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. 2006 Dec;98(6):1179–1187. doi: 10.1093/aob/mcl211

Fig. 1.

Fig. 1

Interaction between AtMBD5 and AtRAN3. (A) Yeast two-hybrid assay. Yeast AH109 cells were co-transformed with GBD-AtMBD5 and AD-AtRAN3 constructs (left panel). The transformants were planted on SD agar supplemented with an amino acid mixture depleted of histidine and tryptophan (SD/–WL) (lane 1), or histidine, tryptophan and leucine in the absence (SD/–HWL) (lane 2) or presence of 40 mm 3-amino-1,2,4-triazole (lane 3). Colonies cultured on the SD/–WL plate were assayed for β-galactosidase by the filter lift method (lane 4). Transformants from the SD/–HWL agar plate were cultured in SD/–WL broth and β-galactosidase activity was estimated using O-nitriphenyl-β-d-galactopyranoside as the substrate and expressed in Miller units (right panel). The value shown is the average of triplicate measurements with standard deviation. (B) Amino acid sequence of AtRAN3 (gene ID: 835612) and its tobacco orthologue, NtRan-A1 (accession no. P41918). Alignment was performed with the Clustal W program. Identical residues are shaded, and a fragment initially identified by the yeast two-hybrid screening is underlined. Specific motifs conserved among Ran GTPases are indicated; G1–G3 and P-loop are for GTP binding, and Switch 1 and Switch 2 are for interaction with RanBD1 and karyopherin-β2, respectively (Macara, 1999). (C) Pull-down assay. Interactions between AtMBD5 and AtRAN3 in the absence or presence of GTP[γS] or GDP[βS] were examined. Approximately 5 μg GST-AtMBD5 (47 kDa) proteins or GST proteins as the control were bound to a glutathione column and 25 μg His-tagged AtRAN3 (45 kDa) was added to each column. After elution with reduced glutathione, proteins were separated on SDS–PAGE (CBB staining, left panel). Loaded samples were molecular markers (lane 1), input His-tagged AtRAN3 (lane 2) (indicated by an open arrowhead), GST protein (lane 3) and GST-AtMBD5 (indicated by a closed arrowhead) (lanes 4–6). His-tagged AtRAN3 was added prior to elution in the absence (lanes 3 and 4) or presence of GTP[γS] (lane 5) or GDP[βS] (lane 6). Proteins were then blotted onto nylon membrane, and AtRAN3 was detected with anti-His-tag antibodies (indicated by an open arrowhead) (right panel).