Essential for Mid1 Function—To determine whether H3 is essential for Mid1 function, a mutant Mid1 protein lacking the whole H3 segment from Ile337 to Phe356, H3De, was constructed by site-directed mutagenesis using the low copy plasmid YCpMID1-23 [1] as a template and expressed in the mid1-⌬5 mutant
Summary
Synthetic presporulation medium contained 6.7 g of yeast nitrogen base and 10 g of potassium acetate/liter. These media were supplemented with the appropriate nutrients as described previously [23]. The resulting plasmid EpMID1-⌬5 was used as a template to synthesize a DNA fragment containing the mid1-⌬5 gene by PCR using a set of synthetic primers, 5Ј-GATATAGTTTCGCTGCCATC-3Ј and 5Ј-CGTATCGTCCAATGGATGAATTCACATCAAGGATGAGTTACC-3Ј. Transformation of Saccharomyces cerevisiae Cells—The S. cerevisiae strain H311 was transformed by various plasmids whose selection marker was LEU2 (Table I) according to the method described previously [27], with minor modifications. Activity of the Mutant Mid Proteins—To examine the activity of the mutant Mid proteins, the viability and Ca2ϩ accumulation of cells producing the proteins were measured by the methods described previously [1]. Statistical Analysis—Statistical significance was determined using an unpaired Student’s t test, with a maximum p value of Ͻ 0.05 required for significance
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