Figure 6.

Tim50a directly interacts with coilin, SMN and SmB'. GST-pulldown assays were conducted on bacterially purified proteins. (A) Tim50a interacts with coilin. Soluble coilin was incubated with beads loaded with GST or GST fused to Tim50a. After incubation and washes, the reactions were subject to Western blotting and probing with antibodies to coilin. The input lane accounts for 26% of the coilin used in the pulldown reactions. The bottom panel is a coomassie stained gel demonstrating the levels of GST-Tim50a (lane 2) and GST (lane 3) used in the pulldown assay. (B) Tim50a interacts with the C-terminus of coilin. Soluble TV-tagged Tim50a was incubated with beads loaded with GST or GST fused to the C-terminal 214 aa of coilin (C214, the same portion of coilin used in the two-hybrid screen). After incubation and washes, the proteins were subject to Western blotting and probing with antibodies to T7. The input lane accounts for 20% of the Tim50a used in the pulldown reactions. (C) SmB' interacts with Tim50a and mutants thereof. T7-tagged SmB' was incubated with GST, GST-CAP50, GST-Tim50a or GST-Tim50aSac, followed by washing and probing with anti-T7 antibodies. The input lane accounts for 20% of the SmB' used in the pulldown reactions. (D) SMN interacts with GST-CAP50, GST-Tim50a and GST-Tim50aSac. Soluble T7-SMN was incubated with various GST fusions, followed by Western blotting and probing with anti-T7 antibodies. The input lane accounts for 10% of the SMN used in the reactions. (E) The mitochondrial Tim50 protein interacts with the C-terminus of coilin in vitro. Soluble T7-tagged Tim50 was incubated with GST or GST-C214 beads, followed by Western blotting and probing with antibodies to T7. The input lane (lane 1) accounts for 20% of the Tim50 used in the pulldown reactions. The membrane was stained with Ponceau S to verify that equivalent amounts of GST proteins were used in the assay.