Fig. 1. Interactions between SEU and AP1/SEP3 in yeast and in vitro. (A) A yeast two-hybrid interaction assay showing positive interactions between the SEU-BD bait and the AP1-AD or SEP3-AD prey. A truncated SEU without its Q2 and C-terminal domains was used in constructing SEU-BD, which no longer self-activates in yeast (Sridhar et al., 2004). Full-length AP1, SEP3, AP3 and PI were fused to GAL4-AD. Activation of HIS3 and lacZ is indicated by growth on -HIS media and by the blue color, respectively. Red colonies indicate a lack of ADE2 reporter activation (James et al., 1996). The relative level of lacZ (ß-galactosidase) activity is shown to the right. (B) A similar yeast two-hybrid interaction assay showing positive interactions between SEU-BD and the C-terminal domain of AP1 and SEP3 (AP1-C and SEP3-C). (C) An in vitro pull-down assay showing ³⁵S-labeled AP1 and SEP3 proteins retained by GST-SEU (right). Equal amounts of in vitro translated products were loaded onto the NuPAGE gel (INPUT lanes). GST alone failed to retain any of the ³⁵S proteins (data not shown). (D) A three-protein pull-down assay with SEU-GST serving as a bridging protein. The ability of MBP-LUFS/amylose beads to retain ³⁵S-labeled AP1, SEP3 or AP3 was tested in the presence (+) or absence (-) of SEUGST. MBP was used as a negative control.