Figure 1

AtFKBP are unable to complex with rapamycin and AtTOR. A. Multiple alignment, using the Clustal program, of the AtFKBPs protein sequences with HsFKBP12 and ScFKBP12. Sequences are numbered according to HsFKBP12. Amino-acids involved in ternary complex formation are boxed. B. Two-hybrid analysis of the interaction between AtTOR FRB and AtFKBPs with ScFKBP as positive control. The yeast two hybrid strain AMY87-4 co expressing the GAL4(BD)::FKBP (were the FKBP used is indicated on the left of the picture) and the GAL4(AD)::AtFRB fusion proteins was spread on medium lacking adenine. Formation of the FKBP-rapamycin-FRB complex induces expression of the GAL-ADE2 reporter gene and is revealed by growth around the rapamycin disc (right). C. Pull down of native AtTOR with recombinant His-tagged ScFKBP. Track 1: Recombinant His-tagged ScFKBP. Track 2: Soluble Arabidopsis cell extract. Track 3: Recombinant His-tagged ScFKBP incubated with soluble Arabidopsis cell extract in the presence of rapamycin. Track 4: Recombinant His-tagged ScFKBP12 incubated with soluble Arabidopsis cell extract without rapamycin. Upper panel proteins were incubated with anti-AtTOR antibody (see methods). Lower panel proteins were incubated with anti-HisTag antibody.