Figure 3

Mapping the rab1 binding domain (R1BD) of Iporin. (A) Overview of deletion mutants used for yeast co-transformation assays. The deletion mutants ΔNx or ΔCx do not contain the N-terminus or the C-terminus, respectively. We were able to map the rab1 binding domain (R1BD) to a RUN domain containing region. (B) Results obtained from yeast co-transformations. The bait construct rab1b Q67R 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). (C) GST-pulldown with coupled GST-Iporin ΔN847ΔC1239 indicated an interaction with Iporin, whereas GST-Iporin ΔN847ΔC992 and GST were negative. The lower panel shows the Coomassie blue stained blot membrane with equal amounts of GST fusion protein. - = no growth on selection plates or no β-galactosidase activity; +/- = β-galactosidase activity appears overnight; + = slow growth or β-galactosidase activity appears after several hours; ++ = strong growth; +++ = very fast growth or high β-galactosidase activity; pP = polyproline; pE = polyglutamic acid.