Fig. 4. The sumoylation motifs of LIN-1 are necessary for the interaction with MEP-1. (A) Schematic of MEP-1 with zinc-finger motifs (black) and glutamine-rich region (gray) (Belfiore et al., 2002). (B) The interactions between LA:LIN-1 fusion proteins and MEP-1(155-859) fused to the GAL4 activation domain (GAL4AD) were measured qualitatively using the yeast two-hybrid system. A (+) indicates robust activation of a LexA-dependent lacZ reporter gene. (C) The interactions between wild-type LA:LIN-1(1-64) or the indicated mutant and MEP-1 were measured quantitatively. Bars represent the average LexA-dependent ß-galactosidase activity and lines indicate the standard deviation of at least six independent yeast transformants. The signal with LA:LIN-1(1-64) was set to 100 relative light units (RLU); the signals with mutant proteins are proportional. (D) To monitor expression of LA:LIN-1 proteins, we analyzed protein extracts from transformed yeast by western blotting using an anti-LA antibody. Lanes 1-14 correspond to LA fusion proteins listed as 1-14 in C. (E) The interaction of MEP-1 with the indicated LA:LIN-1 fusion protein was measured quantitatively. Bars represent the average of six independent yeast transformants and lines indicate the standard deviation. The signal with LA:LIN-1(158-180) was set to 100 RLU and signals with mutant proteins are proportional.