JCS010215 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF
• Supplemental Table S2 - Adobe PDF
• Supplemental Table S3 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Alignment of AtMinE1 and higher-plant MinE proteins. At, Arabidopsis thaliana; Os, Oryza sativa; Pp, Physcomitrella patens; Zm, Zea mays; Ec, E. coli. Filled black boxes represent identical amino acid residues and filled grey boxes represent conservative substitutions. Residues involved in the interaction of MinE with MinD (Ma et al., 2003) are indicated by asterisks. Residues crucial for the topological specificity function in E. coli MinE are indicated by dots, and residues that contribute to the hydrophobic core of the E. coli TSD dimer are indicated by triangles (King et al., 2000). The alignment was created using ClustalW and viewed using BioEdit.

• Supplemental Figure S2 -

  Fig. S2. Interactions that mediate the formation of the Min complex in Arabidopsis. HF7c cells were co-transformed with (A) BD-AtMinE1, (B) BD-AtMinD1, (C) AD-AtMinE1 or (D) AD-AtMinD1 and the indicated truncations of AtMinE1. Growth of the yeast was monitored on synthetic drop-out media lacking tryptophan and leucine (−TL) and synthetic drop-out media lacking tryptophan, leucine and histidine (−HTL) and the ratio of growth of yeast on media −HTL to media −TL calculated as an indicator of the strength of interaction. Standard deviations of the mean are shown. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.

• Supplemental Figure S3 -

  Fig. S3. Confirmation of coexpression of the cDNA fusion cassettes in BiFC assays. AtMinE1, AtMinD1 and GC1 were fused to the nonfluorescent YFP_155-238 (CY) and were coexpressed in tobacco chloroplasts with truncations of AtMinE1 fused to YFP_1-154 (NY). The interactions of AtMinE1_1-141-NY plus AtMinD1-CY, and AtMinE1_142-229-NY plus AtMinE1-CY, were used as positive controls. BiFC assays were carried out as described in Materials and methods. After microscopic analysis, RNA was extracted from infiltrated tissue and RT-PCR conducted using fusion-specific primers to the NY or CY construct. +, positive reconstitution of YFP in tobacco leaf sample; −, no reconstitution of YFP in tobacco leaf sample; M, marker.

• Supplemental Figure S4 -

  Fig. S4. A highly conserved region of AtMinE1 is required for the interaction with AtMinD1. HF7c cells were co-transformed with the indicated combinations of bait (BD; schematic on left) and prey (AD) vectors and grown on selection plates at 30°C for three days. Growth of the yeast was monitored on synthetic drop-out media lacking tryptophan and leucine (−TL) and synthetic drop-out media lacking tryptophan, leucine and histidine (−HTL) and the ratio of growth of yeast on media −HTL to media −TL calculated as an indicator of the strength of interaction. Standard deviations of the mean are shown. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain; D, AtMinD1; E, AtMinE1; EcD, MinD.

• Supplemental Figure S5 -

  Fig. S5. Analysis of the effects of amino acid substitutions in AtMinE1_35-141 on the interaction with AtMinD1. HF7c cells were co-transformed with AD-AtMinD1 (A) or AD-MinD (B) and the indicated combinations BD-AtMinE1_35-141 (AMD) constructs. Growth of the yeast was monitored on synthetic drop-out media lacking tryptophan and leucine (−TL) and synthetic drop-out media lacking tryptophan, leucine and histidine (−HTL) and the ratio of growth of yeast on media −HTL to media −TL calculated as an indicator of the strength of interaction. Standard deviations of the mean are shown. AD, GAL4 activation domain; AMD, anti-MinCD domain; BD, GAL4 DNA-binding domain; D, AtMinD1; EcD, MinD.