Figure 6

Interaction of Iporin with GM130. (A) Yeast co-transformations revealed an interaction between Iporin and GM130. Using different GM130 deletion mutants, we were able to map the binding site to the coiled-coil region 6 (Figure 6A). The bait construct IporinΔN847ΔC1239 was cloned into the pAS2-1 vector and the prey constructs into pGADT7. Clones were cultivated on selection plates as described previously (see Table 1). = no growth on selection plates or no β-galactosidase activity; ++ = strong growth or β-galactosidase activity appears after a few hours; +++ = very fast growth or high β-galactosidase activity (B) GST-pulldown of endogenous GM130 with GST-IporinΔN847ΔC1239 or GST-Iporin ΔN1430. After incubation with HeLa cell extracts, GST-IporinΔN847ΔC1239 showed an interaction with GM130, whereas GST-Iporin ΔN1430 did not bind to GM130. The lower panel shows the Coomassie blue stained blot membrane with equal amounts of GST fusion protein.