Fig. 5. Polo boxes play a crucial role in determining the protein interactions. (A) A schematic diagram of the experimental design to identify plo1 mutants that disrupt a subset of interactions (see Materials and Methods for detail). The plo1⁺ gene is randomly mutagenised by an error-prone PCR (about one mutation per kb). Yeast strain L40 was co-transformed with the PCR product and a gapped bait vector. Short sequences shared between the ends of the bait vector and the PCR products allow gap repair in vivo to recreate bait plasmids with various plo1 mutations. Each strain carrying mutagenised plo1 bait constructs was mated with Y187 strains carrying prey plasmids. The two-hybrid interaction was assessed by expression of the HIS3 reporter gene. Plasmids were isolated from strains of interest, re-transformed to confirm the interaction pattern by expression of another reporter lacZ, and sequenced to determine the mutation site. (B) Sequence analysis of some Plo1 mutants that show differential interactions. Most of the mutations mapped in the polo boxes, which suggests that polo boxes are crucial for determining protein interaction. — (no or marginal interaction) < + (weak) < ++ (intermediate) < +++ (interaction at same levels as wild-type Plo1).

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