Fig. 1.

Function in yeast cells and structural features of Mca1 and Mca2. (A) Complementation of lethality of the yeast mid1 mutant by MCA1. Cell viability of the mid1 mutant strain bearing pFL61-MCA1 or empty vector pFL61 and wild-type strain bearing pFL61 was determined after being exposed to the mating pheromone α-factor. Data are means ± SD from three independent experiments. ∗, P < 0.01 versus mid1/pFL61. (B) Ca²⁺ uptake activity. Exponentially growing cells of the above strains were suspended in Hepes/Tris buffer containing 74 kBq/ml ⁴⁵CaCl₂ (74 MBq/nmol) and aliquots of the suspension were taken at 1-min intervals over 8 min and filtered through a Millipore filter (type HA; 0.45 μm). The radioactivity retained on the filters was counted. Data are means ± SD from three independent experiments. ∗, P < 0.01 versus mid1/pFL61. (C) Genomic organization of the MCA1 and MCA2 genes. Boxes represent exons and their black areas show the ORF. T-DNA (drawn to an arbitrary size) is inserted into the site 28 bp upstream of the fourth exon, producing an mca1-null allele. (D) Multiple amino acid sequence alignment of the Mca1, Mca2, and rice OsMca1 with Clustal W (version 1.8). Amino acid sequence identity (and similarity) between Mca1 and Mca2 is 72.7% (89.4%), that between Mca1 and OsMca1 is 65.0% (90.3%), and that between Mca2 and OsMca1 is 57.2% (86.4%). Asterisk indicates identical amino acid; colon indicates amino acid with strong similarity; dot indicates amino acid with weak similarity. The ARPK domain (for amino-terminal domain of rice putative protein kinases) is boxed. The line under each sequence shows a potential transmembrane segment (TM) predicted by TopPred (19). The lines above the Mca1 sequence represent an EF-hand-like structure, a coiled-coil region, and a C-terminal, cysteine-rich region similar to the PLAC8 motif found in plant and animal proteins of unknown function. (E) Schema of Mca1 and the hydropathy profile of Mca1. The bars indicate the position of the potential transmembrane segments (TM) described in D. (F) MCA1 has the ability to increase Ca²⁺ accumulation even in the mid1 cch1 double mutant. MCA1 cDNA on a plasmid was expressed under the control of the TDH3 promoter in each yeast mutant (mid1, cch1, or mid1 cch1) as well as the parental strain (MID1 CCH1). The MID1 gene was expressed from the plasmid YEpMID1 (25). Exponentially growing cells were incubated for 2 h in the low Ca²⁺ medium SD.Ca100 (15) containing 185 kBq/ml ⁴⁵CaCl₂ (1.8 kBq/nmol) and aliquots of the culture were taken and filtered through a Millipore filter (type HA; 0.45 μm). ∗, P < 0.05 versus vector in each mutant.