Figure 6.

Functional analysis of MEB1 and MEB2 in yeast. A, Phylogenetic tree of DUF125 domains. DUF125 regions were extracted, and the dendrogram was drawn with ClustalW2 and iTOL. Plant DUF125-containing proteins group into three subfamilies. The MEB1/MEB2 subfamily (red branch) contains MEB1, MEB2, At4g27870, grape CAO17728 (Vitis), rice Os06g0103800, and moss XP_001760792 (Physco). The VIT1 subfamily (blue branch) contains Arabidopsis VIT1 (At2g01770), tulip VIT1 (TgVIT1; BAH98154), and rice VIT1 (Os04g0463400) and VIT2 (Os09g0396900). The SEN1/NODULIN21 subfamily (green branch) contains L. japonicus SEN1 (BAL46698), soybean NODULIN21 (P16313), rice Os04g0538400, and Arabidopsis At3g25190, At1g76800, At1g21140, At3g43630, and At3g43660. The dendrogram also includes DUF125 domain-containing proteins from yeast (S. cerevisiae [CCC1] and Schizosaccharomyces pombe [PCL1]) and unicellular bacteria ([Sulfolobus solfataricus [Q97VP8 and Q97TY3], Thermotoga maritima [Q9WYW7], Aeropyrum pernix [Q9YAY1], Pyrobaculum aerophilum [Q8ZYB1], Pseudomonas aeruginosa [Q9HXF5], and Lactococcus lactis [Q9CFK1]). MEB1 and MEB2 are highlighted in red. ScCCC1 and AtVIT1, which have iron/manganese transporter activities, are highlighted in blue. The horizontal scale represents the evolutionary distance expressed as the number of substitutions per amino acid. B, MEB1 and MEB2 suppress the toxicity of iron in the ccc1 mutant. Four yeast strains, the wild type harboring empty vector (WT/vector), ccc1 mutant harboring empty vector (ccc1/vector), MEB1 (ccc1/MEB1), and MEB2 (ccc1/MEB2), were grown on synthetic minimal medium with (left) or without (right) 2 mm iron. The same amount of cells were spotted and germinated for 4 d. C, Three yeast strains, the wild type harboring empty vector (vec; black circles), MEB1 (black triangles), and MEB2 (black squares) were germinated in liquid medium containing 15 mm MnCl₂, and the growth curves were plotted. White circles indicate the growth curve of yeast without manganese.